Artykuły w czasopismach: „Tumoroïde”

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Yankaskas, Chris, Brittany Balhouse, Colin Paul, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Mark Kennedy, Matt Dallas i David Kuninger. "Abstract 160: Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium". Cancer Research 83, nr 7_Supplement (4.04.2023): 160. http://dx.doi.org/10.1158/1538-7445.am2023-160.

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Abstract In vitro cancer research often fails to translate to the clinic, in part due to the use of traditional 2D cancer cell lines as models, which fail to resemble primary cancer cells by a variety of measures, including high mutational burden. An emerging solution to this problem is to replace traditional cell lines with patient tissue-derived cells expanded in 3D, also known as tumoroids or cancer organoids. We developed a defined, serum- and conditioned medium-free system, GibcoTM OncoProTM Tumoroid Culture Medium, that can be used to derive stable tumoroid lines from a variety of tissue sources and maintains the phenotype and genotype of patient-derived tumor cells. By supplementing the base medium with indication-specific growth factors, tumoroid lines were derived from colorectal, lung, and endometrial cancers from both fresh surgical resections and cryopreserved primary cancer cells. To demonstrate the utility of these patient-derived cells as long-term in vitro models, colorectal and lung tumoroid lines were derived from multiple donors and cultured for up to 50 passages. Brightfield microscopy, cell counts, and next-generation sequencing were used to assess maintenance of tumoroid morphology, growth rate, gene expression patterns, and genomic mutations. Patient-derived tumoroid cultures adopted donor-specific morphologies that were maintained during long-term culture. Cell doubling time tended to stabilize within the first few passages as cultures established, was donor-dependent, and averaged around 65 hours for colorectal tumoroids - on par with that of traditional 2D cancer cell lines - and 90-100 hours for lung and endometrial tumoroids, respectively. Importantly, tumoroid lines maintained their gene expression pattern for over 20,000 human RefSeq genes during long-term culture, with correlation between initial tumor material and late-passage samples of R>0.8. Distinct molecular subtypes of colorectal cancer were preserved in cultured tumoroids. The allelic frequency of single nucleotide variations (SNVs) in 161 highly relevant cancer genes was also highly correlated (R>0.9) between uncultured tissue and late-passage tumoroids. Within SNVs, transition/transversion mutation ratios were conserved. Tumoroids were cryopreserved and recovered during this study, demonstrating that biobanking of colorectal and lung tumoroids should not impact their long-term stability. Finally, a subset of the derived colorectal and lung tumoroids were tested and shown to be tumorigenic in mice, where subsequent histology of the tumor was similar to that of in vitro cultures. Altogether, tumoroid derivation and culture in this novel medium enables the long-term preservation of patient-specific cellular genotype and phenotype, which should allow for expansion, biobanking, and performance of experimental repeats within the same patient tissue-derived cultures across labs and over time. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Mark Kennedy, Matt Dallas, David Kuninger. Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 160.

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Alvarez, Janet, Wini Zambare, Chao Wu, Paulina Bleu, Aron Bercz, Baby Satravada, Ritika Kundra i in. "An online rectal cancer tumoroid biorepository: A resource to facilitate multimodal data integration." Journal of Clinical Oncology 42, nr 3_suppl (20.01.2024): 159. http://dx.doi.org/10.1200/jco.2024.42.3_suppl.159.

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159 Background: Advancing genetic and computational technologies have increased the complexity and quantity of data available in the field of rectal cancer research. As such, there is a need for a centralized source of data to aid and organize current research efforts. To this end, our team has created a unique online biorepository of rectal cancer tumoroids which may serve as a resource for a community of researchers. Methods: The rectal cancer tumoroid biorepository was created in cBioPortal in 2015. Organoids were derived from patient tumor samples (tumoroids). These samples represented multiple treatment timepoints and included primary, recurrent, and metastatic tumors. These tumoroids were further analyzed by various assays such as MSK-IMPACT and IC50 (to fluorouracil, FOLFOX, and FOLFIRI) to obtain patient-specific genomic and chemosensitivity data. A tumoroid profile was then created in cBioPortal for each sample. Clinical information was collected by chart review and annotated into the tumoroid profiles using the unique features of cBioPortal. These included interactive filters and graphical depictions of patient demographics, clinical and pathologic staging, treatment course, clinical response, overall survival, and disease-free survival. Results were organized in an intuitive visual display for convenient analysis. Novel features such as chemosensitivity plots and an interactive patient timeline were incorporated as well. Results: In total, 401 rectal cancer tumoroids have been derived from 177 unique patients. Approximately 32% of the tumoroids were from patients 50 years or younger, and 63% of patients have a treatment naïve sample in our repository. Ten percent of tumoroids were derived from T1-T2 tumors, 56% from T3 tumors, and 17% from T4 tumors. Additionally, 14% of tumoroids were derived from primary tumor recurrences while 6% were derived from metastases. Of the 78% of tumoroids with completely annotated clinical data, 8% were MSI-H, 11% were clinical complete responders, and 5.5% were pathologic complete responders. Lastly, complete IC50 data with clinical correlates has been established for 20% of tumoroids. These data show that tumoroids with a high sensitivity to chemotherapy (FOLFOX) correlated with a 3.3-fold increase in clinical complete response rates in the corresponding patients (p=0.0263). Conclusions: Integrating histopathological, clinicogenomic, and tumoroid response data, this expanding online resource provides a platform to identify valid biomarkers for patient treatment response. This publicly available biorepository built within cBioPortal is intuitive and flexible to accommodate and organize a wide range of data and has the potential to serve as an important resource in rectal cancer research.

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Danen, Erik H. J. "Abstract 5226: A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology". Cancer Research 84, nr 6_Supplement (22.03.2024): 5226. http://dx.doi.org/10.1158/1538-7445.am2024-5226.

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Abstract We developed a screening platform for immune oncology using an assay based on automated image guided injection of tumoroids in wells of multi-well plates preloaded with a collagen-rich ECM. The identical x-y-z position and size of the ECM-embedded tumoroids in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. The model is used to generate quantitative data for T cell recruitment to tumoroids and killing of tumoroids by T cells. We have applied this to screening a panel of CD3:Her2 bispecific antibodies (BsAb) binding with different affinities to CD3 or Her2 or displaying high affinity interactions with different epitopes on Her2. Exposure to fresh, non-activated peripheral blood mononuclear cell (PBMC) derived T cells shows an initial phase of random T cell movement throughout the ECM followed by a BsAb-dependent phase of active T cell recruitment to tumoroids (day 2-4) and a subsequent phase of tumoroid killing (day 4-6). We show that the wave of T cell recruitment following initial T cell-tumoroid contact involves chemotactic signaling. Decreased affinity at the Her2 or CD3 arm can be compensated for by increasing BsAb concentrations. However, we detect major differences in efficacy for different high affinity epitopes. I.e., of two BsAbs interacting with high affinity with distinct Her2 epitopes and each causing effective tumor cell killing in 2D co-culture, only one was able to trigger a wave of T cell recruitment and subsequent tumoroid killing in 3D. We have applied the same setup to testing the efficacy of T cells expressing engineered TCRs aimed at application in adoptive T-cell therapy. Kinetics of experiments using these activated engineered T cells are considerably shorter (24 hours instead of ~6 days) but show a similar pattern of T cell recruitment and subsequent tumoroid killing. We demonstrate successful generation of quantitative data for T cell recruitment and tumoroid killing for engineered T cells targeting tumor antigens expressed on TNBC and uveal melanoma tumoroids. Citation Format: Erik H. J. Danen. A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5226.

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Zambare, Wini, Chao Wu, Hannah Kalvin, Hanchen Huang, Sara Yoder, Michael Del Latto, Maria Kierstead i in. "Abstract A012: Development of a translational tumoroid-organoid platform revealing tumor-specific radiosensitization in rectal cancer using matched patient-derived models". Clinical Cancer Research 31, nr 2_Supplement (26.01.2025): A012. https://doi.org/10.1158/1557-3265.targetedtherap-a012.

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Abstract Background: Radiation therapy in rectal cancer treatment is limited by variable tumor responses among patients and harmful effects on normal tissues. We developed a translational human tumoroid-organoid platform to assess novel tumor-specific strategies for radiation sensitization using matched patient-derived models. Methods: Fifteen rectal cancer-derived tumoroids and three matched normal tissue-derived organoids were established from patients, representing primary tumors, metastases, and recurrences. Four tumoroids were derived from a single patient, encompassing different disease stages: primary tumor and splenic metastasis (pre-progression), and rectal and vaginal recurrences (post-progression). To evaluate tumor-specific radiation sensitizers, both tumoroids and their matched normal organoids were treated with 5-fluorouracil (5-FU), one of four DNA damage repair inhibitors (DDRis; ATMi, DNA-PKi, PARPi, or ATRi), or a DMSO control. Following treatment, samples were irradiated, and cell viability was measured. We assessed intrinsic radiation sensitivity under control conditions and radiosensitizer efficacy using a linear regression model with log-transformed cell viability as the outcome. Whole-exome sequencing characterized the mutation profiles of the tumoroids. Results: Intrinsic radiosensitivity was heterogeneous among tumoroids, with a 10–28% decrease in cell growth per unit increase in radiation dose. Critically, when comparing tumoroids to their matched normal organoids, we observed greater sensitization in tumoroids across all cases and for all DDRis, demonstrating tumor-specific radiosensitization. The most potent radiosensitizer relative to DMSO was DNA-PKi in 7 tumoroids, ATMi in 4 tumoroids, and PARPi in 1 tumoroid. Tumoroids derived after disease progression exhibited increased resistance to radiation and a diminished degree of sensitization with DDRi treatment compared to pre-progression tumoroids. Despite variations in radiation sensitivity and DDRi responses, the genetic profiles of the tumoroids remained largely unchanged. Conclusion: We developed a translational ex vivo tumoroid-organoid platform using matched patient-derived models to test tumor-specific radiation sensitizers in rectal cancer. This platform allowed us to determine tumor-specific sensitization by directly comparing tumoroids with matched normal organoids, demonstrating greater sensitization in tumoroids. Our findings highlight the potential of this platform to uncover precise, tumor-selective treatment options, improve patient responses, reduce toxicity, and address resistant tumors in patients with disease progression. Citation Format: Wini Zambare, Chao Wu, Hannah Kalvin, Hanchen Huang, Sara Yoder, Michael Del Latto, Maria Kierstead, Satoru Meguro, Xi Steven Chen, Mithat Gonen, J. Joshua Smith, Paul B. Romesser. Development of a translational tumoroid-organoid platform revealing tumor-specific radiosensitization in rectal cancer using matched patient-derived models. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A012.

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Truelsen, Sarah Line Bring, Nabi Mousavi, Haoche Wei, Lucy Harvey, Rikke Stausholm, Erik Spillum, Grith Hagel i in. "The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids". PLOS ONE 16, nr 7 (7.07.2021): e0253258. http://dx.doi.org/10.1371/journal.pone.0253258.

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The treatment response to anti-angiogenic agents varies among cancer patients and predictive biomarkers are needed to identify patients with resistant cancer or guide the choice of anti-angiogenic treatment. We present “the Cancer Angiogenesis Co-Culture (CACC) assay”, an in vitro Functional Precision Medicine assay which enables the study of tumouroid induced angiogenesis. This assay can quantify the ability of a patient-derived tumouroid to induce vascularization by measuring the induction of tube formation in a co-culture of vascular cells and tumoroids established from the primary colorectal tumour or a metastasis. Furthermore, the assay can quantify the sensitivity of patient-derived tumoroids to anti-angiogenic therapies. We observed that tube formation increased in a dose-dependent manner upon treatment with the pro-angiogenic factor vascular endothelial growth factor A (VEGF-A). When investigating the angiogenic potential of tumoroids from 12 patients we found that 9 tumoroid cultures induced a significant increase in tube formation compared to controls without tumoroids. In these 9 angiogenic tumoroid cultures the tube formation could be abolished by treatment with one or more of the investigated anti-angiogenic agents. The 3 non-angiogenic tumoroid cultures secreted VEGF-A but we observed no correlation between the amount of tube formation and tumoroid-secreted VEGF-A. Our data suggests that the CACC assay recapitulates the complexity of tumour angiogenesis, and when clinically verified, could prove a valuable tool to quantify sensitivity towards different anti-angiogenic agents.

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Yankaskas, Chris, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas i David Kuninger. "Abstract 4251: Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium". Cancer Research 84, nr 6_Supplement (22.03.2024): 4251. http://dx.doi.org/10.1158/1538-7445.am2024-4251.

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Abstract Tumoroids, also known as cancer organoids, are self-assembled three-dimensional cultures of patient-derived tumor cells. Compared to traditional 2D cancer cell lines, tumoroids better maintain key characteristics of the original tumor including the genotype and transcriptome, which lead to better in vitro modeling of clinically relevant functional responses such as drug sensitivity. An under-studied aspect of tumoroid cultures has been their ability to recapitulate dependence on hormone signaling in vitro, with some reports indicating hormone dependence is lost over time in culture. To investigate this, we utilized Gibco™ OncoPro™ Tumoroid Culture Medium and defined tissue-specific supplements, FGF10 and beta-estradiol, that when added to the medium enabled the derivation of endometrial tumoroids from tumor resections. Derivation of tumoroid lines compatible with long-term growth depended on a variety of factors, including tissue quality and time from resection to culture initiation. Each of the 15 samples processed formed tumoroids when initially seeded into culture. 11/15 samples reformed tumoroids through multiple passages (typically 1-2 weeks per passage) over 1-3 months in culture. 7/15 samples met our high criteria for stable tumoroid line growth of >5 passages and reaching >5 cumulative population doublings (from initial number of cells seeded post-resection) in culture. Next-generation sequencing of the established tumoroids and the uncultured tumor material demonstrated a high degree of correlation (pearson’s r>0.9) of genomic mutations in a targeted panel; few differentially expressed genes were detected beyond down-regulation of immune-related genes, presumably because the medium is optimized for outgrowth of cancer epithelial cells, and immune cells do not persist during long-term culture. Endometrial tumoroids were capable of growing embedded in basement membrane extract (BME) or in suspension with dilute BME added and could be cryopreserved and recovered. Endometrial tumoroids retained gene expression levels (both presence and absence) of estrogen receptor and progesterone receptor for up to the 20 passages tested in vitro. A subset of the models was tested and showed increased growth rates in response to increased concentrations of beta-estradiol. Of those tested, 2/3 tumoroid cultures stopped growing within 1-2 weeks after removal of beta-estradiol from the medium, demonstrating dependence on this hormone for growth. Maintenance of hormone receptor expression and dependency should enable in vitro studies to understand the signaling mechanisms and potential ways to modulate them in the context of cancer. Overall, this method provides a toolset for studying endometrial cancer, particularly hormone dependency, and may be more broadly applicable to other gynecological or hormone dependent cancers. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas, David Kuninger. Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4251.

