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1
Maier, Christopher Fabian, Lei Zhu, Lahiri Kanth Nanduri, Daniel Kühn, Susan Kochall, May-Linn Thepkaysone, Doreen William, et al. "Patient-Derived Organoids of Cholangiocarcinoma." International Journal of Molecular Sciences 22, no. 16 (August 12, 2021): 8675. http://dx.doi.org/10.3390/ijms22168675.
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Cholangiocarcinoma (CC) is an aggressive malignancy with an inferior prognosis due to limited systemic treatment options. As preclinical models such as CC cell lines are extremely rare, this manuscript reports a protocol of cholangiocarcinoma patient-derived organoid culture as well as a protocol for the transition of 3D organoid lines to 2D cell lines. Tissue samples of non-cancer bile duct and cholangiocarcinoma were obtained during surgical resection. Organoid lines were generated following a standardized protocol. 2D cell lines were generated from established organoid lines following a novel protocol. Subcutaneous and orthotopic patient-derived xenografts were generated from CC organoid lines, histologically examined, and treated using standard CC protocols. Therapeutic responses of organoids and 2D cell lines were examined using standard CC agents. Next-generation exome and RNA sequencing was performed on primary tumors and CC organoid lines. Patient-derived organoids closely recapitulated the original features of the primary tumors on multiple levels. Treatment experiments demonstrated that patient-derived organoids of cholangiocarcinoma and organoid-derived xenografts can be used for the evaluation of novel treatments and may therefore be used in personalized oncology approaches. In summary, this study establishes cholangiocarcinoma organoids and organoid-derived cell lines, thus expanding translational research resources of cholangiocarcinoma.
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Lee, Chansu, Sung-Noh Hong, Eun-Ran Kim, Dong-Kyung Chang, and Young-Ho Kim. "Epithelial Regeneration Ability of Crohn’s Disease Assessed Using Patient-Derived Intestinal Organoids." International Journal of Molecular Sciences 22, no. 11 (June 2, 2021): 6013. http://dx.doi.org/10.3390/ijms22116013.
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Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn’s disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.
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Weeber, Fleur, Marc van de Wetering, Marlous Hoogstraat, Krijn K. Dijkstra, Oscar Krijgsman, Thomas Kuilman, Christa G. M. Gadellaa-van Hooijdonk, et al. "Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases." Proceedings of the National Academy of Sciences 112, no. 43 (October 12, 2015): 13308–11. http://dx.doi.org/10.1073/pnas.1516689112.
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Tumor organoids are 3D cultures of cancer cells. They can be derived from the tumor of each individual patient, thereby providing an attractive ex vivo assay to tailor treatment. Using patient-derived tumor organoids for this purpose requires that organoids derived from biopsies maintain the genetic diversity of the in vivo tumor. In this study tumor biopsies were obtained from 14 patients with metastatic colorectal cancer (i) to test the feasibility of organoid culture from metastatic biopsy specimens and (ii) to compare the genetic diversity of patient-derived tumor organoids and the original tumor biopsy. Genetic analysis was performed using SOLiD sequencing for 1,977 cancer-relevant genes. Copy number profiles were generated from sequencing data using CopywriteR. Here we demonstrate that organoid cultures can be established from tumor biopsies of patients with metastatic colorectal cancer with a success rate of 71%. Genetic analysis showed that organoids reflect the metastasis from which they were derived. Ninety percent of somatic mutations were shared between organoids and biopsies from the same patient, and the DNA copy number profiles of organoids and the corresponding original tumor show a correlation of 0.89. Most importantly, none of the mutations that were found exclusively in either the tumor or organoid culture are in driver genes or genes amenable for drug targeting. These findings support further exploration of patient-derived organoids as an ex vivo platform to personalize anticancer treatment.
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Valeri, Nicola. "Abstract IA010: Patient derived organoids in precision oncology." Cancer Research 82, no. 23_Supplement_1 (December 1, 2022): IA010. http://dx.doi.org/10.1158/1538-7445.crc22-ia010.
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Abstract Limits in the predictive power of molecular profiling and shortcomings of some pre-clinical models used in drug development represent important obstacles hampering the success of personalized medicine and drug discovery. LGR5+ stem cells can be isolated from a number of organs and propagated as epithelial organoids in vitro. Mouse and human organoids have been used to study the physiology and neoplastic transformation of the liver, pancreas, bowel and prostate among other organs. During my talk, I will highlight opportunities, limitations and potential clinical applications of patient-derived organoids in personalized oncology, emphasizing strengths and hurdles in the use of the organoid technology in forward and reverse translational cancer research. In particular, I will stress the importance of patient-derived organoids as pre-clinical tools to define mechanisms of drug resistance and to design novel drug combinations. Citation Format: Nicola Valeri. Patient derived organoids in precision oncology [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA010.
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McQueeney, Kelley E., Patrick Bhola, Sarah J. Hill, and Anthony Letai. "Abstract 4309: Early apoptotic measurements of patient-derived organoids predict patient response to therapy." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4309. http://dx.doi.org/10.1158/1538-7445.am2023-4309.
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Abstract The use of imperfect models and ex vivo culture systems to try to predict patient drug response represents an enormous bottle neck in cancer treatment. Nonetheless, determining how effective an approved drug will be for an individual cancer patient, as well as identifying novel compounds that may be beneficial to a specific population often requires the use of primary tumor cells. Patient-derived organoids represent an intermediate between primary tumor cells, whose limited supply may hinder reliable drug testing, and cell lines, which often do not reflect what happens in vivo. Herein, we describe the development of a novel assay platform, termed 3D-DBP (3D dynamic BH3 profiling), to detect early apoptotic measurements in ovarian cancer patient-derived organoids and present evidence that this method can be used to predict patient response to therapy. We have optimized the use of patient-derived organoids from 16 individual tumors in a microscopy-based imaging assay. We image the BH3 peptide-induced release of cytochrome c from mitochondria, which indicates permeabilization of the outer mitochondrial membrane, in intact organoids. The less cytochrome c retained in each organoid, the more primed that organoid is for apoptosis. By comparing results of drug-treated and untreated cells, we can identify drugs that cause a significant increase in apoptotic priming in organoids. In the 16 patient-derived organoids investigated this 3D DBP technique was an effective means of predicting patient response to carboplatin therapy. In summary, we have not only created a means of visualizing drug response in intact organoids, but also have demonstrated its clinical utility. Citation Format: Kelley E. McQueeney, Patrick Bhola, Sarah J. Hill, Anthony Letai. Early apoptotic measurements of patient-derived organoids predict patient response to therapy. [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 4309.
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Sondorp, Luc H. J., Vivian M. L. Ogundipe, Andries H. Groen, Wendy Kelder, Annelies Kemper, Thera P. Links, Robert P. Coppes, and Schelto Kruijff. "Patient-Derived Papillary Thyroid Cancer Organoids for Radioactive Iodine Refractory Screening." Cancers 12, no. 11 (October 31, 2020): 3212. http://dx.doi.org/10.3390/cancers12113212.
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Patients with well-differentiated thyroid cancer, especially papillary thyroid cancer (PTC), are treated with surgical resection of the thyroid gland. This is followed by post-operative radioactive iodine (I131), resulting in total thyroid ablation. Unfortunately, about 15-33% of PTC patients are unable to take up I131, limiting further treatment options. The aim of our study was to develop a cancer organoid model with the potential for pre-treatment diagnosis of these I131-resistant patients. PTC tissue from thirteen patients was used to establish a long-term organoid model. These organoids showed a self-renewal potential for at least five passages, suggesting the presence of cancer stem cells. We demonstrated that thyroid specific markers, a PTC marker, and transporters/receptors necessary for iodine uptake and thyroid hormone production were expressed on a gene and protein level. Additionally, we cultured organoids from I131-resistant PTC material from three patients. When comparing PTC organoids to radioactive iodine (RAI)-refractory disease (RAIRD) organoids, a substantial discordance on both a protein and gene expression level was observed, indicating a treatment prediction potential. We showed that patient-derived PTC organoids recapitulate PTC tissue and a RAIRD phenotype. Patient-specific PTC organoids may enable the early identification of I131-resistant patients, in order to reduce RAI overtreatment and its many side effects for thyroid cancer patients.
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Koedoot, Esmee, Inez van Weersch, Gakuro Harada, Masahiko Watanabe, Hamdy Warda, Hideaki Kyan, Yasmine Abouleila, et al. "Abstract 157: Patient in the lab: Down-scaling patient-derived organoid screening for diagnostic purposes." Cancer Research 83, no. 7_Supplement (April 4, 2023): 157. http://dx.doi.org/10.1158/1538-7445.am2023-157.