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Kim, Sung Min, Yoo Ri Ko, Hye Seon Park i Se Jin Jang. "Abstract 218: Deciphering tumoroid-CAF interactions through a spatially segregated coculture model". Cancer Research 84, nr 6_Supplement (22.03.2024): 218. http://dx.doi.org/10.1158/1538-7445.am2024-218.

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Abstract In the context of lung cancers, cancer-associated fibroblast (CAF) plays a pivotal role as a key component of the tumor microenvironment (TME), affecting tumor growth, invasion, metastasis, immune modulation, and drug resistance. Despite the recognized impact of CAFs, the precise interaction between the CAF and tumor cell remains elusive. Previous studies have reported that CAFs stimulate tumor cell growth both in vitro and in vivo. However, our tumoroid models reveal an intriguing phenomenon where CAFs inhibit tumoroids growth within Matrigel. This led us to hypothesize that paracrine signaling might be the primary mode of interaction between CAFs and tumor cells. To unravel the intricacies of this interaction, we developed an innovative coculture method. Using hydrophobic barrier, we segregated the coculture of nine sets of CAFs and tumoroids within a single well, preventing CAFs from infiltrating the tumoroid culture area. This setup enabled interaction solely through paracrine signaling. Surprisingly, cocultured tumoroids exhibited no significant differences compared to individually cultured tumoroids. Conversely, cocultured CAFs displayed a remarkable increase in growth compared to their individually cultured counterparts. This suggests that tumoroids influence CAF growth, while CAFs do not significantly impact tumoroid growth. Furthermore, in three coculture sets, tumor cells within the Matrigel migrated towards the CAF culture area, indicating that CAFs induce tumor cell metastasis. Immunofluorescence staining confirmed this metastatic phenomenon. To identify the paracrine factors influencing CAFs and tumoroids, we employed RNA-sequencing, single-cell RNA-sequencing, and proteomic analysis of culture soup. Proteomic analysis revealed a substantial increase in proteins and signaling pathways related to the extracellular matrix in cocultures compared to single cultures. Our findings suggest that tumor cells recruit CAFs, promoting CAF growth to establish and fortify their TME, ultimately leading to drug resistance, metastasis, and immune modulation Citation Format: Sung Min Kim, Yoo Ri Ko, Hye Seon Park, Se Jin Jang. Deciphering tumoroid-CAF interactions through a spatially segregated coculture model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 218.

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Taha, Eman, Chiharu Sogawa, Yuka Okusha, Hotaka Kawai, May Oo, Abdellatif Elseoudi, Yanyin Lu i in. "Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles". Cancers 12, nr 5 (16.05.2020): 1260. http://dx.doi.org/10.3390/cancers12051260.

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The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Paul, Colin, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas i David Kuninger. "Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models". Cancer Research 84, nr 6_Supplement (22.03.2024): 2047. http://dx.doi.org/10.1158/1538-7445.am2024-2047.

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Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV<2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2047.

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Sogawa, Chiharu, Takanori Eguchi, Yuri Namba, Yuka Okusha, Eriko Aoyama, Kazumi Ohyama i Kuniaki Okamoto. "Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer". Cells 10, nr 2 (6.02.2021): 344. http://dx.doi.org/10.3390/cells10020344.

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Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

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Scholtes, Mathijs P., Maryam Akbarzadeh, Shahla Romal, Miranda van Dijk, Tsung Wai Kan, Tokameh Mahmoudi i Tahlita C. Zuiverloon. "Abstract 2852: Targeting NPEPPS overcomes cisplatin resistance in patient-derived bladder cancer tumoroids". Cancer Research 83, nr 7_Supplement (4.04.2023): 2852. http://dx.doi.org/10.1158/1538-7445.am2023-2852.

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Abstract Introduction and Objectives: Cisplatin-based neoadjuvant chemotherapy (NAC) is the recommended treatment for muscle-invasive bladder cancer (MIBC) patients. Patients with a pathological complete response after NAC (pCR), have a good 5-yr OS of 80%. However, due to NAC resistance, only 25% of patients achieve a pCR. Our aim is to increase the number of patients reaching a pCR by overcoming cisplatin resistance. Previously we identified NPEPPS as a key player in cisplatin resistance that prevents cisplatin uptake via volume-regulated anion channels (VRAC) in cisplatin-resistant cell lines. Studies have demonstrated that patient-derived bladder cancer tumoroids recapitulate the originating tumor at the molecular level, presenting a model system to study chemotherapy resistance ex vivo. Here, we used MIBC tumoroids to functionally interrogate NPEPPS’ role in cisplatin resistance. Material and Methods: Ex vivo interrogation of NPEPPS function in cisplatin resistance was performed in tumoroid cultures generated from MIBC patients (N=10). Tumor tissue was obtained either at transurethral resection of bladder tumor (TURBT, N=5) or RC (N=5), and clinical resistance to NAC was defined as ypT≥2, nodal positive disease at RC or progression during NAC. Molecular characterization of tumor specimens and corresponding tumoroids was performed by bladder cancer-specific SNaPshot mutation analysis, copy-number aberrations analysis, immunohistochemistry (IHC), and hematoxylin & eosinophilic staining (HE). The effect of NPEPPS on cisplatin uptake was investigated using cisplatin treatment combined with the pharmacological NPEPPS-inhibitor tosedostat, shRNA-mediated NPEPPS knock-down or lentiviral NPEPPS over-e xpression followed by cytometry by time of flight (CyTOF) analysis. CellTiter-Glo cell viability assays and caspase 3/7 apoptosis assays were used to investigate whether NPEPPS inhibition overcomes cisplatin resistance ex vivo. Results: Five out of ten patients received NAC, all whom had tumors that were clinically resistant to NAC. Tumoroid cultures from MIBC patients unresponsive to NAC showed resistance to serum concentrations of cisplatin as well as increased NPEPPS mRNA expression. Meanwhile, molecular characterization of tumor and tumoroid pairs confirmed that patient-specific tumor traits were maintained in tumoroids. Pharmacological NPEPPS inhibition or NPEPPS depletion by shRNA-mediated knock-down increased cisplatin influx into the cell and resensitized NAC resistant tumoroids to serum concentrations of cisplatin. Furthermore, NPEPPS overexpression increased cisplatin resistance in cisplatin-sensitive tumoroids. Conclusion: We confirm that NPEPPS is associated with cisplatin-resistance in an ex vivo MIBC tumoroid model. These findings have potential for rapid translation into the clinic and invite trials investigating tosedostat to overcome chemoresistance. Citation Format: Mathijs P. Scholtes, Maryam Akbarzadeh, Shahla Romal, Miranda van Dijk, Tsung Wai Kan, Tokameh Mahmoudi, Tahlita C. Zuiverloon. Targeting NPEPPS overcomes cisplatin resistance in patient-derived bladder cancer tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2852.

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Balhouse, Brittany, Chris Yankaskas, Colin Paul, Shyanne Salen, Sylvia Beam, Pradip Shahi Thakuri, Matt Dallas i David Kuninger. "Abstract 227: Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture". Cancer Research 84, nr 6_Supplement (22.03.2024): 227. http://dx.doi.org/10.1158/1538-7445.am2024-227.

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Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses in the United States. Patients with metastatic TNBC have a low survival rate, driven in part by limited treatment options for the disease. Tumoroids, also known as cancer organoids, have been shown to better maintain tumor-specific mutational and gene expression profiles over long-term culture, and evidence is growing that the functional response of tumoroids to therapeutics better recapitulates in vivo response. These improved preclinical models of TNBC may facilitate the development of new therapies, but tumoroids are yet to be widely adopted due to challenging culture methods. In this study, we show that two TNBC tumoroid models publicly available through the National Cancer Institute Patient-Derived Models Repository can be expanded in suspension culture using Gibco™ OncoPro™ Tumoroid Culture Medium supplemented with FGF10 and beta-estradiol. We compared the bulk tumoroid morphologies, growth rates, maintenance of mutational and gene expression profiles, and molecular phenotypes of cells grown in the NCI-recommended medium in embedded culture, and the OncoPro medium in embedded culture and suspension culture. We saw comparable morphologies and growth rates in all culture conditions. A multivariate analysis of the allelic frequencies of single nucleotide variants and ploidy values of copy number variants for cells expanded in all experimental conditions revealed >95% and >88% correlation, respectively, with the initial material. Interestingly, despite the change in the medium formulation, gene expression was also highly correlated between the initial material and the OncoPro medium conditions for >20,000 RefSeq genes (Pearson r>0.94) and for a cancer-gene specific panel of 1,423 genes (r>0.87). Further analysis showed unchanged expression levels in 97% of total genes analyzed and the basal molecular subtype was maintained for both lines in all conditions. The OncoPro media system was also used to derive a new TNBC tumoroid line, which demonstrates consistent growth rate and maintains the patient-specific mutational profile of the original breast tumor. Differential gene expression analysis revealed that changes in the gene expression profile were largely due to the loss of non-malignant cell populations from the original tumor. Finally, we utilized this new tumoroid line to examine donor-specific killing efficiencies of primary natural killer cells in a co-culture experiment. The discrepancy in the dynamics of killing a tumoroid line versus the standard K562 cell line indicates that tumoroids may offer an improved approach for screening new cell therapies. In summary, the easy-to-adopt OncoPro medium and culture method does not change the characteristics of existing models and may be used to derive new tumoroid lines, both of which can be leveraged for preclinical studies to develop critical new therapies for TNBC. Citation Format: Brittany Balhouse, Chris Yankaskas, Colin Paul, Shyanne Salen, Sylvia Beam, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 227.

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Jensen, Lars Henrik, Anders Kristian Moeller Jakobsen, Birgitte Mayland Havelund, Cecilie Abildgaard, Chris Vagn-Hansen, Claus Dam, Jan Lindebjerg i in. "Functional precision medicine in colorectal cancer based on patient-derived tumoroids and in-vitro sensitivity drug testing." Journal of Clinical Oncology 39, nr 15_suppl (20.05.2021): e15567-e15567. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e15567.

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e15567 Background: Precision oncology based on in-vitro, functional assays has potential advantages compared to the much more common molecular approach, but the clinical benefit is unknown. We here report the results from the largest prospective interventional clinical trial testing the clinical outcome in colorectal cancer patients treated with drugs showing cytotoxic effect in matched patient-derived tumoroids. Methods: This single-center, phase II trial included patients with metastatic colorectal cancer previously exposed to all standard therapies. Specimens from one to three 18-16 G core needle biopsies were manually dissected, enzymatically treated, cultivated, and incubated to form 3D spherical microtumors, i.e. tumoroids. In the assay for in-vitro sensitivity testing, the tumoroids were challenged with single drugs and combinations thereof to determine patient-specific responses. Using tumoroid screening technology (IndiTreat, 2cureX, Copenhagen, Denmark), results were generated by comparing the sensitivity of the individual patient’s tumoroids with a reference panel from other patients. The testing included standard cytostatics and drugs with proven effect in previous early-phase clinical trials, a total of 15 drugs. The primary endpoint was the fraction of patients with progression-free survival (PFS) at two months. Based on placebo arms in randomized last-line trials, a minimal relevant difference of 20% (20% to 40%) was stated. Using Simon's two-stage design, a sample size of 45 patients was calculated with at least 14 PFS at two months (significance 5%, power 90%). Results: Ninety patients were enrolled from 9/2017 to 9/2020. Biopsies from 82 patients were obtained and sent for tumoroid formation of which 44 (54%, 95% CI 42-65) were successful and at least one treatment was suggested. Thirty-four patients initiated treatment according to the response obtained in the drug assays within a median of 51 days from inclusion (IQR 39-63). The primary endpoint, PFS at two months, was met in 17 of 34 patients (50%, 95%CI 32-68). There were no radiological responses. Median PFS was 81 days (95% CI 51-112) and median OS was 189 days (95% CI 103-277). Conclusions: Precision oncology using a functional approach with patient-derived tumoroids and in-vitro drug sensitivity testing seems feasible. The approach is limited by the fraction of patients with successful tumoroid development. The primary endpoint was met, as half of the patients were without progression at two months. Further clinical studies are justified. Clinical trial information: NCT03251612.

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Nikolov, Ekaterina, Oksana Sirenko, Matthew Hammer, Courtney Brock, Anthony Thai, Matthew Burow, Bridgette Collins-Burow i Evan Cromwell. "Abstract 185: Monitoring metabolite dynamics in patient-derived tumoroids using automated microfluidic Pu·MA System". Cancer Research 82, nr 12_Supplement (15.06.2022): 185. http://dx.doi.org/10.1158/1538-7445.am2022-185.

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Abstract Introduction: Cancer cells are characterized by metabolic modifications to meet high bioenergetic and anabolic demand during tumorigenesis. For most tumors aerobic glycolysis is a major driver of cancer progression, resistance to therapy, poor patient outcome. Lactate, a product of glycolysis, plays a role in tumor progression and is linked to drug resistance in breast cancer. Patient-derived 3D cell models are valuable tools for research, drug development and personalized medicine, because they recapitulate features of tumor microenvironment. Therefore, monitoring metabolite dynamics and lactate production in physiologically relevant patient-derived models provides valuable data for understanding metabolic perturbations during disease progression, drug response and resistance. In this study we assayed the glucometabolic changes in response to anti-cancer drugs in triple negative breast cancer (TNBC) tumoroids. Our approach utilizes an automated 3D assay system. The Pu·MA system with microfluidic flowchips allows for in situ supernatant sampling, sensitive luminescence Lactate-Glo assay and high content imaging. Methods: Tumoroids were formed in ULA plate from primary cells from a TNBC biopsy, TU-BcX-4IC (4IC). Single tumoroids were assayed in flowchips in the Pu·MA system. The workflow comprised of a) measuring pre-treatment tumoroid viability with RT-Glo assay b) time-course automated supernatant sampling for measuring lactate using Lactate-Glo assay and c) post-treatment imaging of viability response for IC50 calculation. Results: RT-Glo assay of 4IC tumoroids showed sufficient signal-to-background ratio of 44 and signal-to-noise ratio of 690. Before treatment, the coefficient of variation of signal from samples was 26% indicating high starting uniformity of tumoroid size and viability, which is important for the validity of the conclusions. Treatment of 4IC tumoroids for 48 hr with paclitaxel, romidepsin (HDAC1/2 inhibitor) and trametinib (MEK1/2 inhibitor) showed varying sensitivities to these drugs. IC50 values for romidepsin and trametinib were ~10 nM, as compared to paclitaxel >1 μM, which is much greater than typically reported for breast cancer models. This is consistent with previously published taxane resistance of 4IC primary tumor. Tumoroid treatment of over 9 - 12 hr also resulted in statistically significant increase in lactate secretion for paclitaxel (p < 0.05) and romidepsin (p < 0.01) indicating drug-induced shift toward glycolysis. These results corroborate previous publications linking treatment with paclitaxel to increased lactate production and chemoresistance in breast cancer. Conclusions: Profiling of patient-derived tumoroids using the automated Pu·MA System for dynamic metabolic changes shows great potential for studying disease progression, drug resistance, identifying new therapies and advancing personalized medicine. Citation Format: Ekaterina Nikolov, Oksana Sirenko, Matthew Hammer, Courtney Brock, Anthony Thai, Matthew Burow, Bridgette Collins-Burow, Evan Cromwell. Monitoring metabolite dynamics in patient-derived tumoroids using automated microfluidic Pu·MA System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 185.

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Meulenbroeks, Chris, Phylicia Stathi, Marc van de Wetering, Chio Carranza Villarejo, Norman Mack, Benjamin Schwalm, Neal Geisemeyer i in. "MDB-68. DISCOVERING DRUG SENSITIVITIES IN MEDULLOBLASTOMA USING PATIENT- AND PDX-DERIVED TUMOROIDS". Neuro-Oncology 26, Supplement_4 (18.06.2024): 0. http://dx.doi.org/10.1093/neuonc/noae064.517.

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Abstract BACKGROUND Medulloblastoma (MB) is one of the most common malignant pediatric brain tumor types and is subdivided into four major types: WNT, SHH, Group 3 and Group 4. Treatment, including resection, cranio-spinal radiotherapy, and chemotherapy, often leads to undesirable long-term side effects. MB prognosis depends on type, subtype, and genetic alterations, reflecting the high level of heterogeneity observed in these tumors and indicating that specific patient populations with particularly poor prognosis require alternative treatment approaches. Therefore, preclinical models are essential for recapitulating MB heterogeneity and developing subtype-specific treatments. METHODS Long-term in vitro MB tumoroid models were developed from patient and PDX tumors and molecularly characterized using DNA methylation and transcriptomic analyses. High-throughput single drug screening (225 compounds) and combination screening were performed to identify (subtype-specific) drug sensitivities. RESULTS Fifteen tumoroid lines were established from TP53-mutated SHH (n=6), Group 3 (G3) MYC-amplified (n=4) and non-amplified (n=5) patient (n=6) and PDX (n=9) tumors. DNA methylation analyses confirmed the maintenance of molecular identity in these tumoroids after long-term culture (>5 passages). Transcriptome analyses reveal a more proliferative, progenitor-like phenotype in these tumoroids compared to patient/PDX tumors. Hight-throughput drug screens identified both common and type-specific drug sensitivities within MB. Notably, drugs targeting CDK7, CDK12, CHECK1, BCL2L, and HDAC demonstrated efficacy in both G3 and TP53-mutated SHH-MB, while TOP1/2 and MDM2 inhibitors were specific for G3-MB. Currently, these drugs are being further investigated to select the most effective synergistic drug combinations for in vivo validation in PDX models. CONCLUSIONS Thus far, we have established already fifteen MB tumoroid models representing SHH-MB and G3-MB and identified several drug targets, including cell cycle kinases and HDACs. Tumoroids closely resemble patient tumors, based on methylome and transcriptome, enhancing their relevance for drug discovery applications in a high-throughput fashion and for prioritizing new treatment strategies for MB patients in clinical trials.

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Paul, Colin, Brittany Balhouse, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Matt Dallas i David Kuninger. "Abstract 177: Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system". Cancer Research 83, nr 7_Supplement (4.04.2023): 177. http://dx.doi.org/10.1158/1538-7445.am2023-177.

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Abstract Tumoroid technology enables culture of patient tissue-derived cancer cells in 3D, with retention of key characteristics from the original patient tumor. However, current tumoroid culture relies on labor-intensive media formulations and culture workflows. To address these issues, we have developed the serum- and conditioned medium-free Gibco™ OncoPro™ Tumoroid Culture Medium to enable derivation and expansion of patient-derived lines from multiple cancer indications. The system has been optimized to retain key patient genotypic and phenotypic characteristics during in vitro culture in a Wnt agonist-free system that can be easily adopted and transferred between labs. To test the applicability of our system with previously established tumoroid/cancer organoid models, we procured publicly available cancer models from the National Cancer Institute Patient-Derived Models Repository (NCI PDMR). Multiple colorectal, lung, pancreas, and head and neck tumoroid models were tested, including tumoroids originally derived in conditioned medium containing Wnt-3A, R-spondin 3, and Noggin. Cells were assessed for survival, growth, and fidelity to starting material in both OncoPro Tumoroid Culture Medium and in PDMR-recommended homebrew media. Growth and morphology of tumoroid cells was monitored and was comparable between culture conditions. To test for genetic stability of cultures over time, the mutational status of organoids expanded in each condition was characterized using targeted NGS. Both SNV allelic frequency and ploidy values were conserved from initial starting material following expansion to cryopreservation-competent banks (>10e6 cells). Gene expression levels across over >20,000 human RefSeq genes were compared between culture methods and showed high (>0.9) correlation, and Wnt-related signaling pathways were not differentially regulated between media types. In select cases, tumoroid cells expanded in each condition were also analyzed by flow cytometry using optimized antibody panels and protocols. Total cell viability and expression of EpCAM, CD45, CD31, smooth muscle actin, vimentin, CDX2, CEACAM, and cytokeratin 7 was nearly identical across culture conditions. Taken together, our approach represents a simple and effective method to expand and maintain patient-derived human cancer organoids in vitro, with no clear adverse effects from moving to a Wnt-free system. Citation Format: Colin Paul, Brittany Balhouse, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 177.

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Lê, Hélène, Joseph Seitlinger, Véronique Lindner, Anne Olland, Pierre-Emmanuel Falcoz, Nadia Benkirane-Jessel i Eric Quéméneur. "Patient-Derived Lung Tumoroids—An Emerging Technology in Drug Development and Precision Medicine". Biomedicines 10, nr 7 (12.07.2022): 1677. http://dx.doi.org/10.3390/biomedicines10071677.

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Synthetic 3D multicellular systems derived from patient tumors, or tumoroids, have been developed to complete the cancer research arsenal and overcome the limits of current preclinical models. They aim to represent the molecular and structural heterogeneity of the tumor micro-environment, and its complex network of interactions, with greater accuracy. They are more predictive of clinical outcomes, of adverse events, and of resistance mechanisms. Thus, they increase the success rate of drug development, and help clinicians in their decision-making process. Lung cancer remains amongst the deadliest of diseases, and still requires intensive research. In this review, we analyze the merits and drawbacks of the current preclinical models used in lung cancer research, and the position of tumoroids. The introduction of immune cells and healthy regulatory cells in autologous tumoroid models has enabled their application to most recent therapeutic concepts. The possibility of deriving tumoroids from primary tumors within reasonable time has opened a direct approach to patient-specific features, supporting their future role in precision medicine.

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Ruedlinger, Brittney, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart i Soner Altiok. "Abstract 4557: 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response". Cancer Research 83, nr 7_Supplement (4.04.2023): 4557. http://dx.doi.org/10.1158/1538-7445.am2023-4557.

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Abstract Introduction: Recent studies showed that patients with deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) tumors, have higher sensitivity to immune checkpointinhibitors (ICIs) compared to patients with microsatellite-stable (MSS)/microsatellite instability-low(MSI-L) tumors. However, the mechanisms of treatment responsiveness and resistance is not wellunderstood. Here, we used a novel 3D-EXpress ex vivo fresh patient tumoroid platform to assess theefficacy of nivolumab/ipilimumab combination therapy in tumors with known MSS/MSI-H status andperformed correlative studies. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Tumoroids measuring 150 µm in size retaining tumor cell heterogeneity, tumor-residentimmune cells, stromal components, and cell-extracellular matrix interaction were prepared frompatients with endometrial and colorectal tumors among others. Tumoroid aliquots were cryopreservedin Nilogen’s tumoroid Biorepository for future studies. For the 3D-EXpress studies cryopreservedtumoroids were selected based on MSS/MSI-H status and treated ex vivo with nivolumab andipilimumab alone and in combinations for 72h. Tumor responses to treatments were evaluated by aproprietary tumor cell killing assay and changes in tumor immune microenvironment. Furthermore,tumor PD-L1 expression levels were analyzed on the associated TMA slides. Results: Treatment-induced tumor cell killing activity in intact tumoroids was assessed by a 3D high-content confocal imaging technique using a proprietary algorithm for data analysis. The impact of exvivo treatment by nivolumab and/or ipilimumab on tumor resident immune cell populations wasmonitored by a multiplex cytokine release assay analyzing release of GM-CSF, sCD137, IFNγ, sFas, sFasL,Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin usingculture supernatants isolated from treated tumoroid samples. Based on the ex vivo responses tumorswere assigned to treatment sensitive and resistant groups and correlative analyses were performed withindividual tumors’ innate and adaptive immune cell populations detected by a 21-color flow cytometrypanel, in addition to tumor MSS/MSI-H and p53 status, and detailed clinicopathologic data readilyavailable for the tumors cryopreserved in the Biorepository. Conclusion: The 3D-Express platform, using cryopreserved 3D tumoroids with intact TME is an effectivetool for the assessment of rational combinations which may prove relevant in the treatment of solidtumors. Furthermore, we believe this platform is a useful tool for the pre-clinical assessment of specifictherapeutic regimens designed for individualized patient care. Citation Format: Brittney Ruedlinger, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart, Soner Altiok. 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4557.

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Paul, Colin, Amber Bullock, Anthony Chatman, Brittany Balhouse, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas i David Kuninger. "Abstract 4233: Rapid and robust generation of ex vivo tumoroid samples from fresh patient samples in a serum-free, conditioned medium-free system". Cancer Research 84, nr 6_Supplement (22.03.2024): 4233. http://dx.doi.org/10.1158/1538-7445.am2024-4233.

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Abstract Functional precision oncology describes a method in which an individual patient’s cancer cells are tested for response to various treatments ex vivo to aid in therapy selection. In many cases, these cultures take the form of tumoroids, which are 3D, patient-derived “mini tumors” that retain phenotypic and genotypic characteristics of the patient cancer. While interest in functional precision oncology is increasing due to strong correlation between patient responses and those experimentally observed using matched tumoroid models in the literature, a lack of standardization has precluded its widespread translation to clinical settings. To study the ability to generate short-term tumoroid cultures using a commercially available, serum-free, and conditioned medium-free system, we processed fresh tumor resection samples and performed culture in Gibco™ OncoPro™ Tumoroid Culture Medium. Tumor resection samples spanning a variety of indications were stored in supplemented Hibernate™-A Medium (including antibiotics) and shipped overnight on gel packs to a central tissue processing site. Samples were minced, enzymatically dissociated, and counted. Average yields of ~2000-12,000 cells per mg of tumor resection tissue were observed, with lower yields in lung, head and neck, and breast cancer samples and higher yields in endometrial and colorectal cancer samples. Donor-to-donor variability generated a wide range of yields within a given indication. Similarly, cell viability following dissociation ranged widely, reaching as low as 20% for some samples and exceeding 90% for others. Though yields and initial viability varied, dissociated cells that were plated in OncoPro Tumoroid Culture Medium formed tumoroids in >85% of samples within 7 days of tissue receipt. Both mutational profiles and gene expression patterns were highly conserved (>90%) between initial tumor samples and cells cultured in OncoPro for 7 days. Computational approaches designed to identify cell-type specific signatures from bulk RNA sequencing data indicated that immune cell population signatures were present after one week in OncoPro medium. Additionally, we have developed methods to analyze tumoroid response to compounds and candidate cell therapies by both plate reader- and imaging-based analyses using ~1.2e6 initial tumor cells per 96-well plate. Due to the high success rate of tumoroid formation from fresh tumor samples and retention of donor-specific characteristics in OncoPro Tumoroid Culture Medium, we believe that it will be a valuable tool for functional precision oncology of solid tumor samples. Citation Format: Colin Paul, Amber Bullock, Anthony Chatman, Brittany Balhouse, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Rapid and robust generation of ex vivo tumoroid samples from fresh patient samples in a serum-free, conditioned medium-free system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4233.

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Currlin, Seth, Brittney Ruedlinger, Sharon Camacho, Angie Rivera, Jasmin D'Andrea, Jared Ehrhart i Soner Altiok. "Abstract 4552: 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy". Cancer Research 83, nr 7_Supplement (4.04.2023): 4552. http://dx.doi.org/10.1158/1538-7445.am2023-4552.