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Abstract Colorectal cancer (CRC) is the third leading cause of cancer and cancer-related death. A large fraction of the CRC patients diagnosed with de novo metastatic disease do not benefit from standard of care and experience substantial side effects. Therefore, there is an urgent need for preclinical models that help predict patient response in the clinic. Patient-derived organoids (PDOs or HUB Organoids®) represent a significant breakthrough as preclinical models as they are directly established from patient tissue and faithfully recapitulate patient disease. HUB Organoid Technology can already be applied to preclinical drug screening, however, to provide direct patient benefit the turnaround time between patient diagnosis and PDO-based results must be shortened. In collaboration with Yamaha Motor, the Yamaha CELL HANDLERTM has been validated to automatically pick-and-place with high accuracy a significantly reduced number of organoids per screening plate, compared to standard procedures. In addition, an image-based readout was developed that enables a precise quantification of organoid number ensuring high assay quality. Organoid responses to chemotherapy and targeted agents were validated using this newly automated system, confirming comparable PDO-patient drug sensitivity profiles, and known drug responses based on genetic dependencies. In summary we describe the development of an automated workflow that combines patient-representative HUB Organoids and state-of-the-art robotics by Yamaha CELL HANDLER. This workflow down-scales the number of organoids needed per screening well and allows to efficiently predict patient response, thereby reducing the diagnostic turnaround time and increasing patient benefits. Citation Format: Esmee Koedoot, Inez van Weersch, Gakuro Harada, Masahiko Watanabe, Hamdy Warda, Hideaki Kyan, Yasmine Abouleila, Takahiko Kumagai, Yuichi Hikichi, René Overmeer, Jeanine Roodhart, Kiyotaka Matsuno, Carla Verissimo, Sylvia F. Boj. Patient in the lab: Down-scaling patient-derived organoid screening for diagnostic purposes [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 157.
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Costales-Carrera, Alba, Asunción Fernández-Barral, Pilar Bustamante-Madrid, Orlando Domínguez, Laura Guerra-Pastrián, Ramón Cantero, Luis del Peso, Aurora Burgos, Antonio Barbáchano, and Alberto Muñoz. "Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue." Cancers 12, no. 8 (August 15, 2020): 2302. http://dx.doi.org/10.3390/cancers12082302.
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Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (LGR5, LRIG1, SMOC2, and MSI1) in normal rectum organoids, while it downregulated differentiation marker genes (TFF2 and MUC2). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids.
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Verissimo, Carla S., Lidwien Smabers, Emerens Wensink, Esmee Koedoot, Maarten Huismans, Celia Higuera Barón, Ricardo Korporaal, et al. "Abstract 4112: Patient derived organoids predict clinical response: A patient in the lab." Cancer Research 82, no. 12_Supplement (June 15, 2022): 4112. http://dx.doi.org/10.1158/1538-7445.am2022-4112.
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Abstract Colorectal cancer (CRC) is the third leading cause of cancer and cancer-related death. A large fraction of the CRC patients diagnosed with de novo metastatic disease do not benefit from the standard of care but still experience substantial side effects. Therefore, there is the urgency for a new model to predict clinical response. Adult epithelial stem cell (ASC) -derived organoids are proving to be a major breakthrough in pre-clinical models. ASC-derived organoids can be developed from healthy as well as diseased tissue, including cancer lesions and therefore are often referred to as patient-derived organoids (PDOs or HUB Organoids࣪). These model is established directly from patient tissue, represent the tissue of origin and faithfully recapitulate patient disease in vitro and can be propagated for drug testing in a matter of weeks. PDOs bridges the gap between the lab and the clinic and effectively bring a “patient in the lab.” In this study we aimed to validate the predictive value of PDOs in the stratification of metastatic CRC (mCRC) patients for treatment with chemotherapeutic agents. PDOs from mCRC tissues were established following our optimized procedures and drug screening was performed. Clinical response was compared with PDO drug response and the best predicting drug response parameters (growth rate (GR), GRmax, GR50 and area under the curve) were identified. The patient response was evaluated by the percentage of change in size of the target lesions on response scans (% size change), best Response Evaluation Criteria in Solid Tumors (RECIST) response and progression-free survival (PFS). Importantly, we validated the predictive value of organoids towards fluorouracil (5-FU). We are validating these results in a large trial. Current efforts to further expand our PDO and drug sensitivity biobank will enable the implementation of the personalized HUB Organoid Technology to accurate and fast predict of the treatment response to improve clinical outcome of mCRC patients. Citation Format: Carla S. Verissimo, Lidwien Smabers, Emerens Wensink, Esmee Koedoot, Maarten Huismans, Celia Higuera Barón, Ricardo Korporaal, Emma Teal, Katerina-Chara Pitsa, Edwin van Oosten, Roshni Nair, Liselot Valkenburg, Geert Cirkel, Anneta Brousali, Jorieke Salij, René Overmeer, Manon Braat, Sjoerd Elias, Robert Vries, Onno Kranenburg, Miriam Koopman, Sylvia F. Boj, Jeanine Roodhart. Patient derived organoids predict clinical response: A patient in the lab [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 4112.
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Pernik, Mark N., Cylaina E. Bird, Jeffrey I. Traylor, Diana D. Shi, Timothy E. Richardson, Samuel K. McBrayer, and Kalil G. Abdullah. "Patient-Derived Cancer Organoids for Precision Oncology Treatment." Journal of Personalized Medicine 11, no. 5 (May 17, 2021): 423. http://dx.doi.org/10.3390/jpm11050423.
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The emergence of three-dimensional human organoids has opened the door for the development of patient-derived cancer organoid (PDO) models, which closely recapitulate parental tumor tissue. The mainstays of preclinical cancer modeling include in vitro cell lines and patient-derived xenografts, but these models lack the cellular heterogeneity seen in human tumors. Moreover, xenograft establishment is resource and time intensive, rendering these models difficult to use to inform clinical trials and decisions. PDOs, however, can be created efficiently and retain tumor-specific properties such as cellular heterogeneity, cell–cell and cell–stroma interactions, the tumor microenvironment, and therapeutic responsiveness. PDO models and drug-screening protocols have been described for several solid tumors and, more recently, for gliomas. Since PDOs can be developed in clinically relevant time frames and share many characteristics of parent tumors, they may enhance the ability to provide precision oncologic care for patients. This review explores the current literature on cancer organoids, highlighting the history of PDO development, organoid models of glioma, and potential clinical applications of PDOs.
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Ikezawa, Kenji, Tomoya Ekawa, Shinichiro Hasegawa, Yugo Kai, Ryoji Takada, Takuo Yamai, Nobuyasu Fukutake, et al. "Establishment of organoids using residual samples from saline flushes during endoscopic ultrasound-guided fine-needle aspiration in patients with pancreatic cancer." Endoscopy International Open 10, no. 01 (January 2022): E82—E87. http://dx.doi.org/10.1055/a-1713-3404.
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Abstract Background and study aims In patients with pancreatic cancer (PC), patient-derived organoid cultures can be useful tools for personalized drug selection and preclinical evaluation of novel therapies. To establish a less invasive method of creating organoids from a patient’s tumor, we examined whether PC organoids can be established using residual samples from saline flushes (RSSFs) during endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Methods Five patients with PC who underwent EUS-FNA were enrolled in a prospective study conducted at our institution. RSSFs obtained during EUS-FNA procedures were collected. An organoid culture was considered as established when ≥ 5 passages were successful. Organoid-derived xenografts were created using established organoids. Results EUS-FNA was performed using a 22- or 25-gauge lancet needle without complications. Patient-derived organoids were successfully established in four patients (80.0 %) with the complete medium and medium for the selection of KRAS mutants. Organoid-derived xenografts were successfully created and histologically similar to EUS-FNA samples. Conclusions Patient-derived PC organoids were successfully established using EUS-FNA RSSFs, which are produced as a byproduct of standard manipulations, but are usually not used for diagnosis. This method can be applied to all patients with PC, without additional invasive procedures, and can contribute to the development of personalized medicine and molecular research.
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Xie, Irene Y., Yuanchang Fang, Amy X. Zhang, Karen Ng, Zhen-Mei Liu, Eugenia Flores-Figueroa, Gun Ho Jang, et al. "Abstract 173: Genomic characterization of patient-derived pancreatic cancer organoids." Cancer Research 83, no. 7_Supplement (April 4, 2023): 173. http://dx.doi.org/10.1158/1538-7445.am2023-173.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death with few effective therapies. Patient-derived organoids (PDOs) are a 3-D culture model that allow primary tumour cells to propagate, and have gained considerable traction in many cancer types such as PDAC. However, concerns remain regarding whether these models can predict what occurs in patients. We hypothesize that genomic and transcriptomic drift occurring in PDO models impacts the fidelity of drug response. To investigate, matched WGS and bulk RNAseq was performed on paired PDAC organoids and tumour tissue (n=41). Core biopsies were obtained from in patients with Stage III-IV PDAC enrolled in the COMPASS trial (NCT02750657) and divided for sequencing and organoid generation. Tumour cellularity was enriched by laser capture microdissection. Although alterations in the four major driver genes (KRAS, TP53, SMAD4, CDKN2A) remained consistent, other genomic differences were identified. SNV count was higher in organoids (median 6584 vs 5931, p<0.0001), and enriched in SBS5 mutational signature (p<0.0001). This was not significantly correlated with passage number, and private mutations were identified in both tumours and organoids (median 67% overlap). Two organoids showed significant shifts in ploidy, with both diploid to polyploid shifts and vice versa observed. In the transcriptome, expression of Basal-like genes (KRT5, TP63) was decreased. Bias towards the Classical transcriptomic subtype have previously been observed in PDAC organoid cultures. PDO responses to 5-FU, irinotecan, and oxaliplatin correlated with patient response to FFX (n=22, 72% concordance). However, PDO responses to gemcitabine and paclitaxel were poorly predictive of patient responses to GnP (n=10, 38% concordance), and notably, expression of the biomarker hENT1 was not correlated in matched tumors and PDOs (R=0.17, p=0.40). Despite this, tumour expression of the biomarker hENT1 successfully stratified patient responses to gemcitabine, and organoid hENT1 expression was correlated to gemcitabine response in vitro (R=0.47, p=0.005), indicating that transcriptomic drift may be a major contributor to discrepancies in patient-PDO drug response. In summary, organoids recapitulate major histologic features and driver mutations of patient tumours, but genetic drift and subclonal selection are observed even at lower passages. Further study is required to improve the utility of organoids in translational precision medicine. Citation Format: Irene Y. Xie, Yuanchang Fang, Amy X. Zhang, Karen Ng, Zhen-Mei Liu, Eugenia Flores-Figueroa, Gun Ho Jang, Stephanie Ramotar, Anna Dodd, Julie Wilson, Jennifer J. Knox, Steven Gallinger, Faiyaz Notta. Genomic characterization of patient-derived pancreatic cancer organoids [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 173.