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Abstract Introduction: Cancer associated fibroblasts (CAFs) are a major component of the tumormicroenvironment (TME) and exhibit diverse tumorigenic functions such as immunosuppressionand extracellular matrix (ECM) remodeling. Treatment of solid tumors is often hindered by acomplex TME, which persists despite effective tumor-cell directed therapies. Therefore,treatment of solid tumors in combination with stromal-targeting therapies is essential toovercome CAF-facilitated tumor growth, and immunosuppression. Herein we interrogate theimpact of tumor stroma targeting therapeutics, in combination with nivolumab, on the efficacy oftumor cell killing (TCK) in a 3D human tumoroid ex vivo culture model (3D-EXplore). Resultingdata suggests that impairing the recruitment and activation of CAFs within the TME leads toenhanced TCK when combined with immune checkpoint blockade therapy. Materials and Methods: All patients tumor samples were collected with patient consent andrelevant IRB approval. 3D tumoroids measuring 150 microns in size were generated from freshpatient tumors including endometrial, ovarian, and colorectal cancer tissues. Tumoroids werethen treated with stromal targeting strategies for TGF-beta (galunisertib), the FGF pathway(Dovitinib), FAK inhibitors (defactinib), and cell adhesion modulators (plerixafor) alone or incombination with nivolumab for 72-hours ex vivo. Tumor responses to ex vivo treatments wereassessed using a proprietary tumor cell killing assay and 21-color flow cytometry analysis. Results and Summary: Here we evaluated the impact of stromal targeting therapeutics onCAFs and associated tumor cell killing as well as overcoming immune evasion mechanismspreserved in the tumoroid models. Furthermore, we used a multicolor flow panel to analyzewhether combination of stromal targeting drugs enhances nivolumab’s effect on the activation oftumor resident CD4, CD8 T-cells, NKT, and NK cell populations as well as on macrophagepolarization. Treatment-mediated changes in the tumor immune microenvironment was furthercorroborated by a multiplex cytokine release assay detecting GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α,Perforin in tumoroid culture media and correlated with clinicopathologic findings and PD-L1expression for individual tumnors. Further, this 3D-tumoroid platform provides unique insightinto the microenvironment of both treatment responsive and non-responsive tumors and can aidin the development of patient-centered therapeutic regimens. Citation Format: Seth Currlin, Brittney Ruedlinger, Sharon Camacho, Angie Rivera, Jasmin D'Andrea, Jared Ehrhart, Soner Altiok. 3D-EXplore platform of fresh patient tumoroids with intact TME allows assessment of the efficacy of drugs targeting the tumor stroma on ex vivo tumor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4552.

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Malmros, Karina, Daniel Carlsén i Hans Brunnström. "Abstract 7561: Standardized conditions for growth of lung cancer tumoroids cultured reproducibly in 3D-cultures". Cancer Research 84, nr 6_Supplement (22.03.2024): 7561. http://dx.doi.org/10.1158/1538-7445.am2024-7561.

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Abstract Background: Lung cancer is the most common cause of cancer-related death in Sweden with an estimated mortality of 3500 persons in 2021. Late diagnosis and heterogeneity of the disease lead to difficulty in predicting optimal treatment regime for each case. Further development of personalized therapy would be facilitated by reliable and efficient cultivation of cancer cell samples from patients in a clinical setting. A promising method is to grow the cells as 3D structures known as tumoroids, though the choice of extracellular matrix substrate or solid support most suitable for this purpose should be elucidated. Aim: To assess which extracellular matrix substrates or solid supports which enable lung cancer tumoroid generation reproducibly without affecting the status or detection of genetic alterations and protein expression, using lung cancer cell lines. Methods: Eight lung cancer cell lines, where of three adenocarcinoma and five squamous cell carcinomas, were included in this project. Each cell line was characterized with immunohistochemistry (IHC) for commonly expressed antigens in the two lung cancer types. Also, protein detection with Western Blots was performed. Cell lines found not expressing relevant antigens associated with the specific lung cancer variant were discarded. The remaining cell lines were cultivated in BIOFLOAT TC-Platte 96 well plate, Nunclon Sphera flasks, or GrowDex-T hydrogel, later transferred to a Corning Elplasia 6 well plate, and monitored for tumoroid generation. Results: The lung squamous cell carcinomas HCC-95 and HCC-1588 were both positive for CK5 and p40, while H1703 was negative for both markers. The adenocarcinoma H1975 was positive for TTF-1, also, unexpectedly, seen for NCI-H1703, earlier defined as squamous cell carcinoma. The tested cell lines were successfully cultured as tumoroids in the BIOFLOAT TC- 96 well plate, Nunclon Sphera flasks, and GrowDex-T hydrogel. The mutational status of the tumoroids as compared to the original tumors is currently under evaluation. Conclusion: We here show successful 3D culturing of cell lines with alternative substrates. Further fine-tuning of the chosen reproducible system's simplicity and cost is required before culturing tumoroids from lung cancer patients can be launched. Citation Format: Karina Malmros, Daniel Carlsén, Hans Brunnström. Standardized conditions for growth of lung cancer tumoroids cultured reproducibly in 3D-cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7561.

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Paul, Colin D., Amber Bullock, Anthony Chatman, Km Shams Ud Doha, Harrison Leong, Sylvia Beam, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas i David Kuninger. "Abstract B017: An integrated workflow for multiomic characterization and functional precision medicine testing of solid tumor samples". Cancer Research 85, nr 5_Supplement (11.03.2025): B017. https://doi.org/10.1158/1538-7445.genfunc25-b017.

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Abstract The development of tumoroid (or cancer organoid) technology has enabled the in vitro culture of patient-derived cancer cells that retain key morphological, mutational, and transcriptomic characteristics of patient tumors. In a number of studies, the response of tumoroids to therapies has mirrored that seen in the patients from which the tumoroids were derived. As a result, there is increasing interest in the use of tumoroids in functional precision oncology, in which an individual patient’s cancer cells are used as ex vivo tumor avatars and tested for response to a range of various potential treatments to aid in therapy selection. Here, we explored a workflow to integrate genomic sequencing with functional drug sensitivity testing for precision medicine applications. Excess FFPE tissue from 6 colorectal cancer donors was processed to generate H&E slides and unstained sections. Slides (2 x 10 µm sections per donor) were digested, and RNA and DNA were isolated using an automated nucleic acid purification system. The output samples were analyzed on the Genexus™ Integrated Sequencer for targeted detection of SNVs, CNVs, gene fusions, and indels for each sample using the Oncomine™ Comprehensive Assay v3. Purified RNA was also utilized for bulk RNA sequencing to generate transcriptomic profiles for each donor. In parallel, viably cryopreserved dissociated tumor cells (DTCs) were thawed and plated in Gibco™ OncoPro™ Tumoroid Culture Medium, a commercially available, serum-free, and conditioned medium-free system. OncoPro medium was developed and optimized for the culture of patient-derived tumor cells from solid cancers to provide an easy-to-use, reproducible, cGMP manufactured culture system for these samples. Where sample size allowed, RNA was isolated from DTC samples and sequenced to compare transcriptomic results generated from FFPE vs. viably cryopreserved material. Additionally, DTCs were sampled for untargeted proteomic and metabolomic profiling. DTCs were cultured for 3-7 days in OncoPro medium prior to exposure to chemotherapy drugs used in standard-of-care treatment for colorectal cancer, and cell response was quantified. Dissociated tumoroid cells formed tumoroids at high rates in culture, in line with previous studies showing that patient-derived cancer cells across colorectal, endometrial, lung, breast, and head and neck cancers form tumoroids in OncoPro medium in >85% of samples within 7 days of tissue receipt. We observed heterogeneous genomic, proteomic, transcriptomic, and drug response profiles across donors. Current efforts are focused on development of computational methods to tie multiomic characterization with functional response. Overall, this data presents an integrated workflow to combine genomic approaches with functional testing to inform patient care. Citation Format: Colin D Paul, Amber Bullock, Anthony Chatman, Km Shams Ud Doha, Harrison Leong, Sylvia Beam, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. An integrated workflow for multiomic characterization and functional precision medicine testing of solid tumor samples [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B017.

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Akbaba, Cihat Ediz, Adem Polat i Dilek Göktürk. "Tracing 2D Growth of Pancreatic Tumoroids Using the Combination of Image Processing Techniques and Mini-Opto Tomography Imaging System". Technology in Cancer Research & Treatment 22 (styczeń 2023): 153303382311642. http://dx.doi.org/10.1177/15330338231164267.

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Objectives: In this study, we aimed to trace the 2D growth development of tumoroids produced with MIA PaCa-2 pancreatic cancer cells at different time points. Methods We cultured 3 different tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations and calculated the growth rate of the tumoroids with their images acquired at 9 imaging time points by mini-Opto tomography imaging system applying image processing techniques. We used the metrics contrast-to-noise ratio (CNR), peak signal-to-noise ratio (PSNR), and mean squared error (MSE) to analyze the distinguishability of the tumoroid structure from its surroundings, quantitatively. Additionally, we calculated the increase of the radius, the perimeter, and the area of 3 tumoroids over a time period. Results In the quantitative assessment, the bilateral and Gaussian filters gave the highest CNR values (ie, Gaussian filter: at each of 9 imaging time points in range of 1.715 to 15.142 for image set-1). The median filter gave the highest values in PSNR in the range of 43.108 to 47.904 for image set-2 and gave the lowest values in MSE in the range of 0.604 to 2.599 for image set-3. The areas of tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations were 1.014 mm2, 1.047 mm2, and 0.530 mm2 in the imaging time point-1 and 33.535 mm2, 4.538 mm2, and 2.017 mm2 in the imaging time point-9. The tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations grew up to times of 33.07, 4.33, and 3.80 in area size over this period, respectively. Conclusions The growth rate and the widest borders of the different tumoroids in a time interval could be detected automatically and successfully. This study that combines the image processing techniques with mini-Opto tomography imaging system ensured significant results in observing the tumoroid's growth rate and enlarging border over time, which is very critical to provide an emerging methodology in vitro cancer studies.

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Yang, Xiaoyu, Andrew Tsao, Garrett Wong, Chris Yankaskas, Colin Paul, Brittany Balhouse, Amber Bullock i in. "Abstract 2750: Multiplexed plate-reader based drug screening of 3D-tumoroid models". Cancer Research 83, nr 7_Supplement (4.04.2023): 2750. http://dx.doi.org/10.1158/1538-7445.am2023-2750.

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Abstract Cancer drug development is an extremely challenging and resource-consuming process with a dismal success rate of 5%. The high failure rate is partly due to the inadequacy of traditional 2D cell culture model to predict drug efficacy and toxicity. A highly accurate and representative system is therefore urgently needed to improve cancer drug discovery and development process. The emerging 3D cancer models can better recapitulate the in vivo tumor microenvironment and have shown closer gene expression profiles to clinical samples compared to 2D models. Another crucial element of in vitro cancer models is the source of cancer cells: patient-derived models have shown advantages to better capture tumor heterogeneity and clinically relevant genetic alterations of the tissue origin compared to traditional cancer cell lines. Several studies showed that response of patient-derived models to treatment reflects the response of patients from which the organoids are derived, suggesting they are great platform for studies of personalized medicine. Hereby, we performed a proof-of-concept high throughput multiplexed plate-reader based drug screening assay on 3D patient-derived tumoroids. Methods: Patient-derived tumoroids were sequenced for cancer relevant mutations, copy number variants, and altered gene expression using multi-biomarker targeted next generation sequencing (NGS) and RNA-seq. 3D tumoroid lines derived from three different colorectal cancer patient samples, together with a colorectal cancer cell line were treated with chemotherapy, and targeted therapy agents. The same tumoroid and cell lines were cultured and treated in 2D as experimental control. Drug response readout was multiplexed using three different plate reader-based assays, measuring the reducing power, ATP, and the release of lactate dehydrogenase from treated cells. Results: Comparable drug responses were observed from three different viability cytotoxicity assays. Different patient-derived tumoroids demonstrated differential drug sensitivity that correlates with their clinical cancer stages. Both patient-derived and cancer cell lines cultured in 3D models showed markedly increased drug resistance compared 2D adherent culture. Differential response was also observed between cancer cell lines vs patient-derived lines. Conclusion: Taken together, these findings suggest the potential advantages of 3D patient-derived tumoroid models over 2D culture and cancer cell lines for accurate prediction of drug response. Through targeted NGS and RNA-seq, patient-specific drug targets could be identified for personalized drug screening. Selected drug candidates could be further subjected to in vivo drug validation using patient-derived xenograft model generated from the same donor. In combination with other derived models or large donor banks, this workflow provides a platform for large-scale drug discovery workflows and precision medicine. Citation Format: Xiaoyu Yang, Andrew Tsao, Garrett Wong, Chris Yankaskas, Colin Paul, Brittany Balhouse, Amber Bullock, Anthony Chatman, Shyanne Salen, Sybelle Djikeng, Matt Dallas, David Kuninger. Multiplexed plate-reader based drug screening of 3D-tumoroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2750.

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Sogawa, Chiharu, Takanori Eguchi, Manh Tien Tran, Masayuki Ishige, Kilian Trin, Yuka Okusha, Eman Ahmed Taha i in. "Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3". Cancers 12, nr 2 (24.02.2020): 523. http://dx.doi.org/10.3390/cancers12020523.

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Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

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Balhouse, Brittany N., Colin Paul, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Anthony Chatman, Amber Bullock, Matthew Dallas i David Kuninger. "Abstract 158: How low can you go: Maintenance of tumoroid phenotype with a highly scalable and automation-compatible reduced-ECM suspension culture method". Cancer Research 83, nr 7_Supplement (4.04.2023): 158. http://dx.doi.org/10.1158/1538-7445.am2023-158.