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Schultz, Emily M., TyAnthony J. Jones, Sibei Xu, Dana D. Dean, Bernd Zechmann, and Kelli L. Barr. "Cerebral Organoids Derived from a Parkinson’s Patient Exhibit Unique Pathogenesis from Chikungunya Virus Infection When Compared to a Non-Parkinson’s Patient." Pathogens 10, no. 7 (July 20, 2021): 913. http://dx.doi.org/10.3390/pathogens10070913.
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(1) Background: Arboviruses of medical and veterinary significance have been identified on all seven continents, with every human and animal population at risk for exposure. Like arboviruses, chronic neurodegenerative diseases, like Alzheimer’s and Parkinson’s disease, are found wherever there are humans. Significant differences in baseline gene and protein expression have been determined between human-induced pluripotent stem cell lines derived from non-Parkinson’s disease individuals and from individuals with Parkinson’s disease. It was hypothesized that these inherent differences could impact cerebral organoid responses to viral infection. (2) Methods: In this study, cerebral organoids from a non-Parkinson’s and Parkinson’s patient were infected with Chikungunya virus and observed for two weeks. (3) Results: Parkinson’s organoids lost mass and exhibited a differential antiviral response different from non-Parkinson’s organoids. Neurotransmission data from both infected non-Parkinson’s and Parkinson’s organoids had dysregulation of IL-1, IL-10, and IL-6. These cytokines are associated with mood and could be contributing to persistent depression seen in patients following CHIKV infection. Both organoid types had increased expression of CXCL10, which is linked to demyelination. (4) Conclusions: The differential antiviral response of Parkinson’s organoids compared with non-Parkinson’s organoids highlights the need for more research in neurotropic infections in a neurologically compromised host.
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Buehler, Joseph D., Cylaina E. Bird, Milan R. Savani, Lauren C. Gattie, William H. Hicks, Michael M. Levitt, Mohamad El Shami, et al. "Semi-Automated Computational Assessment of Cancer Organoid Viability Using Rapid Live-Cell Microscopy." Cancer Informatics 21 (January 2022): 117693512211007. http://dx.doi.org/10.1177/11769351221100754.
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The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess in vivo tumor tissue treatment response are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed “Apex Imaging.” We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.
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Rosenbluth, Jennifer M. "Abstract IA011: Patient-derived organoids as models for breast cancer prevention and interception." Cancer Prevention Research 15, no. 12_Supplement_1 (December 1, 2022): IA011. http://dx.doi.org/10.1158/1940-6215.dcis22-ia011.
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Abstract Organoid culture technology can be an efficient means for propagating breast tissues from patients, and we have previously generated organoid biobanks derived from normal and malignant human breast samples. Non-cancerous organoids, which are grown in a hydrogel that mimics the basement membrane and are bathed in a cocktail of growth factors, vitamins, and substances that substitute for stroma, grow in a polarized fashion to re-form acinar- and duct-like structures, and differentiate into the major mammary epithelial lineages, and we have found that all the major mammary epithelial lineages can be propagated in vitro. We have utilized mass cytometry and single-cell RNA sequencing to identify specific cell subtypes that are associated with breast cancer risk factors in organoids. In parallel, in malignant organoid cultures we have utilized RNA sequencing and compound screening approaches to identify potential strategies to overcome therapeutic resistance. We now present our initial efforts to establish a DCIS organoid biobank. Similar to the normal breast, organoids can be propagated from DCIS cases that are resistant to endocrine therapy with the goal of identifying strategies to overcome the resistant state. Citation Format: Jennifer M. Rosenbluth. Patient-derived organoids as models for breast cancer prevention and interception [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA011.
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Kiwaki, Takumi, and Hiroaki Kataoka. "Patient-Derived Organoids of Colorectal Cancer: A Useful Tool for Personalized Medicine." Journal of Personalized Medicine 12, no. 5 (April 26, 2022): 695. http://dx.doi.org/10.3390/jpm12050695.
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Colorectal cancer is one of the most important malignancies worldwide, with high incidence and mortality rates. Several studies have been conducted using two-dimensional cultured cell lines; however, these cells do not represent a study model of patient tumors very well. In recent years, advancements in three-dimensional culture methods have facilitated the establishment of patient-derived organoids, which have become indispensable for molecular biology-related studies of colorectal cancer. Patient-derived organoids are useful in both basic science and clinical practice; they can help predict the sensitivity of patients with cancer to chemotherapy and radiotherapy and provide the right treatment to the right patient. Regarding precision medicine, combining gene panel testing and organoid-based screening can increase the effectiveness of medical care. In this study, we review the development of three-dimensional culture methods and present the most recent information on the clinical application of patient-derived organoids. Moreover, we discuss the problems and future prospects of organoid-based personalized medicine.
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Kiwaki, Takumi, and Hiroaki Kataoka. "Patient-Derived Organoids of Colorectal Cancer: A Useful Tool for Personalized Medicine." Journal of Personalized Medicine 12, no. 5 (April 26, 2022): 695. http://dx.doi.org/10.3390/jpm12050695.
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Colorectal cancer is one of the most important malignancies worldwide, with high incidence and mortality rates. Several studies have been conducted using two-dimensional cultured cell lines; however, these cells do not represent a study model of patient tumors very well. In recent years, advancements in three-dimensional culture methods have facilitated the establishment of patient-derived organoids, which have become indispensable for molecular biology-related studies of colorectal cancer. Patient-derived organoids are useful in both basic science and clinical practice; they can help predict the sensitivity of patients with cancer to chemotherapy and radiotherapy and provide the right treatment to the right patient. Regarding precision medicine, combining gene panel testing and organoid-based screening can increase the effectiveness of medical care. In this study, we review the development of three-dimensional culture methods and present the most recent information on the clinical application of patient-derived organoids. Moreover, we discuss the problems and future prospects of organoid-based personalized medicine.
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Psilopatis, Iason, Amalia Mantzari, Kleio Vrettou, and Stamatios Theocharis. "The Role of Patient-Derived Organoids in Triple-Negative Breast Cancer Drug Screening." Biomedicines 11, no. 3 (March 3, 2023): 773. http://dx.doi.org/10.3390/biomedicines11030773.
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Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, with a grave prognosis and few effective treatment options. Organoids represent revolutionary three-dimensional cell culture models, derived from stem or differentiated cells and preserving the capacity to differentiate into the cell types of their tissue of origin. The current review aims at studying the potential of patient-derived TNBC organoids for drug sensitivity testing as well as highlighting the advantages of the organoid technology in terms of drug screening. In order to identify relevant studies, a literature review was conducted using the MEDLINE and LIVIVO databases. The search terms “organoid” and “triple-negative breast cancer” were employed, and we were able to identify 25 studies published between 2018 and 2022. The current manuscript represents the first comprehensive review of the literature focusing on the use of patient-derived organoids for drug sensitivity testing in TNBC. Patient-derived organoids are excellent in vitro study models capable of promoting personalized TNBC therapy by reflecting the treatment responses of the corresponding patients and exhibiting high predictive value in the context of patient survival evaluation.
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Abdullah, Kalil G., Cylaina E. Bird, Joseph D. Buehler, Lauren C. Gattie, Milan R. Savani, Alex C. Sternisha, Yi Xiao, et al. "Establishment of patient-derived organoid models of lower-grade glioma." Neuro-Oncology 24, no. 4 (November 26, 2021): 612–23. http://dx.doi.org/10.1093/neuonc/noab273.
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Abstract Background Historically, creating patient-derived models of lower-grade glioma (LGG) has been challenging, contributing to few experimental platforms that support laboratory-based investigations of this disease. Although organoid modeling approaches have recently been employed to create in vitro models of high-grade glioma (HGG), it is unknown whether this approach can be successfully applied to LGG. Methods In this study, we developed an optimized protocol for the establishment of organoids from LGG primary tissue samples by utilizing physiologic (5%) oxygenation conditions and employed it to produce the first known suite of these models. To assess their fidelity, we surveyed key biological features of patient-derived organoids using metabolic, genomic, histologic, and lineage marker gene expression assays. Results Organoid models were created with a success rate of 91% (n = 20/22) from primary tumor samples across glioma histological subtypes and tumor grades (WHO Grades 1–4), and a success rate of 87% (13/15) for WHO Grade 1–3 tumors. Patient-derived organoids recapitulated stemness, proliferative, and tumor-stromal composition profiles of their respective parental tumor specimens. Cytoarchitectural, mutational, and metabolic traits of parental tumors were also conserved. Importantly, LGG organoids were maintained in vitro for weeks to months and reanimated after biobanking without loss of integrity. Conclusions We report an efficient method for producing faithful in vitro models of LGG. New experimental platforms generated through this approach are well positioned to support preclinical studies of this disease, particularly those related to tumor immunology, tumor-stroma interactions, identification of novel drug targets, and personalized assessments of treatment response profiles.