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Abstract Tumoroid cultures, also known as cancer organoids, have been shown to maintain patient-specific mutational and gene expression profiles over the course of long-term culture better than traditional cancer lines. Despite the physiological relevance of tumoroid models, they have yet to supplant cancer lines, particularly for high-throughput screening (HTS) applications, in large part due to the relative difficulty of the culture workflow. The current gold standard tumoroid culture method involves embedding the cells into a scaffold (most commonly basement membrane extract or BME), which is highly manual, costly in time and resources, and difficult to implement in HTS workflows. We have developed a novel tumoroid culture medium, named GibcoTM OncoProTM Tumoroid Culture Medium, and method in which the addition of diluted BME to tumoroid suspension cultures preserves apical-in polarity (equivalent to that of embedded cultures) while leveraging the benefits of a suspension culture workflow. Additionally, histology for a subset of suspension cultures have been compared with xenograft tumors formed in mice and demonstrate that the suspension culture maintains similar cellular organization. Our system enables a single user to generate hundreds of millions of cells, a feat that would require hundreds of BME domes using standard tumoroid culture methods, and has been shown compatible with colorectal, lung, pancreatic, and head and neck tumoroid lines that were derived in embedded culture with various complete media formulations. Critically, our data confirm that, when paired with our novel tumoroid culture medium, suspension culture maintains patient-specific characteristics comparably to embedded culture when passaged side-by-side for multiple months. Tumoroid lines representing the four unique cancer indications evaluated maintained patient-specific mutational and gene expression profiles (both ≥ 90% correlated with the original material) over the course of culture. An evaluation of differentially expressed genes from a panel of over 20,000 human RefSeq genes showed that there was no change (greater than a two-fold increase/decrease; p-value < 0.05) in gene expression for ≥ 98% of genes, on average, between embedded and suspension culture. Following scale up in suspension culture, we also show that tumoroids can be plated using automated liquid handling techniques for downstream HTS assays. Taken together, our data indicate that this suspension culture workflow paired with OncoPro Tumoroid Culture Medium maintains all critical characteristics of tumoroid lines grown in the traditional embedded culture workflow, while providing the scalability and compatibility with liquid handling that will enable greater adoption of these more physiologically relevant cancer models. Citation Format: Brittany N. Balhouse, Colin Paul, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Anthony Chatman, Amber Bullock, Matthew Dallas, David Kuninger. How low can you go: Maintenance of tumoroid phenotype with a highly scalable and automation-compatible reduced-ECM suspension culture method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 158.

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Arya, Neha, Viren Sardana, Meera Saxena, Annapoorni Rangarajan i Dhirendra S. Katti. "Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: an improved in vitro tumour model". Journal of The Royal Society Interface 9, nr 77 (12.09.2012): 3288–302. http://dx.doi.org/10.1098/rsif.2012.0564.

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Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grown in vivo for factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grown in vivo . Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wider potential for in vitro tumoroid generation. Overall, the results demonstrated that CG scaffolds can be an improved in vitro tool to study cancer progression and drug screening for solid tumours.

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Yang, Cian-Ru, Chu-Ting Liang, Shih-Chieh Tsai, Yu-Chun Wu, Ching-Wen Liu, Hui-Hua Yang, Ting-Yuan Tu i in. "A Novel 3D Culture Scaffold to Shorten Development Time for Multicellular Tumor Spheroids". International Journal of Molecular Sciences 23, nr 22 (12.11.2022): 13962. http://dx.doi.org/10.3390/ijms232213962.

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Multicellular tumor spheroids and tumoroids are considered ideal in vitro models that reflect the features of the tumor microenvironment. Biomimetic components resembling the extracellular matrix form scaffolds to provide structure to 3-dimensional (3D) culture systems, supporting the growth of both spheroids and tumoroids. Although Matrigel has long been used to support 3D culture systems, batch variations, component complexity, and the use of components derived from tumors are complicating factors. To address these issues, we developed the ACD 3D culture system to provide better control and consistency. We evaluated spheroid and tumoroid formation using the ACD 3D culture system, including the assessment of cell viability and cancer marker expression. Under ACD 3D culture conditions, spheroids derived from cancer cell lines exhibited cancer stem cell characteristics, including a sphere-forming size and the expression of stem cell marker genes. The ACD 3D culture system was also able to support patient-derived primary cells and organoid cell cultures, displaying adequate cell growth, appropriate morphology, and resistance to oxaliplatin treatment. These spheroids could also be used for drug screening purposes. In conclusion, the ACD 3D culture system represents an efficient tool for basic cancer research and therapeutic development.

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Sanders, Karin, Femke C. A. Ringnalda, Marc L. van de Wetering, Hans S. Kooistra, Björn P. Meij, Hans Clevers i Sara Galac. "Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease". Journal of the Endocrine Society 5, Supplement_1 (1.05.2021): A533. http://dx.doi.org/10.1210/jendso/bvab048.1085.

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Abstract Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.

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Waterman-Smith, Jonathan P., Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman i Soner Altiok. "Abstract 288: An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics". Cancer Research 82, nr 12_Supplement (15.06.2022): 288. http://dx.doi.org/10.1158/1538-7445.am2022-288.

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Abstract Background: Molecular changes underlying immune cell signaling in response to new therapy development are crucial to validate the clinical efficacy of immunotherapeutics. There are several preclinical tools including patient derived cell lines, organoid and xenograft (PDX) models to assess the efficacy of drugs. However, it is important to recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. Here we report, an ex vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal and immune cell components to assess treatment-mediated changes in molecular and transcriptional profiles of tumor resident immune cells using a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) platform. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids measuring 150 µm in size with intact tumor immune microenvironment were prepared from fresh tumor samples of colon and lung tissues using proprietary technology developed at Nilogen Oncosystems. No enzymatic digestion, propagation or reassembly was used during the preparation of the tumoroids. Hundreds of tumoroids originated from different parts of each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with or without immunostimulatory agents for 48h. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells. Results: Following sequencing of FACS sorted viable CD45 populations, the FAST-Q output files were uploaded into Cell Ranger for analysis with Loupe Browser. Multimodal analysis of transcriptomes or proteomics at the single-cell level revealed significant changes in the top 100 gene expression levels specific to CD4 and CD8+ T cells, NK cells and B cells signaling and activation upon stimulation with immunostimulatory agents. In addition, several pro and anti-inflammatory chemokines and cytokines were upregulated in response to ex vivo treatment in both colon and lung tumoroids, which coincided with marked changes in the activation status of tumor resident immune cells, as detected by multiparameter flow cytometry analysis. Conclusions: These results demonstrate Nilogen’s 3D fresh tumoroid model reflects the significant impact on activation and proliferation of tumor resident immune cells within the intact tumor microenvironment at the transcriptional level. We believe this model system can serve as a clinically relevant tool for accelerating immuno-oncology drug development. Citation Format: Jonathan P. Waterman-Smith, Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 288.

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Li, Haifeng, Wen Gao, Ruixue Liu i Xiaonan Han. "Abstract 2450: Transforming intestinal stem cells into metastatic cancer Stem cells by aberrantly activated cytokine-STAT5A signaling". Cancer Research 83, nr 7_Supplement (4.04.2023): 2450. http://dx.doi.org/10.1158/1538-7445.am2023-2450.

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Abstract Background: Lgr5 intestinal stem cells (ISC) are augmented by colorectal (CRC), but it is not clear if the aberrant Lgr5 ISCs result in the metastatic phenotypes of CRC, suggesting that epistasis-involved mutations in CRC suppressor genes or oncogene control Lgr5 ISC transformation into cancer stem cells (CSC). Somatic mutations in STAT5A or B are highly associated with CRC. Cytokine-STAT5 action is essential for Lgr5 ISC stemness, and enhanced STAT5 phosphorylation can induce Lgr5 ISC differentiation into secretory niche cells. Using human and mouse tumoroids, and organoid xerograph models, we aim to determine the effects of STAT5 oncogenic activation on Lgr5 ISC malignant and metastatic phenotypes. Methods: Colorectal surgical specimen from CRC and hepatic metastatic CRC patients were collected to perform RNAseq or single cell RNAseq (scRNAseq) analysis. Compound-mutant mice with STAT5 loss or gain of function in Apcmin+/- mice, were generated by crossing Apcmin+/- mice with Lgr5CreER and Rs26CreER, and Stat5f/f (loss of function of Stat5) or icS5f/f (gain of function of STAT5A) mice. CRISPR/Cas9 gene editing was applied to engineer APC mutations in human epithelial cell lines or Apc mutations in Lgr5 ISCs. Apc-mutant Lgr5 ISCs or tumoroids from Apcmin+/- mice combined with STAT5 loss or gain of function were differentiated, then were under intra-abdominal xenotransplantation. APC-mutant intestinal epithelial cell lines were used to determined CSC migration in the presence of STAT5 activation. STAT5 inhibitors were used to treat Apcmin+/- mice or tumoroids. Results: Human subject studies revealed that hepatic metastatic CRC patients exhibited the robustly increased STAT5A and/or Lgr5 colonic cancer cells. Murine studies showed that constitutively STAT5A activation (Ca-pYSTAT5) in colonic Apcmin+/- CSCs promoted CSC migration, invasion and metastasis. In vivo migration assay showed that transient STAT5A activation in the APC-mutant intestinal epithelia accelerated the migration of APC-mutant epithelia. Ex Vivo tumoroid xenotransplantation showed that Ca-pYSTAT5 resulted in tumoroid invasion into liver. Lineage tracing analysis showed that Ca-pYSTAT5 expanded Lgr5 CSCs in tumoroids and drove Lgr5 CSCs to hepatic metastasis. Importantly, Stat5 deletion in Lgr5 ISCs abolishes colon cancer orgnoids invasion. Pharmacological inhibition of STAT5 dimerization leads to Lgr5 cancer organoid degeneration and partially annihilated intestinal adenoma in Apcmin+/-. CHIP-seq analysis showed that Ca-pYSTAT5 increased an aberrant high level of Bcl6 expression by inducing super-enhancer activation in the Bcl-6 locus. Conclusion: Aberrantly activated Cytokine-STAT5A signaling is required for ISC malignancy and CSC hepatic metastasis. STAT5 blockade could be important to impair CRC metastasis. Citation Format: Haifeng Li, Wen Gao, Ruixue Liu, Xiaonan Han. Transforming intestinal stem cells into metastatic cancer Stem cells by aberrantly activated cytokine-STAT5A signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2450.

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Robben, Stijn, Anais Peyron, Ana Rita Ribeiro, Divyasree Prabhakaran, Antoni Homs Corbera, Pierre Gaudriault, Patricia Davidson i Dario Fassini. "Abstract 193: A high throughput vascularized immunocompetent tumoroids model in a standard multiwell plate for precision oncology". Cancer Research 83, nr 7_Supplement (4.04.2023): 193. http://dx.doi.org/10.1158/1538-7445.am2023-193.

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Abstract Recent advances in immuno-oncology have significantly increased the therapeutic arsenal available for clinicians. However, being able to identify the responder to a given treatment remains difficult and time consuming. This is due to the lack of translational preclinical models that recapitulate the complete cellular and physical tumor-immune micro-environment (TME). We report for the first time a high throughput vascularized immunocompetent breast tumoroids model in standard multiwell culture plate (MW). The model uses micro physiological system (MPS) and microfabrication to recapitulate and precisely control the TME. The tumor model includes fibroblasts, tumor cells (MDA-MB-231), immune cells (CD81+, CD64+), endothelial cells (CD31+), and a collagen extra cellular matrix. These are essential for the TME and are often lacking in preclinical models, potentially biasing the observed therapeutic response. The physical microenvironment was recapitulated using Cherry Biotech’s MPS, CubiX, and 4DCell SmartSphero Plates (SSoP). A combined system, which to the best of our knowledge is the only one able to recreate the complete TME in a 24 MW with multiplexed and uniformized tumoroid sizes. The SSoP technology is based on microstructured hydrogels, where microwells with an anchoring point at the bottom, allow the formation of the tumoroids, and to keep them in place, making it easy to trace them. The non-adherent properties of the gels help maintain the tumoroids shape. The CubiX system allowed controlling the cell culture conditions: temperature (37°C) and medium perfusion (150 µL/min). The presence of an enriched gas mix with 5% CO2 and O2, was also provided to the tumoroids, and pH, lactate, glucose, O2 consumption were monitored along all culture periods. We focused on optimizing the recapitulation of the physiopathology of breast cancer in 3 aspects: growth rate; tumor cell migration; and oxygen gradient within the MW plate to mimic different depths of the tumor (normoxic to the hypoxic core). Those features are essential for accurate drug efficacy testing. We were able to grow and maintain up to 91 tumoroids per well in a 24MW. After tumoroids formation, we obtained a fully vascularized and immuno-competent model in 48h, the fastest to the best of our knowledge. The system enabled monitoring of the tumoroids growth rate, the differentiation of the endothelium cells (Kfl 2/4, Van Willebrand Factor, eNOS, -% actin fiber alignment), differentiation of CD81+ and CD64+ cells into Macrophage type 1 or 2. All cell types were kept viable for 7 days, and endothelial cells alignment was found physiological (80%). Furthermore, we were able to induce on demand the invasiveness phenotype of the tumoroids. We envision that this model will evolve into a vascularized immunocompetent patient derived tumor model that can be used routinely in precision oncology to predict the drug response of a given patient. Citation Format: Stijn Robben, Anais Peyron, Ana Rita Ribeiro, Divyasree Prabhakaran, Antoni Homs Corbera, Pierre Gaudriault, Patricia Davidson, Dario Fassini. A high throughput vascularized immunocompetent tumoroids model in a standard multiwell plate for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 193.

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Ehrhart, Jared, Seth Currlin, Sharon Camacho, Samantha Hoffman, Angie Rivera i Soner Altiok. "Abstract 4572: 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade". Cancer Research 83, nr 7_Supplement (4.04.2023): 4572. http://dx.doi.org/10.1158/1538-7445.am2023-4572.