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Yamazaki, Shintaro, Fumiharu Ohka, Masaki Hirano, Yukihiro Shiraki, Kazuya Motomura, Kuniaki Tanahashi, Takashi Tsujiuchi, et al. "TB-2 Patient-derived meningioma organoid model demonstrates FOXM1 dependent tumor proliferation." Neuro-Oncology Advances 3, Supplement_6 (December 1, 2021): vi5—vi6. http://dx.doi.org/10.1093/noajnl/vdab159.020.
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Abstract Recent comprehensive studies have revealed several molecular alterations that are frequently found in meningiomas. However, effective treatment reagents targeting specific molecular alterations have not yet been identified because of the limited number of representative research models of meningiomas. We established 18 organoid models comprising of two malignant meningioma cells (HKBMM and IOMM-Lee), 10 benign meningiomas, four malignant meningiomas, and two solitary fibrous tumors (SFTs). Using immunohistochemistry and molecular analyses consisting of whole exome sequencing, RNA-seq, and DNA methylation analyses, we compared the histological findings and molecular profiling of organoid models with those of parental tumors. The organoids exhibited consistent histological features and molecular profiles with those of the parental tumors. Using a public database of meningioma, we identified that upregulated forkhead box M1 (FOXM1) was correlated with increased tumor proliferation. Overexpression of FOXM1 in benign meningioma organoids increased organoid proliferation; depletion of FOXM1 in malignant organoids decreased proliferation. Additionally, thiostrepton, a FOXM1 inhibitor combined with radiation therapy, significantly inhibited proliferation of malignant meningioma organoid models (P<0.01). An organoid model for meningioma enabled us to elucidate the tumor biology of meningioma along with potent treatment targets for meningioma.
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Park, Misun, Junhye Kwon, Joonseog Kong, Sun Mi Moon, Sangsik Cho, Ki Young Yang, Won Il Jang, Mi Sook Kim, Younjoo Kim, and Ui Sup Shin. "A Patient-Derived Organoid-Based Radiosensitivity Model for the Prediction of Radiation Responses in Patients with Rectal Cancer." Cancers 13, no. 15 (July 27, 2021): 3760. http://dx.doi.org/10.3390/cancers13153760.
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Patient-derived tumor organoids closely resemble original patient tumors. We conducted this co-clinical trial with treatment-naive rectal cancer patients and matched patient-derived tumor organoids to determine whether a correlation exists between experimental results obtained after irradiation in patients and organoids. Between November 2017 and March 2020, we prospectively enrolled 33 patients who were diagnosed with mid-to-lower rectal adenocarcinoma based on endoscopic biopsy findings. We constructed a prediction model through a machine learning algorithm using clinical and experimental radioresponse data. Our data confirmed that patient-derived tumor organoids closely recapitulated original tumors, both pathophysiologically and genetically. Radiation responses in patients were positively correlated with those in patient-derived tumor organoids. Our machine learning-based prediction model showed excellent performance. In the prediction model for good responders trained using the random forest algorithm, the area under the curve, accuracy, and kappa value were 0.918, 81.5%, and 0.51, respectively. In the prediction model for poor responders, the area under the curve, accuracy, and kappa value were 0.971, 92.1%, and 0.75, respectively. Our patient-derived tumor organoid-based radiosensitivity model could lead to more advanced precision medicine for treating patients with rectal cancer.
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Lam, Hung-Ming, Yuzhen Liu, Funda Vakar-Lopez, Lisha Brown, Robert B. Montgomery, Eva Corey, Andrew Caleb Hsieh, and Jonathan L. Wright. "Characterization of human bladder cancer patient-derived xenograft in vivo and in organoids." Journal of Clinical Oncology 36, no. 6_suppl (February 20, 2018): 479. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.479.
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479 Background: To establish and molecularly characterize a human bladder cancer patient-derived xenograft (PDX) in vivo and in an organoid system derived from the PDX for preclinical studies. Methods: Two-mm3 bits of urothelial carcinoma originated from muscle invasive disease excised in cystectomy were implanted subcutaneously into male severe combined immunodeficient mice to establish PDXs. Established PDXs (CoCaB 1) were passaged subcutaneously in SCID mice and histopathology of each passage was compared with the originating tumor. Tumor size was measured weekly by caliper to determine the growth rate of PDXs from early (P1/P2) through late passage (P8/P9). Representative early and late passages were collected for organoid establishment. For both early and late passages, proliferation was assessed by Ki67 in PDXs and organoids, and cell cycle analysis and MTS assay specifically in organoids. RNA sequencing was performed to compare the fidelity of PDX and organoids vs. primary tumor. Results: Histologically, 16 of the 16 (100%) PDXs generated from early through late passage (1-2 tumors per passage) were similar to the original high-grade urothelial carcinoma. In vivo, the latency of PDX establishment decreased upon passage (9 weeks to take in early P1/2 vs. 2 weeks to take in late P8/9) and the growth rate increased upon passage. Concordantly, Ki67 proliferation index increased from 40% in P1 to 95% in P8 and was positively correlated with increasing passage (Spearman R=0.804, p=0.001). Similarly, in organoids, late passage demonstrated a shorter growth doubling time, higher Ki67 proliferation index, and faster progression through cell cycle. Transcriptional analysis showed that the PDX contained 81-92% human transcripts, whereas organoids contained >99% human transcripts. Conclusions: Bladder cancer PDXs histologically represented the originating disease. PDX and organoid systems demonstrated concordant increase in proliferation upon serial passages, suggesting clonal selection may take place in this aggressive tumor type. Despite more mouse stromal content in PDX, PDX and organoid represent two independent model systems with highly similar biological responses that allow therapeutic studies.
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Shiihara, Masahiro, and Toru Furukawa. "Application of Patient-Derived Cancer Organoids to Personalized Medicine." Journal of Personalized Medicine 12, no. 5 (May 13, 2022): 789. http://dx.doi.org/10.3390/jpm12050789.
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Cell models are indispensable for the research and development of cancer therapies. Cancer medications have evolved with the establishment of various cell models. Patient-derived cell lines are very useful for identifying characteristic phenotypes and susceptibilities to anticancer drugs as well as molecularly targeted therapies for tumors. However, conventional 2-dimensional (2D) cell cultures have several drawbacks in terms of engraftment rate and phenotypic changes during culture. The organoid is a recently developed in vitro model with cultured cells that form a three-dimensional structure in the extracellular matrix. Organoids have the capacity to self-renew and can organize themselves to resemble the original organ or tumor in terms of both structure and function. Patient-derived cancer organoids are more suitable for the investigation of cancer biology and clinical medicine than conventional 2D cell lines or patient-derived xenografts. With recent advances in genetic analysis technology, the genetic information of various tumors has been clarified, and personalized medicine based on genetic information has become clinically available. Here, we have reviewed the recent advances in the development and application of patient-derived cancer organoids in cancer biology studies and personalized medicine. We have focused on the potential of organoids as a platform for the identification and development of novel targeted medicines for pancreatobiliary cancer, which is the most intractable cancer.
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Liu, Feiyang, Dandan Ma, Qingsong Liu, and Xiaowei Qi. "A novel patient-derived organoid model for triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy for screening personalized drugs." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e12625-e12625. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e12625.
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e12625 Background: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy (NAC) have high risk of recurrence. In this study, we developed a novel organoid culture method for growing TNBC patient samples after NAC, with the goal of generating models that more faithfully recapitulates the key features of the primary tumor for screening personalized drugs. Methods: We established organoid lines derived from TNBC patients who had residual tumors after anthracycline and taxane±platinum based NAC as well as normal human mammary tissues. The histopathology signatures of organoid lines were characterized by H&E staining and immunohistochemistry. The genetic and transcriptional features of organoids were analyzed by targeted sequencing and RNA sequencing. A set of clinical drugs were screened for their ability to suppress cancer organoids in vitro and the consistency was also analyzed between clinical response and drug efficacy of organoid models. Results: A total of 10 cancer organoid lines were successfully established, and cultured stably for more than 4 months. For comparison, 2 organoids derived from normal mammary tissues were generated as well. Our success rate for establishing cancer organoids from TNBC patient samples after NAC was 83% (10 out of 12 samples), and for organoids from normal mammary tissues, it was 100% (2 out of 2 samples). These organoid lines closely recapitulated the histopathology, genetic and transcriptional signature, and intratumor heterogeneity of the primary tissues. We found that active PI3K/AKT/mTOR pathways signaling is always required for cancer organoids from TNBC patient samples after NAC, while Wnt pathway activation is dispensable for generation of them in comparison to the organoid cultures from normal breast tissues. More importantly, we found that the anti-Trop-2 antibody-drug conjugate sacituzumab govitecan (IMMU-132) showed higher sensitivity to most cancer organoids from TNBC patient samples after NAC (8 out of 10 samples) with IC50 ranging from 0.3 nM to 86 nM in contrast to 5-FU (5 out of 10 samples) with IC50 ranging from 0.9 μM to 10.2 μM in vitro, whose prodrug capetabine is considered as a standard option in the postneoadjuvant setting. These results were also confirmed in patient-derived tumor xenograft models. The consistency of drug efficacy in the established cancer organoids and the patient responses in the clinic reached to 70% (7 out of 10 samples). Conclusions: We established a novel organoid culture system for TNBC patient samples after NAC, which was potentially a useful platform to explore molecular characteristics of TNBC patients after NAC and discover personalized drugs for intensive treatment.