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Abstract Introduction: Nilogen Oncosystems’ 3D-EXpress platform allows for the detailed characterization offreshly resected patient tumor tissue for inclusion in studies investigating rational combinations of IOtherapeutics. Incorporating data on immune cell composition, tumor cell target expression, mutationalstatus, and HPV infection within tumoroids generated from HNSCC tissues provides tremendous input toidentify candidate tissues for therapeutic testing. The 3D-EXpress platform employed here providesdetailed observations investigating tumor cell viability using confocal microscopy and immune cellactivation indicated by increases in cytokine release. Materials and Methods: 3D tumoroids measuring 150 microns in size were generated from fresh patientHNSCC resection samples collected with proper patient consent and relevant IRB approval. Tumoroidswere never enzymatically dissociated, propagated, or reassembled to maintain the intact tumormicroenvironment (TME). The resulting tumoroids were cryopreserved in the Biorepository, where eachsample had associated patient demographic data, tumor grade, stage, mutational and HPV status inaddition to a detailed tumor immune profile and a FFPE representative sample in TMA format for targetselection. Pooled tumoroids for each patient tumor were treated ex vivo both singly with a selectiveWEE1 inhibitor, AZD1775 and an ATR blocker AZD6738 alone and in combination with a PD-1 immunecheckpoint inhibitor antibody nivolumab for 72h. Results and Summary: To quantify treatment-mediated tumor cell killing we used 3D high-contentconfocal imaging and a proprietary algorithm for data analysis. Additionally, culture supernatantsisolated from treated tumoroids were used for multiplex cytokine detection to monitor changes in theTME upon ex vivo treatment. Responses to treatments were further correlated with the composition ofinnate and adaptive TIL populations within each tumor as assessed by 21-color flow cytometry inaddition to tumor mutational status, HPV infection, smoking status, as well as EGFR and PD-L1expression levels analyzed by multiplex IF on associated TMA slides. Conclusion: These results demonstrate that targeting DNA damage response and immune checkpointblockade may provide a potential combination strategy for the treatment of HNSCC. Furthermore, weshowed that our 3D-EXpress ex vivo tumoroid model provides a unique platform to rapidly assess theefficacy of drugs and drug combinations in fresh patient tumor samples with intact TME. Correlation ofex vivo drug responses with characteristics of each tumor’s immune microenvironment and detailedclinicopathological data may allow to identify clinically relevant biomarkers to enable the most effectivetreatment strategies for individual patients in the clinic. Citation Format: Jared Ehrhart, Seth Currlin, Sharon Camacho, Samantha Hoffman, Angie Rivera, Soner Altiok. 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4572.

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Kodumudi, Krithika Nandakumar, Brittany Bunch, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman i Soner Altiok. "Abstract 239: Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment". Cancer Research 82, nr 12_Supplement (15.06.2022): 239. http://dx.doi.org/10.1158/1538-7445.am2022-239.

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Abstract Background: Entinostat, a selective histone deacetylase 1/3 inhibitor has been shown to promote a robust anti-tumor response and increased neoantigen expression and antigen-specific T cell activation in preclinical tumor models. Entinostat plays a role in neutralizing myeloid derived suppressor cells (MDSC) to improve anti-tumor immune response. Entinostat is currently being tested in later-stage clinical trials in various types of cancer. Using a novel 3D ex-vivo platform with fresh patient tumor samples we assessed the therapeutic efficacy of Entinostat with intact stromal components and tumor immune microenvironment. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples from renal cell carcinoma (RCC) patients using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Entinostat for 48h to detect treatment-mediated changes in tumor immune cell composition including lymphoid and myeloid markers and their activation status. Results: Tumoroids treated with Entinostat increased NK, NKT, CD4 and CD8+T cells compared to untreated control. Ex vivo treatment with Entinostat led to an increase in CD69 and Granzyme B expression in tumor resident CD4+ T cells. Immune checkpoint receptors, 41BB and PD-1 expression was increased in CD8+T cells followed by Entinostat treatment. Decreased monocytic MDSC was seen in Entinostat treated tumoroids compared to untreated control and this was associated with decreased GM-CSF, IL-4, IL-6 cytokine levels and increased IFN-γ and IL-2, quantified by multiplex cytokine assay. Interestingly, the tumor cell killing study revealed a higher percentage of death in early stage MDSC (eMDSC) indicating a direct effect of Entinostat on MDSC survival. Conclusions: These results demonstrate that Entinostat treatment leads to a broad range of immune cell activation and MDSC inhibition in RCC. These data provide a mechanistic rationale utilizing our 3D tumoroid platform for discovery of potential biomarkers of drug sensitivity and to identify novel immunotherapeutic approaches. Clinical applications of 3D-EXplore may help to identify RCC patients who may likely benefit from Entinostat treatment. Citation Format: Krithika Nandakumar Kodumudi, Brittany Bunch, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Assessment of immunotherapeutic efficacy of entinostat by using the 3d-explore ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 239.

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Leto, Simonetta M., Elena Grassi, Marco Avolio, Valentina Vurchio, Francesca Cottino, Martina Ferri, Eugenia R. Zanella i in. "Abstract 219: Molecular and therapeutic characterization of a large-scale collection of metastatic colorectal cancer patient-derived xenografts and matched organoids for translational oncology". Cancer Research 84, nr 6_Supplement (22.03.2024): 219. http://dx.doi.org/10.1158/1538-7445.am2024-219.

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Abstract Strong evidence supports the use of patient-derived xenografts (PDXs) as faithful models reflecting patient outcomes. Since not all research can support the use of animals, exploiting patient-derived in vitro models such as organoids (also know as tumoroids) is essential. However, current biobanks of metastatic colorectal cancer (mCRC) tumoroids have important limitations, such as small sample size and a lack of systematic in vivo validation with paired xenografts, which decreases the translational value of tumoroid-based pipelines. Here we describe XENTURION, a unique open-science resource of XENografts and Tumoroids for Research In ONcology that encompasses 129 sibling pairs of mCRC PDXs and PDX-derived tumoroids (PDXTs) with accompanying molecular and therapeutic characterization.The vast majority of XENTURION models were analyzed for mutations (targeted next-generation sequencing of 116 relevant CRC genes), gene copy number architecture (DNA shallow sequencing) and global transcriptomics (RNAseq) and benchmarked against large patient datasets (TCGA and MSK-IMPACT). Results showed that the PDX-PDXT collection proved to be representative of the genetic heterogeneity of mCRCs and displayed high genetic and transcriptional similarity between matched pairs. A PDXT-based population trial with the clinically approved anti-EGFR antibody cetuximab was performed in 116 PDXTs, revealing variable sensitivities that were consistent with clinical response biomarkers and with tumor growth changes in 79 matched PDXs. Cetuximab response profiles also recapitulated the outcome of EGFR knock-out by CRISPR-Casp9 technology in 13 representative PDXTs. Adaptive signals upregulated by EGFR blockade both in tumoroids and PDXs were computationally and functionally prioritized. Top candidates were screened in PDXTs and 3 surviving compounds were identified as actionable co-extinction targets with cetuximab in 12 PDXT models. Results were finally validated in PDXs.To our knowledge, this is the first large-scale study in which a systematic comparison of molecular and therapeutic profiles between PDXT-PDX pairs was attempted. As a publicly available resource, XENTURION will offer a knowledge base of disseminatable methods, resources and information to streamline preclinical studies and accelerate new treatments for patients with mCRC. Citation Format: Simonetta M. Leto, Elena Grassi, Marco Avolio, Valentina Vurchio, Francesca Cottino, Martina Ferri, Eugenia R. Zanella, Laura Di Blasio, Alberto Puliafito, Luca Primo, Andrea Bertotti, Livio Trusolino. Molecular and therapeutic characterization of a large-scale collection of metastatic colorectal cancer patient-derived xenografts and matched organoids for translational oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 219.

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Mora-Lagos, Bárbara, María Elena Reyes, Lorena Lobos-Gonzalez, Matías del Campo, Carmen Gloria Ili, Kurt Buchegger, Yuselin Mora, Louise Zanella, Ismael Riquelme i Priscilla Brebi. "Abstract 1749: The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation". Cancer Research 83, nr 7_Supplement (4.04.2023): 1749. http://dx.doi.org/10.1158/1538-7445.am2023-1749.

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Abstract Introduction: Gastric cancer (GC) is an important cause of death worldwide. Cisplatin (CDDP) is the most used drug in the chemotherapy of advanced GC. However, CDDP resistance reduces GC survival. The CCR5/CCL5 axis has been associated with GC development, but its role in CDDP resistance in GC has not been elucidated. The aim of this study was to determine the effects of CCR5/CCL5 axis blockade by a chemokine receptor antagonist (CRA), alone and in combination with CDDP, on in vitro and 3D tumorid models from AGS R-CDDP cells. Methodology: AGS R-CDDP cells (CDDP-resistant human gastric adenocarcinoma cells) were previously established using a stepwise drug dosing protocol and characterized. CCL5 was selected through transcriptomic analysis and its expression level was validated by qRT-PCR. Cytotoxicity assays were determined by using MTT. Apoptosis and cell cycle assays were evaluated by flow cytometry. Tumoroid formation was performed from AGS R-CDDP cells in low adhesion plates (NunclonTMSpheraTM). After 10 days in culture, tumoroid >150 µm in diameter were scored and pharmacological stimuli were added on days 14, 17 and 20. CRA was used alone and in combination with CDDP in all assays. Results: CRA/CDDP combination triggered a re-sensitized phenotype in AGS R-CDDP cells, decreasing cell viability but not increasing apoptosis compared to independent treatments. AGS R-CDDP cells treated with CRA/CDDP were mainly arrested in S phase. CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination compared to separate treatments. AGS R-CDDP cells showed a higher potential in the ability to form tumoroids compared to AGS WT cells. Finally, CRA/CDDP combination inhibited tumoroid formation compared to independent treatments. Discussion and Conclusion: CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination, possibly correlating with allosteric inhibition of the CCR5 receptor. CRA/CDDP combination has a synergic effect that sensitizes AGS R-CDDP cells and inhibits tumoroid formation. This combination could be used as a potential coadjuvant in GC therapy allowing to reduce the doses of CDDP and therefore to reduce side effects. Citation Format: Bárbara Mora-Lagos, María Elena Reyes, Lorena Lobos-Gonzalez, Matías del Campo, Carmen Gloria Ili, Kurt Buchegger, Yuselin Mora, Louise Zanella, Ismael Riquelme, Priscilla Brebi. The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1749.

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Horowitz, Nina Beryl, John Hickey, Garry P. Nolan i John B. Sunwoo. "Overcoming barriers to solid tumor immunotherapy using natural killer cell therapies designed to mimic intraepithelial group 1 innate lymphoid cells". Journal of Immunology 208, nr 1_Supplement (1.05.2022): 122.10. http://dx.doi.org/10.4049/jimmunol.208.supp.122.10.

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Abstract Our work aims to design cell-based immunotherapies for maximum solid tumor efficacy by leveraging insights from tissue-resident cells and single-cell RNA sequencing of innate immune cell subsets present within patient tumors. We discovered that by co-culturing peripheral blood natural killer cells (pbNKs) with irradiated epithelial tumor cells, we induced high expression of surface integrins, increased cytotoxicity and IFNg production, decreased sensitivity to TGFb, and significantly enhanced solid tumoroid infiltration. The cells were profiled using CyTOF and we determined that they closely resembled intraepithelial group 1 innate lymphoid cells, so we refer to them as ieILC1-like cells. We quantified the cytotoxicity of ieILC1-like cells against a variety of target cell lines and determined that they are broadly cytotoxic and capable of antibody-dependent cellular cytotoxicity, which was assessed using cetuximab. Tumor-infiltrating capacity was modeled using 3D tumoroids grown from single-cell suspensions of epithelial tumor cell lines in basement membrane extracts; ieILC1-like NK cells or pbNK cells were fluorescently labeled, added to tumoroids, and imaged using confocal microscopy. We stimulated the cells in the presence or absence of overnight TGFb exposure to determine the extent of TGFb-mediated immunosuppression. ieILC1-like cells were more resistant to TGFb and produced significantly more IFNg after stimulation than their pbNK counterparts. Preliminary in vivo work indicates that ieILC1-like cells perform comparably to K562-expanded NK cells. Thus, ieILC1-like NK cells represent a novel class of cell therapy that are more capable of infiltrating solid tumors and resisting their immunosuppressive cues. Supported by NIH R35DE030054.

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Sinenko, Irina, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier i Paul Dyson. "Abstract 3541: Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma". Cancer Research 83, nr 7_Supplement (4.04.2023): 3541. http://dx.doi.org/10.1158/1538-7445.am2023-3541.

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Abstract Current treatments for retinoblastoma, the most common intraocular malignancy, rely on a limited number of drugs repurposed from other pediatric cancer therapies. Drug toxicity and relapse of the disease necessitate new therapeutic strategies. The implementation of drug candidates in the clinic is usually followed by in vitro validation, which requires accurate and clinically relevant in vitro models. We report here a robust 3D tumoroid-based platform, for testing various clinical protocols, e.g. therapeutic agents following a single or repeated exposure, alone or in combination with focal therapy (thermotherapy). The platform, generated from the Y79 retinoblastoma cell line, consists of matrix-embedded tumoroids, which retain the key features of the disease and allow prolonged culture time. Following the optimization of growth conditions, the effect of single and repeated exposure to chemotherapeutic agents has been measured by high-throughput automated fluorescence imaging followed by quantification. The clinical conditions of chemothermotherapy have been recapitulated with a setup consisting of: an infrared diode laser (810 nm, 0.3 W) that heats the tumoroids in the presence of indocyanine green (50 µg/mL); a thermal camera to enable real-time temperature measurement; and an environment-controlled inverted microscope to ensure physiological conditions and precise focusing of the laser. Melphalan and carboplatin, two drugs currently used to treat retinoblastoma in clinical protocols of chemotherapy and chemothermotherapy respectively, have been applied to validate the system.Tumoroid viability and size progressively decreased over 4 weeks of repeated drug exposure with melphalan, reflecting the response of retinoblastoma in advanced clinical cases. Carboplatin treatment showed an activity enhancement in the tumoroid model similar to that observed in the clinic, when combined with focal therapy, demonstrating that the platform system closely recapitulates the clinical conditions. Inversely, the thermotherapy experimental protocol designed for 2D in vitro cell culture and commonly used to date, i.e. an incubation temperature increased to 42 °C for 1 h, did not show an improvement of the carboplatin cytotoxicity, confirming that our laser-based thermotherapy protocol provides a more accurate and clinically relevant drug response. Such an approach, combining physiologically relevant in vitro cancer models and investigation of drugs in well-established treatment modalities, can significantly reduce the number of new drug candidates that give false-positives or overestimated efficacies when evaluated in relevant in vivo models. It is expected that the platform will contribute to the implementation of new therapeutic strategies to treat retinoblastoma, and could also be adapted to other diseases for which repeated drug exposure and/or chemothermotherapy is relevant. Citation Format: Irina Sinenko, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier, Paul Dyson. Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3541.

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Deems, Liam, Maria Ivanova, Cheryl Murphy, Amit Shahar, David Deems i Dmitry Shvartsman. "Abstract PO-074: Identification of C-MET receptor as a therapeutic target in patient-specific tumoroid models of metastatic pancreatic adenocarcinoma allows identification of a new mode of action for its inhibitors". Cancer Research 81, nr 22_Supplement (15.11.2021): PO—074—PO—074. http://dx.doi.org/10.1158/1538-7445.panca21-po-074.

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Abstract Creating patient-specific models with a high level of characterization and functionality in tumoroid modeling enables a better understanding of the disease drivers in metastatic cancers. It presents the opportunity for personalized treatment with novel therapeutic reagents in underrepresented cancers such as pancreatic ductal adenocarcinoma. Using a defined and treatment-naive patient-specific model cohort of nine patients diagnosed with pancreatic adenocarcinoma, we aimed to represent the diversity of progression grade and assay profiles. These models were tested with a panel of common, broad-range chemotherapeutic agents and characterized for genetic, proteomic, and tumoroid drug response profiles. A detailed analysis of gene expression values from the original tissue specimen, compared with cancer cultures in hypoxic, normoxic planar, and spheroid (3D) culture conditions, enabled the identification of C-MET as a critical gene that shows upregulation closely resembling the expression profile observed in the original patient biopsy and was modulated by tissue culture format. Once this potential target was identified, the pancreatic models were treated with five C-MET-targeting chemotherapeutic agents with different modes of action. Proto-oncogene receptor C-MET (HGF receptor) is a potential therapeutic target in many metastatic cancers due to its integral role in angiogenesis, proliferation, and cancer cell survival¹. We propose that these reproducible and easily scaled tumoroid models can be used to find new therapeutic targets in-vitro and provide the example of C-MET in our pancreatic cancer patient cohort. Moreover, only 3D hypoxic models closely resembled original patient tissues in their genomic expression profiles. Using these model formats, we distinguished differences between various C-MET targeting compounds and their ability to induce cell death, which correlated with their mode of action on C-MET neutralization. Identifying these targets in 3D produces new and potentially more effective drug targets where they may not have been observed solely in planar conditions with legacy cell lines. Our models of metastatic pancreatic cancer tumoroids can be readily utilized to benefit researchers and clinicians seeking new targets for patient treatment. Citation Format: Liam Deems, Maria Ivanova, Cheryl Murphy, Amit Shahar, David Deems, Dmitry Shvartsman. Identification of C-MET receptor as a therapeutic target in patient-specific tumoroid models of metastatic pancreatic adenocarcinoma allows identification of a new mode of action for its inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-074.

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Ayehunie, Seyoum, Megan Groves, Bryda Bryda, Alex Armento i Anthony Tolcher. "Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies". Cancer Research 84, nr 6_Supplement (22.03.2024): 221. http://dx.doi.org/10.1158/1538-7445.am2024-221.

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Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 221.

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Yang, Xiaoyu, Maryam Mafreshi, Garrett Wong, Vivek Chandra, Colin Paul, Chris Yankaskas, Brittany Balhouse i in. "Abstract 6787: Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids". Cancer Research 84, nr 6_Supplement (22.03.2024): 6787. http://dx.doi.org/10.1158/1538-7445.am2024-6787.

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Abstract Introduction: Over the last decade, chimeric antigen receptor T cell (CAR-T)-based immunotherapy has achieved unprecedented clinical benefits for the treatment of hematological malignancies. However, application of CAR-T therapy to solid tumors remains challenging, in part due to tumor antigen heterogeneity and microenvironmental complexity present in these tumors. Therefore, selecting physiologically-relevant models is critical for validation of the on-target efficacy and off-target effect of CAR-T therapy. Emerging 3D patient-derived tumoroid models better recapitulate the morphological organization, mutational status, and gene expression levels of patient tumors. Hereby, we propose a screening platform to evaluate mesothelin (MSLN)-targeted CAR-T therapy for solid tumors using patient-derived tumoroids. Methods: T cells were isolated from peripheral blood mononuclear cells of healthy human donors. Meso3 (region III of MSLN) or CD19 CARs (served as control) were knocked-in (KI) to activated T cells using electroporation-based CRISPR/Cas9 system followed by CAR Cassette containing adeno-associated virus (AAV) transduction. T-cell receptor (TCR) knockout (KO) and CAR-KI efficiency were validated by flow cytometry. Patient-derived tumoroids with high and low MSLN expression were co-cultured with various effector-to-target ratios of Meso3 CAR, CD19 CAR and Control (TCR KO without CAR KI) T cells. Cytotoxicity was evaluated by caspase-based live cell imaging. Key cytokine secretion was measured using a multiplexing platform 2-3 days post co-culture. Results: T-cell electroporation of CRISPR/Cas9 yielded a high KO efficiency (>90%) of TCR as well as high AAV-mediated CAR cassette-KI efficiency (>70%). The killing efficiency of Meso3 CAR-T cells with different effector to target ratios was successfully captured in a ratio-dependent manner via live cell imaging and analysis platform in the MSLN-high tumoroids, while minimum killing was observed in MSLN-low tumoroids. As expected, control and CD19 CAR-T cells showed no killing towards MSLN-high or -low tumoroids, which do not express CD19. Moreover, the observed killing ability correlated with secretion of cytokines associated with T cell-mediated cytotoxicity. Conclusion: These findings demonstrated the ability of 3D patient-derived models as a screening platform for validations of CAR-T cell therapy in vitro. This platform has potential advantages over commonly used impedance-based and flow cytometry-based killing assay since it better captures the 3D architecture and microenvironment of the tumor origin and allows real-time quantification. With the ability of accurately detecting on and off-target killing, quick turn-around time, and cost-effectiveness compared to in vivo systems, this platform allows for high-throughput screening workflows for CAR-T cell therapies. Citation Format: Xiaoyu Yang, Maryam Mafreshi, Garrett Wong, Vivek Chandra, Colin Paul, Chris Yankaskas, Brittany Balhouse, Jason Sharp, Matt Dallas, Erik Willems, David Kuninger. Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6787.

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Ehrhart, Jared, Seth Currlin, Michelle Ataya, Alliyah Humphrey i Rikhia Chakraborty. "Abstract 951: 3D-PREDICT - An ex vivo precision oncology and clinical trial enrichment platform by Nilogen Oncosystems". Cancer Research 84, nr 6_Supplement (22.03.2024): 951. http://dx.doi.org/10.1158/1538-7445.am2024-951.

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Abstract Background: Immuno-oncology (IO) therapy with checkpoint inhibitors has demonstrated a higher response rate, duration of response, and overall survival than chemotherapy. However, predicting which FDA approved IO therapies or pharmaceutical agents, as well as best combinations and delivery sequence, will have optimal patient response remains a significant challenge for personalized medicine. Nilogen Oncosystem’s 3D-PREDICT is a novel ex vivo therapeutic investigative platform that provides a functional model of a patient’s tumor to investigate susceptibility to different therapeutic approaches directly. Derived from core biopsies, the 3D-PREDICT model captures the tumor heterogeneity while preserving the tumor microenvironment by retaining stromal components and cell-cell and cell-extracellular matrix interactions. Here, we employed the 3D-PREDICT platform to compare the efficacy of different immunotherapeutic approaches in various solid tumor indications ex vivo. Methods: Tumoroids from 18-gauge core biopsies from solid tumors were generated using Nilogen Oncosystems’s proprietary mechanical process forgoing any enzymatic digestion or propagation while keeping the spatial contexture of stroma intact. Pooled tumoroids were treated ex vivo for 72 hours with various standard-of-care mono or combo therapy (carboplatin, anti-programmed death-ligand 1 (PD-L1) antibody atezolizumab, anti-CD47 antibody magrolimab, anti-programmed cell death receptor-1 (PD-1) antibody nivolumab, or a combination of magrolimab and atezolizumab). Treatment-mediated immune modulation in tumor microenvironment were analyzed to predict patient response to subjected therapies. Results: Treatment-mediated tumor cell killing activity was evaluated using 3D high throughput confocal imaging and Nilogen Oncosystems’s proprietary algorithm for data analysis. The ex vivo response to checkpoint inhibitors was correlated with retrospective patient clinical information. Tumoroid samples treated with atezolizumab and magrolimab demonstrated enhanced tumor cell killing. Approximately 20% of tumors showed increased CD8 T-cell activation upon ex vivo treatment, correlating with proinflammatory cytokine release in conditioned media. Conclusions: The 3D-PREDICT ex vivo tumoroid platform provides a unique resource to predict patients’ response to treatment, potentially serving as a clinical diagnostic to guide clinical care and stratifying patient procurement into clinical trials. This would allow prospective assessment of therapeutic efficacy ex vivo to match each patient to the most effective and least toxic, to improving outcomes, reducing costs, and assigning therapeutics on a more rational basis. Citation Format: Jared Ehrhart, Seth Currlin, Michelle Ataya, Alliyah Humphrey, Rikhia Chakraborty. 3D-PREDICT - An ex vivo precision oncology and clinical trial enrichment platform by Nilogen Oncosystems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 951.

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Kreahling, Jenny, Jared Ehrhart, Mibel Pabon, Stephen Iwanowycz, Tina Pastoor i Soner Altiok. "619 Evaluating the effectiveness of targeted ADC therapy in a patient-derived ex vivo tumoroid model, 3D-EX, for quantitative tumor cell killing". Journal for ImmunoTherapy of Cancer 8, Suppl 3 (listopad 2020): A655. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0619.

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BackgroundAntibody drug conjugates (ADCs) are an effective tool for site directed delivery of cytotoxic agents to cancer cells. Tailoring of ADC-specificity to the uniqueness of a patient‘s tumor can aid in direct-targeting of tumor cells and potentially improve drug responsiveness. Here we evaluate the potential of using an ADC therapy for targeted tumor cell death and immune cell activation in combination with checkpoint inhibitors in 3D tumoroids.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue of various histologic types including CRC and NSCLC were processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a conjugated ADC therapeutic antibody alone or in combination with PD-1/PD-L1 inhibitors. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells in combination with high-content confocal imaging to determine extent of tumor cell death in the intact tumor extracellular matrix.ResultsUsing fresh patient-derived tumoroids, we observed ADC-mediated cell death and activation of immune cells within the tumor microenvironment. Production of pro-inflammatory cytokines correlated with increased activation of tumor infiltrating immune cell populations. The improved immune response led to increased tumor cell killing within the 3D tumor microenvironment observed by high-content confocal imaging.ConclusionsIn this study we demonstrate that our physiologically relevant 3D tumoroid model is an effective system to assess novel antibody drug conjugates and to develop rational drug combinations with other immuno-oncology agents. Furthermore, implementation of 3D-EX platform, in the clinical setting, may also allow for determination of the most effective combinatorial immuno-oncology treatment strategies for individualized patient care.Ethics ApprovalThe study was approved by Chesapeake IRB Pro00014313.

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Ehrhart, Jared, Brittney Ruedlinger, Angie Rivera, Romanus Ezeoke, Sharon Camacho, Seth Currlin i Soner Altiok. "Abstract 4571: A novel ex vivo platform, 3D-EXpress, to rapidly assess the efficacy of KRAS targeting drugs alone and in combination with nivolumab using a biorepository of fresh patient tumoroids with intact tumor microenvironment". Cancer Research 83, nr 7_Supplement (4.04.2023): 4571. http://dx.doi.org/10.1158/1538-7445.am2023-4571.

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Abstract Introduction: 3D-EXpress is a novel ex vivo drug testing platform using a Biorepository of neverdissociated, propagated, or reassembled fresh patient tumoroids with intact tumor microenvironment.Tumoroids measuring 150 µm in size retain tumor cell heterogeneity, tumor-resident innate andadaptive immune cells, stromal components, and cell-extracellular matrix interaction allowing to rapidlytest the efficacy of drugs and drug combinations targeting various components of the TME includingtumor cells, stroma, and immune cell populations. Here we employed the 3D-EXpress platform tocompare the efficacy of different drugs and drug combinations targeting KRAS and PD-1/PD-L1 immunecheckpoint ex vivo. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Cryopreserved tumoroids with known KRAS mutation status, PD-L1 expression, detailedimmune profiles and clinicopathologic data were selected from Nilogen’s tumoroid Biorepository for theex vivo assays. 3D tumoroids were treated with vehicle only, a SOS1::KRAS inhibitor, BI 1701963, apotent, selective, and covalent KRASG12C inhibitor, MRTX849, and a KRAS G12C inhibitor, sotorasib,alone and in combination with nivolumab for 72h. Treatment-mediated changes in tumor cell killing andtumor immune microenvironment were analyzed. Results: To quantify treatment-mediated tumor cell killing activity we employed 3D high-contentconfocal imaging using a proprietary algorithm for data analysis. To monitor how ex vivo drugtreatments affect the tumor immune microenvironment culture supernatants isolated from treatedtumoroid samples were used for multiplex cytokine detection including GM-CSF, sCD137, IFNγ, sFas,sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin.We observed a significant difference in tumor responses among patient samples to ex vivo tested drugsand drug combinations, which were further correlated with tumor resident innate and adaptive immunecell populations detected by a 21-color flow cytometry panel in addition to tumor KRAS mutation status,patient smoking history, tumor characteristics as well as PD-L1 expression levels analyzed on theassociated TMA slides. Conclusion: Our data demonstrate that the 3D-Express platform, using cryopreserved 3D tumoroidswith intact TME, is an effective tool to assess the efficacy of KRAS and immune checkpoint inhibitorstargeting drugs to identify rational combination therapies and to develop clinically relevant biomarkersfor individualized patients in the future. Citation Format: Jared Ehrhart, Brittney Ruedlinger, Angie Rivera, Romanus Ezeoke, Sharon Camacho, Seth Currlin, Soner Altiok. A novel ex vivo platform, 3D-EXpress, to rapidly assess the efficacy of KRAS targeting drugs alone and in combination with nivolumab using a biorepository of fresh patient tumoroids with intact tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4571.

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Daszkiewicz, Lidia, Maarten Klop, Kuan Yan i Leo Price. "A 3D image-based quantification of immune cell-tumor spheroid interactions in the presence of checkpoint inhibition." Journal of Clinical Oncology 35, nr 7_suppl (1.03.2017): 82. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.82.