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Oh, Ju Hee, Hyang Sook Seol, Eun Hye Choi, Yong Jae Lee Lee, Jung-Yun Lee, Sang Wun Kim, Sunghoon Kim, et al. "Abstract 200: Patient-derived cervical cancer organoids as a pre-clinical model predicting drug and radiation response." Cancer Research 83, no. 7_Supplement (April 4, 2023): 200. http://dx.doi.org/10.1158/1538-7445.am2023-200.
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Abstract Objective: Advanced cervical cancer is still difficult to treat and in the case of recurrent cancer, it is desirable to utilize personalized treatment rather than uniform treatment because the type of recurrence is different for each individual. Therefore, this study aimed to establish patients-derived organoid platform to determine the effects of chemotherapy, radiation therapy, and targeted therapy in cervical cancer. Methods: We established organoids from four patients with various types of cervical cancer. The histopathological and gene profiles of these organoid models were compared to determine their characteristics and the maintenance of the patient phenotype. Each type of organoid was also subjected to anticancer drug screening and radiation therapy to evaluate its sensitivity. Results: We established patient-derived organoids recapitulated the main elements of the original patient tumors, including the DNA copy number and mutational profile. We screened seven drugs that showed growth inhibition in cervical cancer organoids using two analytical tools (individual drugs and an FDA-approved drug library). Moreover, squamous cell carcinoma and villoglandular carcinoma showed significant response to radiation therapy, however, adenocarcinoma and large-cell neuroendocrine carcinoma showed relative resistance to radiation therapy. Conclusion: Our results showed patient-derived cervical cancer organoid can be used as a pre-clinical model for drug and radiation sensitivity test. These findings suggest that patient-derived cervical cancer organoids could be used as a personalized medicine confirmation tool and to improve treatment options for patients with various subtypes of cervical cancer. Citation Format: Ju Hee Oh, Hyang Sook Seol, Eun Hye Choi, Yong Jae Lee Lee, Jung-Yun Lee, Sang Wun Kim, Sunghoon Kim, Young Tae Kim, Hyun Ki Kim, Young Joo Lee, Eun Ji Nam. Patient-derived cervical cancer organoids as a pre-clinical model predicting drug and radiation 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 200.
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Kuraoka, Shohei, Shunsuke Tanigawa, Atsuhiro Taguchi, Akitsu Hotta, Hitoshi Nakazato, Kenji Osafune, Akio Kobayashi, and Ryuichi Nishinakamura. "PKD1-Dependent Renal Cystogenesis in Human Induced Pluripotent Stem Cell-Derived Ureteric Bud/Collecting Duct Organoids." Journal of the American Society of Nephrology 31, no. 10 (August 3, 2020): 2355–71. http://dx.doi.org/10.1681/asn.2020030378.
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BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor–derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD.MethodsCRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation.ResultsCysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation.ConclusionsCysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.
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von Schlichting, Jennifer, Stefan Peidli, Markus Morkel, Nils Blüthgen, and Leona Simon. "Abstract 4644: Modeling the effects of niche specific microenvironmental changes on patient-derived organoids." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4644. http://dx.doi.org/10.1158/1538-7445.am2023-4644.
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Abstract Here, we present an approach to study the effects of microenvironmental changes on inter- and intratumoral heterogeneity using colorectal cancer (CRC) patient-derived organoids. We identified relevant paracrine interactions between stromal and tumor cells based on the expression of ligand-receptor pairs in single cell-RNAseq data of 12 colorectal cancer patients. Physiological relevance was tested by adding CRC stroma-derived ligands to patient-derived organoids. A systematic screening of different conditions was enabled by multiplexed mass cytometry and scRNAseq analysis. We also aimed to model the effects of a changing extracellular matrix composition, by supplementing the laminin/collagen IV rich environment with other known ECM proteins such as collagen I to identify the impact of a changing substrate on cell plasticity. We found that ECM parameters as well as niche specific paracrine factors affect proliferation, differentiation, and developmental trajectories of colorectal cancer patient-derived organoids. We hypothesize that supplementing patient-derived organoids in vitro with physiologically relevant factors and substrates may expand the phenotypic space in which organoid cells differentiate, resulting in a more versatile preclinical model system.Our data provide insights into non-genetic sources of inter- and intratumoral heterogeneity resulting in differential drug responses. Additionally, they can serve as guidelines to improve existing patient-derived colorectal cancer models and provide a feasible approach to address common limitations in organoid culture. Citation Format: Jennifer von Schlichting, Stefan Peidli, Markus Morkel, Nils Blüthgen, Leona Simon. Modeling the effects of niche specific microenvironmental changes on patient-derived organoids. [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 4644.
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Kim, Hyunjin, So-Youn Jung, Sung Hoon Sim, Hee Jung Chae, Yun-Hee Kim, Yena Kim, Eun Gyeoung Lee, et al. "Abstract 6038: Target therapeutics evaluation using patient-derived organoids from breast cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6038. http://dx.doi.org/10.1158/1538-7445.am2022-6038.
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Abstract Purpose: Patient-derived organoid (PDO) is emerging tool for drug sensitivity evaluation. Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Therefore, we set up conditions for breast cancer PDOs and establish an in vitro drug evaluation system for future therapeutic approaches. Methods: All specimens were collected from histologically confirmed breast cancer patients after obtaining IRB approval from the National Cancer Center (NCC2017-0146, NCC2020-0299) under patient’s informed consent. Fifty-four patient samples obtained from surgery or biopsy were delivered to the laboratory within 1-2 hours for organoid establishment. All process for cell isolation from human tissues, criteria of successful culture and passaging, quality control (QC), producing genomic and histologic data were adjusted to make standard operating procedure. For long-term cultured PDOs, we tested 30 drugs including kinase inhibitor or standard therapeutic drugs with concentration from 0.001uM to 100uM using 384well plate and the results were analyzed with AUC data with bortezomib as a control. Results: We optimized organoids culture conditions for breast cancer tissues. Total of 54 tissues included disease subtypes as following: hormone positive (n=32), HER2 positive (n=2), triple-negative (n=19), and other (n=1). Nine organoids were successfully long term cultured over 5 passages and 4 were successfully cryopreserved. Clinic characteristics (TNM stages, subtypes, and disease status) showed no significant association with success of organoid establishment. Also, we confirmed that organoids had similar characteristics to the patients tissue through histological data (H&E, ER, PR, HER2, Ki-67, and CK-19). Measurement of AUC values for tested drugs showed variety range which implicates different drug sensitivity of each organoid. Currently, genetic information of long-term cultured organoid is ongoing with comparative analysis of drug sensitivity. Conclusions: We have established breast cancer organoids which reflect molecular characteristics of the original tumor. This model will serve as the system that can recapitulate drug response profiles of human cancer, and pave the way for screening innovative drugs, identifying novel targets, and stratifying patients for pertinent therapeutic options. (This work was supported by National Cancer Center, Korea (No.2010120) and National Research Foundation of Korea grant, funded by the Korea government (MSIT) (No. 2020M3A9A5036362)) Citation Format: Hyunjin Kim, So-Youn Jung, Sung Hoon Sim, Hee Jung Chae, Yun-Hee Kim, Yena Kim, Eun Gyeoung Lee, Seeyoun Lee, Jai Hong Han, Young Mi Kwon, Eun Sook Lee, Keun Seok Lee, Sun-Young Kong. Target therapeutics evaluation using patient-derived organoids from breast cancer [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 6038.
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Navarro-Serer, Bernat, Kenna Sherman, and Laura D. Wood. "Abstract PO-121: Investigating the role of human cancer-associated fibroblasts in pancreatic cancer invasion using patient-derived PDAC organoids." Cancer Research 81, no. 22_Supplement (November 15, 2021): PO—121—PO—121. http://dx.doi.org/10.1158/1538-7445.panca21-po-121.
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Abstract Organoid cultures have emerged as a promising research model as they more accurately recapitulate in vivo tumor features and provide a system to study cancer invasion, among others. We have previously reported the development of a human pancreatic cancer organoid model using surgically resected PDAC tumors. This model has allowed us to characterize molecular alterations critical for invasion. Culturing PDAC organoids in collagen I gels identified the ability of organoids to invade using two distinct, morphologically defined invasive phenotypes. Interestingly, invasion of PDAC organoids in collagen I decreased after culturing and passaging them in Matrigel, suggesting the microenvironment plays a crucial role in promoting invasion. To identify factors that increase PDAC organoid invasion, we sought to investigate the relationship between human PDAC organoids and patient-derived cancer-associated fibroblasts (CAFs). CAFs are a key component of the PDAC microenvironment, characterized by their ability to perform a variety of functions, including deposition and remodeling of ECM as well as promoting tumor growth, among others. CAFs may induce tumor-suppressive or tumor-promoting effects, and the role of CAFs subtypes in PDAC invasion has not been studied extensively. CAFs are also a heterogeneous group of cells: two mutually exclusive and reversible subtypes of CAFs have been discovered (inflammatory and myofibroblastic, also known as iCAFs and myCAFs respectively) driven on paracrine and juxtacrine signaling mechanisms. We have investigated the impact of patient-derived CAFs on PDAC organoid invasion by using conditioned media of CAFs in monolayers, which expand as myCAFs. After 2-3 days of culture, media was collected and human PDAC organoids (100-150 total) from 5 patients were allowed to grow and invade in collagen I gels with or without the addition of CAF conditioned media. Our results show conditioned media of CAFs increases the invasiveness of PDAC organoids but does not increase the percentage of organoids invading. In order to identify potential heterogeneity between primary CAF cultures from different patients, we performed time-lapse analyses of organoids in collagen I gels with conditioned media from 3-4 patient-derived CAFs. Interestingly, we did not find any significant differences between primary CAF cultures in inducing PDAC organoid invasion, suggesting that the ability of CAFs to induce tumor cell invasion does not vary widely between different patients. However, we did observe some patient-derived organoids did not increase invasiveness upon addition of conditioned media from any of the CAF cultures, showing heterogeneity in tumor cells from different patients in response to invasion-inducing factors secreted by CAFs. Future studies will examine the ability of iCAFs to induce invasion in our organoid model and the differences between the secretomes of myCAFs and iCAFs. Citation Format: Bernat Navarro-Serer, Kenna Sherman, Laura D. Wood. Investigating the role of human cancer-associated fibroblasts in pancreatic cancer invasion using patient-derived PDAC organoids [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-121.
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Chen, Dong, Yawen Tan, Zhichao Li, Wujiao Li, Lei Yu, Wei Chen, Yuchen Liu, et al. "Organoid Cultures Derived From Patients With Papillary Thyroid Cancer." Journal of Clinical Endocrinology & Metabolism 106, no. 5 (February 1, 2021): 1410–26. http://dx.doi.org/10.1210/clinem/dgab020.
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Abstract Context Papillary thyroid cancer (PTC) has been one of the most frequent endocrine malignancies around the world. Although most PTC patients have a favorable prognosis, a subgroup of patients die, especially when disease recurrence occurs. There is a pressing need for clinically relevant preclinical thyroid cancer models for personalized therapy because of the lack of in vitro models that faithfully represent the biology of the parental tumors. Objective To understand thyroid cancer and translate this knowledge to clinical applications, patient-derived PTC organoids as a promising new preclinical model were established. Methods Surgically resected PTC primary tissues were dissociated and processed for organoid derivation. Tumor organoids were subsequently subjected to histological characterization, DNA sequencing, drug screen, and cell proliferation assay, respectively. Results We describe a 3-dimensional culture system for the long-term expansion of patient-derived PTC organoid lines. Notably, PTC organoids preserve the histopathological profiles and genomic heterogeneity of the originating tumors. Drug sensitivity assays of PTC organoids demonstrate patient-specific drug responses, and large correlations with the respective mutational profiles. Estradiol was shown to promote cell proliferation of PTC organoids in the presence of estrogen receptor α (ERα), regardless of the expression of ERβ and G protein–coupled ER. Conclusion These data suggest that these newly developed PTC-derived organoids may be an excellent preclinical model for studying clinical response to anticancer drugs in a personalized way, as well as provide a potential strategy to develop prevention and treatment options for thyroid cancer with ERα-specific antagonists.
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Acklin-Wehnert, Scarlett, Divya Dayanidhi, Brian G. Czito, Manisha Palta, Christopher Willett, Christine Elissa Eyler, Christopher Mantyh, et al. "Feasibility of establishing and drug screening patient-derived rectal organoid models from pretreatment rectal cancer biopsies." Journal of Clinical Oncology 41, no. 4_suppl (February 1, 2023): 176. http://dx.doi.org/10.1200/jco.2023.41.4_suppl.176.
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176 Background: Response to neoadjuvant chemotherapy and radiation therapy in the treatment of locally advanced rectal cancer is heterogenous and prognostic of clinical outcomes, necessitating the need for predictive biomarkers to guide personalized treatment recommendations. Sensitivity to a given chemotherapy in patient-derived organoids predicts patient response to that chemotherapy, establishing it as a promising model for efforts to ascertain predictive biomarkers and personalize treatment decisions. This study assessed the feasibility of obtaining patient-derived rectal organoids from standard of care pre-treatment proctoscopy biopsies. Methods: In this clinical trial (NCT04371198), biopsies were obtained from patients with stage II rectal adenocarcinoma prior to receipt of neoadjuvant therapy. Tissue samples were mechanically and enzymatically dissociated to obtain a single cell suspension. Cells were then mixed with matrigel at a ratio of 2,000 cells:5 µL Matrigel in a 50ul dome and plated on a 24 well tissue culture plate with colorectal cancer organoid media at 37oC/5% CO2. Established patient-derived organoids were then used to perform drug screens with clinically-applicable chemotherapeutics including oxaliplatin, irinotecan and 5-FU, followed by high throughput drug screen using our recently published MicroOrganoSpheres platform using the NCI Approved Oncology Drugs Set VI* library. Results: Of the 20 patients enrolled, 17 (85%) patient-derived organoids were created from pre-treatment specimens. 15 (88%) of these samples were successfully established as defined by the ability to passage organoids for at least two passages. All established samples were used to perform standard of care drug screens and high throughout drug screens, which demonstrated differences in drug sensitivities among the samples. Moreover, within two weeks of receiving the sample, four established quickly enough to complete drug screening with oxaliplatin, SN38, and 5-Fluorouracil. Conclusions: These results demonstrate the feasibility of establishing patient-derived rectal organoids from biopsy specimens obtained by proctoscopy, and reinforce the utility of patient-derived organoids as a tractable ex vivo platform to personalize rectal cancer treatment. Planned future directions include in vitro determination of radiation therapy sensitivity as well as systematic assessment of the correlation between individual patients and their organoid model. Clinical trial information: NCT04371198 .
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Koch, Michael, Sandra Nickel, Ruby Lieshout, Susanna M. Lissek, Martina Leskova, Luc J. W. van der Laan, Monique M. A. Verstegen, Bruno Christ, and Francesco Pampaloni. "Label-Free Imaging Analysis of Patient-Derived Cholangiocarcinoma Organoids after Sorafenib Treatment." Cells 11, no. 22 (November 15, 2022): 3613. http://dx.doi.org/10.3390/cells11223613.
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Monitoring tumor growth dynamics is crucial for understanding cancer. To establish an in vitro method for the continuous assessment of patient-specific tumor growth, tumor organoids were generated from patients with intrahepatic CCA (iCCA). Organoid growth was monitored for 48 h by label-free live brightfield imaging. Growth kinetics were calculated and validated by MTS assay as well as immunohistochemistry of Ki67 to determine proliferation rates. We exposed iCCA organoids (iCCAOs) and non-tumor intrahepatic cholangiocyte organoids (ICOs) to sub-therapeutic concentrations of sorafenib. Monitoring the expansion rate of iCCAOs and ICOs revealed that iCCAO growth was inhibited by sorafenib in a time- and dose-dependent fashion, while ICOs were unaffected. Quantification of the proliferation marker Ki67 confirmed inhibition of iCCAO growth by roughly 50% after 48 h of treatment with 4 µM sorafenib. We established a robust analysis pipeline combining brightfield microscopy and a straightforward image processing approach for the label-free growth monitoring of patient-derived iCCAOs. Combined with bioanalytical validation, this approach is suitable for a fast and efficient high-throughput drug screening in tumor organoids to develop patient-specific systemic treatment options.
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Pinho, Diana, Denis Santos, Ana Vila, and Sandra Carvalho. "Establishment of Colorectal Cancer Organoids in Microfluidic-Based System." Micromachines 12, no. 5 (April 28, 2021): 497. http://dx.doi.org/10.3390/mi12050497.
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Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase in global health care costs. Patient-derived tumour organoids are considered preclinical models with the potential for preclinical drug screening, prediction of patient outcomes, and guiding optimized therapy strategies at an individual level. Combining microfluidic technology with 3D tumour organoid models to recapitulate tumour organization and in vivo functions led to the development of an appropriate preclinical tumour model, organoid-on-a-chip, paving the way for personalized cancer medicine. Herein, a low-cost microfluidic device suitable for culturing and expanding organoids, OrganoidChip, was developed. Patient-derived colorectal cancer organoids were cultured within OrganoidChip, and their viability and proliferative activity increased significantly. No significant differences were verified in the organoids’ response to 5-fluorouracil (5-FU) treatment on-chip and on-plate. However, the culture within the OrganoidChip led to a significant increase in colorectal cancer organoid-forming efficiency and overall size compared with conventional culture on a 24-well plate. Interestingly, early-stage and late-stage organoids were predominantly observed on-plate and within the OrganoidChip, respectively. The OrganoidChip thus has the potential to generate in vivo-like organotypic structures for disease modelling and drug screening applications.