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82 Background: Delivering on the promises of cancer immunotherapy is hampered by a lack of in vitro testing platforms that enable the early selection of promising drugs candidates. While quantification of the number of cytotoxic T cells and IFNγ secretion can give an indication of the drug effect, it is often critical to assess the functional cytotoxicity and infiltration capacity. In addition, immunomodulatory effects of drug combinations need to be carefully assessed before these treatment modalities reach patients. However there are unlimited possibilities of new combinations, especially in the view of synergy effects observed with double checkpoint inhibition blockade and combination with standard of care drugs. To specifically address the needs of immunotherapy cancer drug developers, we developed robust assays that incorporate the human immune system into tumor spheroid cultures, with functional read-outs in a high throughput (384well plate) format. Methods: Breast cancer cells were grown in a hydrogel to form 3D tumoroids. HLA-matched PBMCs with and without pre-activation/exhaustion were added and the infiltration of T cells and subsequent tumoroid killing was quantified. The effect of immune checkpoint blockade was also assessed with the addition of pembrolizumab. Quantification of effects of stimulators and checkpoint inhibition was achieved with 3D imaging and morphometric analysis with OMiner software. Results: Automated 3D image and data analysis enabled discrimination of immune-tumour cell interactions depending on activation status of T cells. Activated T cells more efficiently infiltrated and killed tumoroids than non-activated ones. Cytotoxic activity of overactivated/exhausted T cells was supressed but this could be reversed by the presence of checkpoint inhibitor in the coculture. Conclusions: A 3D environment allows the different cell types to engage in a more realistic setting than when cells are grow in a monolayer. Using image-based analysis, immune-tumor interactions can be dissected. This represents a new, highly powerful tool for cancer immunotherapy drug developers to select the most promising compounds, allowing faster progression towards the clinic.

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Tsao, Andrew, Xiaoyu Yang, Garrett Wong, Vivek Chandra, Jacob Delgadillo, Lindsay Bailey Steinitz, Brittany Balhouse i in. "Abstract 2753: Engineering patient-derived tumors to enable high-throughput screening: Immuno-oncology workflows". Cancer Research 83, nr 7_Supplement (4.04.2023): 2753. http://dx.doi.org/10.1158/1538-7445.am2023-2753.

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Abstract Aim/Introduction: In recent years, cancer immunotherapy has become one of the fastest-growing areas in cancer research. Selecting suitable and cost-effective experimental models for developing and validating immunotherapies is one of the major obstacles researchers face today. To overcome this, patient-derived tumor models are of increasing interest because they can better recapitulate many of the properties and the heterogeneity exhibited by the tumor microenvironment at a relatively low cost. Hereby, we propose a high throughput screening platform for an effective and efficient evaluation of cancer immunotherapies in patient-derived tumor models. Methods: Tumor models were established in vitro from patient-derived tumor biopsies. Established tumoroids were engineered using a luciferase-green fluorescent protein (GFP) lentivirus to generate a reporter pool. Transduced pools were enriched for GFP via flow cytometry and characterized using RNA/scRNA-seq and biomarker-based sequencing. Natural Killer (NK) cells were co-cultured with the enriched pool in various effector-to-target ratios and recorded using a live cell imaging and analysis platform. Cytotoxicity and cell health were measured by GFP intensity, luciferase activity, and caspase-based live staining. Results: A patient-derived tumoroid reporter pool was successfully generated through GFP enrichment using a flow cytometer. The killing efficiency of immune cells with various effector(E) to target(T) ratios has been successfully captured in a ratio-dependent manner via the live cell imaging and analysis platform. NK cell-mediated cytotoxicity was successfully measured through GFP intensity, luciferase, and caspase activity. Conclusions: Traditional cell line generation can be used in patient-derived tumoroid models to generate enriched reporter cell pools without selection pressure. Outside of establishing screening platforms, scientists can use this approach to efficiently engineer patient-derived tumoroid models to meet their specific research goals. Here, we used the reporter pool to develop a multiplex-killing assay to measure cell viability and toxicity. This platform can be used in a variety of immune cell workflows, providing a method that can predict tissue-specific responses, and evaluate solid tumor immunotherapies in high throughput cell-based assays. Citation Format: Andrew Tsao, Xiaoyu Yang, Garrett Wong, Vivek Chandra, Jacob Delgadillo, Lindsay Bailey Steinitz, Brittany Balhouse, Colin Paul, Jakhan Nguyen, Sybelle Djikeng, Shyanne Salen, Jason Sharp, Matt Dallas, David Kuninger. Engineering patient-derived tumors to enable high-throughput screening: Immuno-oncology workflows [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2753.

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Horowitz, Nina, John Hickey i John Sunwoo. "181 Intraepithelial group 1 innate lymphoid cells generated in vitro exhibit enhanced cytotoxicity and infiltration into solid tumoroids". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (listopad 2021): A193. http://dx.doi.org/10.1136/jitc-2021-sitc2021.181.

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BackgroundImmunotherapy approaches have shown striking success in the liquid tumor setting but have been unable to demonstrate similar efficacy against solid tumors. Cell-based therapies, in particular, struggle to overcome the harshly immunosuppressive tumor microenvironment. Additionally, cells for adoptive therapy are often generated from immune cells circulating in the peripheral blood of patients or healthy donors, rather than isolating them from solid tissues. Designing immunotherapies using insights from tissue-resident cells represents a novel method for enhancing trafficking into and retention within solid tumors.1 To this end, we developed methodology for differentiating peripheral blood natural killer cells (pbNKs) into cells resembling intraepithelial group 1 innate lymphoid cells (ieILC1s) in vitro and assessed their potential for immunotherapy.MethodsWe co-cultured irradiated squamous cell carcinoma (SCC) cells and pbNKs, isolated from blood of healthy human donors, to generate cells exhibiting an ieILC1 phenotype. We assessed the differentiation using traditional flow cytometry and further profiled the cells using cytometry by time of flight (CyTOF)2 to obtain higher-dimensional data about their surface and intracellular phenotypes. We then tested the cells for their cytotoxicity against a variety of target cell lines using the xCELLigence platform. Next, we grew three-dimensional tumoroids from single-cell suspensions of SCC cell lines in basement membrane extracts and added fluorescently labeled pbNKs and ieILC1s to them. We assessed their infiltration capacity into the tumoroids using confocal microscopy.ResultsThe ieILC1-like cells generated in vitro had a comparable surface and intracellular phenotype to ieILC1s in healthy tissue. These cells exhibited significantly increased cytotoxicity against multiple SCC cell lines and were also capable of antibody-dependent cellular cytotoxicity, which we tested using anti-epidermal growth factor receptor (EGFR) antibody (figure 1). Importantly, the ieILC1-like cells efficiently infiltrated the tumoroids in a manner consistent with their tissue-resident phenotype (figure 2A) and at higher rates than the conventional pbNKs (figure 2B).Abstract 181 Figure 1Cytolysis of pbNKs and ieILC1-like cells was compared using the xCELLigence platform to monitor killing of adherent SCC target cells over 36 hours. Cells were cultured at a 1:4 E:T ratio with or without 10 ug/mL cetuximab.Abstract 181 Figure 2Tumoroid infiltration rates of pbNKs and ieILC1-like cells were compared using confocal microscopy. A: Representative cross-sections of tumoroids, with nuclei labeled with DAPI (blue) and infiltrating cells labeled with CellTrace Far Red (pink). B: Infiltrating cells within each tumoroid were counted and then normalized to the area of the tumoroid.ConclusionsOur data show that ieILC1-like cells can be generated from pbNKs using a co-culture system with irradiated epithelial tumor cells. These ieILC1-like cells provide a novel platform for adoptive cell therapy, as they exhibit strong natural cytotoxicity and ADCC against multiple cell lines. Finally, the ieILC1-like cells have an enhanced capacity for infiltration into solid tumoroids. Future work will include in vivo modeling of tumor infiltration and efficacy as well as optimization of the co-culture platform to maximize expansion and cytotoxicity of the cells as they differentiate.AcknowledgementsN.B.H. is funded by the Stanford Bio-X Fellowship. The laboratory of J.B.S. is funded by NIH R35 DE030054.ReferencesMilner J, Toma C, Yu B, Zhang K, Omilusik K, Phan A, Wang D, Getzler A, Nguyen T, Crotty S, Wang W, Pipkin M, Goldrath A. Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours. Nature 2017;552:253–257.Bendall S, Simonds E, Qiu P, Amir E, Krutzik P, Finck R, Bruggner R, Melamed R, Trejo A, Ornatsky O, Balderas R, Plevritis S, Sachs K, Pe’er D, Tanner S, Nolan G. Single-Cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 2011;332:687–696.Ethics ApprovalThe studies reported here were approved by the Stanford Institutional Review Board (IRB 11402) and the Stanford Administrative Panel on Laboratory Animal Care (APLAC 20547).

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Gowrikumar, Saiprasad, Mark Primeaux, Kristina Pravoverov, Chao Wu, Bryan C. Szeglin, Charles-Etienne Gabriel Sauvé, Ishwor Thapa i in. "A Claudin-Based Molecular Signature Identifies High-Risk, Chemoresistant Colorectal Cancer Patients". Cells 10, nr 9 (26.08.2021): 2211. http://dx.doi.org/10.3390/cells10092211.

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Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.

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Bunch, Brittany, Autumn Joerger, Nino Mtchedlidze, Olivia Hoff, Kelly Guzman, Jared Ehrhart i Soner Altiok. "759 Single-Cell Proteogenomics (Cite-seq) analysis of cGAS-STING pathway activation alone and in combination with nivolumab using a patient-derived 3D ex vivo tumoroid platform". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (listopad 2021): A794. http://dx.doi.org/10.1136/jitc-2021-sitc2021.759.

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BackgroundThe tumor immune microenvironment comprises a heterogeneous collection of adaptive and innate immune cells that play a critical role in immune evasion and response to immunotherapeutic agents. cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway results in activation of various immune cells promoting innate immunity in addition to senescence of cancer cells. However, the mechanisms involved in response and resistance to cGAS-STING pathway activation is not well understood. Using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), we explored immunological heterogeneity of tumor microenvironment in colorectal cancer and analyzed transcriptional and compositional changes of the immune landscape in response to cGAS-STING pathway activation alone and in combination with a PD-1 inhibitor nivolumab.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue was processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a STING agonist, ADU-S100, alone or in combination with nivolumab. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells.ResultsMultimodal analysis of transcriptomes or proteomics at the single-cell level provided an unprecedented view of cellular diversity and enabled better understanding of how activation of STING pathway alone and in combination with nivolumab affects the TME in colorectal cancer. Flow cytometric analysis of immune cell populations isolated from 3D tumoroids demonstrated treatment mediated activation of tumor resident T-cells and changes in the innate immune cells, which coincided with marked changes in pro-and anti-inflammatory cytokine profiles determined by multiplex analysis.ConclusionsThese results demonstrate that the 3D-EXplore ex vivo tumoroid model provides a unique platform to assess the efficacy of immunotherapeutic agents and to develop novel therapeutic combinations. Furthermore, implementation of this platform in the clinical studies may also allow identifying clinically relevant biomarkers to enable the most effective treatment strategies for individual patients.

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Singh, Surya P., Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao i Venkateshwar Madka. "Abstract 5255: Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models". Cancer Research 83, nr 7_Supplement (4.04.2023): 5255. http://dx.doi.org/10.1158/1538-7445.am2023-5255.

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Abstract Bladder cancer (BC) is a lethal genitourinary malignancy associated with frequent recurrence and poor survival due to metastatic potential. Identification of key cancer cell signaling networks and developing promising agents is critical for effectively inhibiting tumor growth and progression. In many cancers, including bladder cancer (BC), signal transducer and activator of transcription 3 (STAT3) has emerged as an important molecular pathway due to its role in promoting proliferation, invasion, and chemoresistance. Thus, developing STAT3 targeting, orally bioavailable small molecule inhibitors may be helpful for the prevention of BC progression and improving the survival rate of patients with muscle invasive BC. Monolayer culture has limitations for drug testing. Therefore, spheroid and organoid culture are used extensively as they may mimic in-vivo drug response more accurately. The aim of our study is to examine the preclinical anticancer efficacy of STAT3 inhibitors [TTI-101 (C188-9) and SH5-07] in 3D (spheroid and tumoroid) invitro models of BC. We optimized the spheroid growth using various BC cell lines [human (J82), rat (NBT-II), and mouse (MB49) BC cells]. Similarly, tumoroids from rat (BBN-induced bladder tumors) and transgenic mice (UPII-SV40T) bladder tumors were developed. These spheroids and tumoroids were treated with various concentrations (0 - 50 μM range) of STAT3 inhibitors and evaluated for their viability [Calcein AM (CA) and EtBr staining], ATP production (CellTiter-Glo™ 3D), and ROS production (MitoSOXTM). Effect of drug treatment on biomarkers of cell proliferation, apoptosis, stemness, STAT3 signaling, and immune modulation was determined using western blotting and immunofluorescence. Treatment with TTI-101 (0 - 50 μM or SH5-07 (0 - 50 μM) for 144 hrs resulted in significant reduction in the spheroids size (39-45% smaller Vs untreated; p<0.0001), along with decreased ATP levels (20%-40%, p<0.05). MitoSOXTM staining suggested that STAT3 inhibitors treatment increased ROS production in BC cells. CA and EtBr staining revealed that TTI-101 and SH5-07 treatment resulted in increased cell death in BC spheroids compared to control. Decreased spheroids and organoids size also correlated with increased apoptotic marker (cleaved caspase-3) along with decreased cyclin D1, PCNA, and pSTAT3 protein expression. Drug treated BC spheroids/tumoroids also showed reduction in CD44 (BC stemness and invasion marker) and induction of cGAS-STING pathway (cGAS, STING, TBK1, and IRF3) in comparison to the control. These findings indicate that STAT3 inhibitors, TTI-101 and SH5-07, could inhibit bladder cancer by suppressing STAT3 pathway activation and therefore warrant further study in vivo. (Supported by P30 CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P. Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5255.

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