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Kang, Sumin, Mi Rim Lee, Sun-Young Kong, Jong-Ho Lee, Dohyun Kwon, Ikjae Kwon, Soung-Min Kim, et al. "Abstract 6234: Development of a patient-derived organoid platform for predicting responses to standard treatments in oral cavity cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6234. http://dx.doi.org/10.1158/1538-7445.am2022-6234.
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Abstract Oral cancer is a rare cancer that occurs in the oral cavity, in areas such as the tongue, roof of mouth, floor of mouth, gum, and inside cheek. Oral cancer has frequent local recurrence and lymph node metastasis even after curative surgery with radiation to prevent recurrence. However, more than 50% of patients show a relapse pattern, and it is difficult to improve treatment due to the lack of experimental models that reflects the heterogeneity of the tumors as well as markers to predict treatment response. Although the patient-derived organoid (PDO) system is a good preclinical model that reflects the characteristics and heterogeneity of the original tumor, there are only small numbers of patients with oral cancer, so it has been relatively difficult to secure clinical samples. Therefore, oral cancer organoid development has so far been insufficient. Here, we have established a normal organoid library as well as cancer using primary tissue from many oral cancer patients. Seventy patients were enrolled in this study between Jan 2021 and Sep 2021. Fifty tumor organoids and fifty-five normal organoids were generated and sustained. Among them, 29 tumor organoids and 28 normal organoids were successfully cultured over 4 passages and cryopreserved. Moreover, 15 pairs of normal-tumor organoids were established. The success rate of organoids has no significant relationship with clinical information such as TNM stages, disease status and differentiation. These organoids recapitulated genomic features and histopathological characteristics of the patient tissue was examined through copy number variation (CNV) and IHC with TP53, P16, CK8, and so on, respectively. Normal organoids derived from tissue adjacent to the tumor of each patient showed typical morphology such as multilayer epithelium. Moreover, we set the assay system to evaluate the responses to radiation and the drug treatment using AUC value. Each organoid including normal and tumor showed different sensitivity to radiation. This oral cancer organoids platform is the largest repository in the field so far, apart from head and neck cancers. It provides a valuable platform for personalized treatment. It may also contribute to the discovery of factors associated with resistance or sensitivity to radiotherapy and chemotherapy in oral cavity cancer treatment. Citation Format: Sumin Kang, Mi Rim Lee, Sun-Young Kong, Jong-Ho Lee, Dohyun Kwon, Ikjae Kwon, Soung-Min Kim, Hye Won Shon, Yu-Sun Lee, Hyun-jin Kim, Joo Yong Park, Sung Weon Choi, Yun-Hee Kim. Development of a patient-derived organoid platform for predicting responses to standard treatments in oral cavity cancer [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 6234.
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Lee, Huisong, Hyeon Kook Lee, and Ja Lok Ku. "Establishment of Patient-derived Cholangiocarcinoma Organoids." European Journal of Surgical Oncology 49, no. 2 (February 2023): e159. http://dx.doi.org/10.1016/j.ejso.2022.11.444.
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Nguyen, Huyen T., Emily Kohl, Jessica Bade, Stefan E. Eng, Anela Tosevska, Ahmad Al Shihabi, Jenny J. Hong, et al. "Abstract 196: A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening." Cancer Research 83, no. 7_Supplement (April 4, 2023): 196. http://dx.doi.org/10.1158/1538-7445.am2023-196.
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Abstract Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by Neurofibromatosis Type 1 syndrome, a common disorder driven by alterations in the NF1 gene. cNF tumors are heterogenous and comprised of different cell types such as Schwann cells, fibroblasts, and mast cells. While cNFs do not undergo malignant transformation, they carry significant co-morbidities, including itching, pain, and socio-emotional repercussions. There is currently no therapy for cNF beside surgical removal or desiccation, which are typically unable to eliminate all lesions due to their number, with patients developing as many as several hundred cNFs, as well as additional concerns of scarring and potential re-growth. A lack of models to investigate cNFs growth and perform drug screenings has hindered drug discovery and development studies thus far. We set out to develop high throughput screening-compatible patient-derived cNF organoids to perform translational studies. Tumor organoids are promising ex vivo models to recapitulate a patient’s tumor histology, molecular features, and drug responses. To support drug discovery efforts focused on identifying effective systemic therapies for cNF, we have developed an approach to routinely establish and screen cNF tumor organoids. Here we present of our systematic characterization of media conditions together with molecular and functional analysis to validate cNF organoids as a model system. We enrolled n=12 Neurofibromatosis Type 1 syndrome patients in this study and procured cNF tumors that we dissociated to single cells to establish organoids in nine different media compositions. We performed a detailed comparison to the tumor of origin using a combination of immunohistochemistry, flow cytometry, DNA methylation and RNAseq for a subset of samples (n=6 tumors from 5 patients). cNF organoids were successfully established and grown in all cases, regardless of the NF1 alteration present. We determined the optimal medium that had the highest combined correlation (parental vs organoid) across all assays as well as promoted the highest rate of growth ex vivo. We also demonstrated feasibility of screening cNF organoids using our established mini-ring pipeline (Phan et al, 2019; Nguyen and Soragni, 2020; Al Shihabi et al, 2022). In summary, we have optimized conditions for ex vivo growth of cNF organoids that closely recapitulate the molecular and cellular heterogeneity of these tumors as measured by immunohistopathology, DNA methylation, RNAseq and flow cytometry. Our tractable patient-derived cNF organoid platform enables the rapid screening of hundreds of FDA-approved drugs in a patient- and tumor-specific manner. Citation Format: Huyen T. Nguyen, Emily Kohl, Jessica Bade, Stefan E. Eng, Anela Tosevska, Ahmad Al Shihabi, Jenny J. Hong, Sarah Dry, Paul C. Boutros, Andre Panossian, Sara Gosline, Alice Soragni. A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening [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 196.
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Costales-Carrera, Alba, Asunción Fernández-Barral, Pilar Bustamante-Madrid, Laura Guerra, Ramón Cantero, Antonio Barbáchano, and Alberto Muñoz. "Plocabulin Displays Strong Cytotoxic Activity in a Personalized Colon Cancer Patient-Derived 3D Organoid Assay." Marine Drugs 17, no. 11 (November 19, 2019): 648. http://dx.doi.org/10.3390/md17110648.
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Plocabulin is a novel microtubule-disrupting antitumor agent of marine origin that is currently undergoing phase II clinical trials. Plocabulin has potent antiproliferative and antiangiogenic actions in carcinoma cell lines and has antitumor activity in xenografted mice. Here, we used three-dimensional (3D) tumor organoids derived from three colorectal cancer (CRC) patients to study the effect of plocabulin in a personalized assay system that ensures dose dependence and high reproducibility. The cytotoxicity of plocabulin was an order of magnitude higher than that of the active irinotecan derivative SN38 (7-ethyl-10-hydroxy-camptothecin) in tumor organoids at different passages. Moreover, plocabulin maintained its strong cytotoxic activity in wash-out experiments, in which a short pulse treatment of tumor organoids was as efficient as continuous treatment. Our data show that plocabulin has a very potent cytotoxic action in CRC patient-derived tumor organoids, supporting ongoing clinical trials with plocabulin and the use of organoid assays to provide personalized validation of antitumor drugs.
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Habič, A., B. Majc, A. Porčnik, R. Bošnjak, J. Mlakar, T. Lah Turnšek, B. Breznik, and M. Novak. "P02.07.B Patient-derived glioblastoma organoids: Elucidating the mechanisms of glioblastoma therapeutic resistance in the context of tumor microenvironment." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii30—ii31. http://dx.doi.org/10.1093/neuonc/noac174.100.
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Abstract Background Intratumoral heterogeneity plays an important role in glioblastoma (GB) resistance to standard therapy consisting of irradiation and chemotherapy with temozolomide (TMZ). However, classical in vitro GB models fail to represent the complex cellular composition of tumors in vivo, which hinders relevant examination of GB therapeutic response. To overcome these limitations, we studied the effects of irradiation and TMZ in a novel patient-derived organoid model. Material and Methods We established a patient-derived GB organoid model by a protocol recently published by Jacob et al. Original tumor tissue and tissue-derived organoids were compared by immunofluorescence staining of selected cell type markers and qPCR analysis of expression levels of a panel of selected target genes, including 15 genes defining GB subtypes. To analyze GB therapeutic response, organoids from 11 patients were exposed to a single dose of irradiation (10 Gy), one-week treatment with TMZ (50 µM) or their combination. The effects of therapy were assessed by viability and invasion assays. Expression levels of a number of genes related to GB subtypes, epithelial-mesenchymal transition, stemness, DNA damage responses, cell cycle, cytokines, and cell markers of the tumor microenvironment (TME) were compared between treated organoids and untreated controls. In addition, the heterogeneity of the TME and its responses to treatment were investigated by spatially resolved transcriptomics with in situ sequencing (ISS) methodology. Results Organoids recapitulate inter-patient variability and reflect the cellular composition and gene expression levels of the tumor tissue from which they were derived. GB stem cells and differentiated cancer cells are present in organoids along with various cells of the TME, e.g., macrophages and microglia, lymphocytes, and endothelial cells. Irradiation and TMZ showed no significant effects on organoid viability and invasion. However, some target genes were differentially expressed in the treated organoids, such as E3 ubiquitin-protein ligase MDM2 and cyclin-dependent kinase inhibitor 1A (CDKN1A). To our knowledge, we are the first to apply spatially resolved transcriptomics (ISS) to formalin-fixed, paraffin-embedded sections of (un)treated GB organoids. Our results elucidate the role of the TME in GB therapeutic response and shed light on potential mechanism underlying GB therapy resistance. Conclusion Patient-derived GB organoids recapitulate the key characteristics and complex composition of patient’s tumor tissue, providing a valuable platform for studies of GB therapeutic response and resistance.
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Nie, Lei, Yang Liu, Yuxuan Che, Yijing Li, Wei Wang, Jingling Jin, Qingsong Cai, et al. "Establishment of Patient-Derived Organoid Culture Platform of Mantle Cell Lymphoma." Blood 138, Supplement 1 (November 5, 2021): 3508. http://dx.doi.org/10.1182/blood-2021-152994.
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Abstract Background Mantle Cell Lymphoma (MCL) is a rare incurable non-Hodgkin's lymphoma despite remarkable recent therapeutic innovations such as CAR T cell therapies. Drug sensitivity and immunotherapy efficacy assays using preclinical tumor models are important in new therapy development. As for preclinical tumor models, in addition to the commonly used mouse model such as patient-derived xenograft (PDX) and genetically engineered mouse models, a 3 rd tumor model, the patient-derived organoid (PDO) in 3D culture model has been established in many types of solid tumors. However, no PDO models have been generated from MCL yet. We have established MCL PDO model by optimizing cell isolation, culture add-ons, spatial and temporal conditions. Our protocol presents a versatile MCL PDO platform, suitable for quick drug screens and applicable for rapid immunotherapy evaluation. For proof of concept demonstration, we tested the therapeutic efficacy of CD19-targeted CAR T-cell in MCL-specific PDOs. The established PDO procedures can be used for diverse biopsies including whole blood, apheresis, bone marrow, lymph node and previously established PDX tumors. Methods For human blood, bone marrow and apheresis biospecimens, we first eliminated red blood cells using RBC lysis buffer. The cells were then spun down and cell pellets were resuspended in culture medium and aliquoted and spun-down into V-shaped 96-well plates at 1-6´10 6 cells per well. After overnight incubation, the cell culture medium was replaced by 60 ml of precooled 60% Matrigel per well. The 3D formed aggregates were gently transferred into 24-well plate and feed the solidified domes with the medium containing an in-house cytokine cocktail. The developed organoids reach the drug screening optimal phase within 3-6 days or it can be further expanded for future use. For PDX tumor and human lymph node biospecimens, we first sliced tumor tissue using surgical scalpel blade, which resulted in small tissue pieces that were then resuspended in HBSS. Debris (>100 mm) were removed by brief gravity sedimentation. The resultant tissue pieces were then resuspended in 60% Matrigel in culture medium and dispensed onto pre-warmed 24-well plates. The primary tumor organoids were observed for 3-5 days before drug testing on processed for further expansion. For organoid passaging, primary organoids were extracted in pre-cooled medium by mechanically breaking the gel domes. The extracted organoids were digested by collagenase/dispase. The resultant MCL and stromal cells were enforced to aggregate in V-shape wells as described in the apheresis procedure. Organoid cell composition was examined using FACS. Drug sensitivity and T-cell activity against the MCL organoids were assessed using CellTiter-Glo 3D kit. Results We have successfully established and optimized the PDO procedure from diverse MCL biopsies (Fig. 1A). The success rate of MCL organoid from apheresis was > 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples (> 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although <5% of total cell population is composed of macrophages, circulating fibroblast, epithelial and endothelial cells and they are indispensable for organoid formation and expansion. Addition of hematopoietic cytokines in culture medium significantly improved the organoid formation and expansion from diverse biopsies. FACS with lineage markers revealed that the cell populations of the primary and secondary organoids were not significantly different (Fig. 1B). Tumor-killing activity of the CD19-targeting CAR T cells was assessed by co-culturing the CAR T cells with MCL organoids. Conclusion We have established an MCL PDO platform which is time-efficient, labor-saving, cost-effective and highly reproducible. This platform provides a rapid approach for immune cell activity assays and drug screening. The organoids have been successfully used to generate PDX models. This platform can also be used for investigating the mechanism of drug resistance in the context of different TMEs. Figure 1 Figure 1. Disclosures Wang: Acerta Pharma: Consultancy, Honoraria, Research Funding; BioInvent: Research Funding; Pharmacyclics: Consultancy, Research Funding; Lilly: Research Funding; CStone: Consultancy; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Anticancer Association: Honoraria; VelosBio: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Juno: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Bayer Healthcare: Consultancy; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Imedex: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; Dava Oncology: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Clinical Care Options: Honoraria; Chinese Medical Association: Honoraria; BGICS: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria.
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Wang, Yue, Glauco R. Souza, and Robert J. Amato. "3D Organoids from Milligrams of Genitourinary Cancer Patients Tissue Retain Key Features of Original Tumors." Journal of Clinical Oncology 36, no. 6_suppl (February 20, 2018): 268. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.268.
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268 Background: 3D organoid culture, especially that generated from patient samples, is becoming prevalent as 2D cell culture models fail to provide clinical relevance. However, a relatively large amount of starting tissue is still required for this process, and the turnover rate and overall success rate vary greatly if relying solely on spontaneous aggregation. Methods: Here, we report the successful use of magnetic 3D bioprinting to drive organoid formation derived from fresh tumor samples of patients with prostate cancer, renal cell cancer, and urothelial cancer. Results: Only milligrams to tens of milligrams of starting tissues (from fine-needle biopsies, core biopsies, or surgical excision) were used. Most organoids formed overnight, with a very high overall success rate ( > 95%, n = 25). We have demonstrated that these cultures can be maintained, passed, and cryopreserved like conventional 2D cultures. More importantly, we characterized the organoids derived from prostatic adenocarcinomas and neuroendocrine tumors by the means of immunostaining and showed that the organoids expressed the same signature proteins as the originating tumors. This clinical concordance is crucial for downstream translational and biological research. We also showed that these adenocarcinoma cells responded to enzalutamide, an androgen receptor inhibitor that is used clinically to treat prostatic adenocarcinoma. Conclusions: In summary, we have established an efficient and robust system to create patient-derived 3D organoids; molecular characterization shows clinical concordance with original patient tumors; and our system shows great potential in drug screening with patient-derived 3D cultures.
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Senkowski, Wojciech, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, et al. "Abstract 5779: A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine." Cancer Research 83, no. 7_Supplement (April 4, 2023): 5779. http://dx.doi.org/10.1158/1538-7445.am2023-5779.
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Abstract High-grade serous ovarian cancer (HGSC) is the most prevalent and lethal ovarian cancer type. While HGSC usually responds well to primary treatment, most cases eventually relapse. Functional precision medicine - tailoring individualized treatments based on functional in vitro assays on patient-derived cells - has been recently employed in cancer clinical trials. Cancer organoids - three-dimensional, self-organizing, self-renewing cell cultures that recapitulate original tissue structure and function - have been applied as cellular models in these trials. However, in case of HGSC, organoid derivation has proven time consuming and inefficient, hindering their application in functional precision medicine due to a short time window, in which therapy for each patient needs to be selected. To address this problem, we aimed to establish whether drug vulnerabilities at HGSC relapse could be predicted using organoids derived from the primary disease cells. We derived sequential organoid models from material sampled during primary treatment and at relapse. Then, for organoid pairs (primary-relapse), we performed large-scale drug response profiling of a library of 370 compounds (approved drugs or drugs in clinical development), in 384-well microplate format, alone or in combination with a standard HGSC chemotherapeutic agent carboplatin. First, we found that HGSC organoid responses to standard chemotherapeutics retrospectively correlated to observed clinical treatment outcomes. But further, for each patient we identified compounds with pronounced cytotoxicity both in the primary and in the relapsed model, amounting to 66% of all hits (7% were primary-specific and 27% relapse-specific). We then focused on identifying patient-specific hits rather than compounds displaying general toxicity in all patient models. Based on a potential clinical applicability, for three patients we selected compounds for validation in organoid outgrowth assay, with prolonged (>1 month) drug-free period post-treatment. In two patients, AZD4573, a selective CDK9 inhibitor in clinical development for hematological malignancies, at nanomolar concentrations caused eradication of organoids when combined with carboplatin. Organoids from the third patient were vulnerable to nitazoxanide, an approved anti-helminthic agent and an inhibitor of mitochondrial oxidative phosphorylation. Importantly, the selected final hits were identified solely based on screening in organoid models from primary disease. In summary, we here demonstrate that HGSC organoids derived from primary disease material predict a majority of patient-specific drug vulnerabilities of organoids derived from the relapsed HGSC lesions. This indicates that patient stratification in functional precision medicine for treatment of HGSC relapse could be prospectively performed at the primary disease stage. Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Yan-Jun Chen, Erdogan P. Erkan, Mia K. Høg, Ida M. Larsen, Tarja Lamminen, Katja Kaipio, Jutta Huvila, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine. [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 5779.
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Baubet, Valerie, David Beale, Santi Mariarita, Angela Viaene, Peter Madsen, Fadi Jacob, Guo-li Ming, et al. "MODL-28. Patient-derived, three-dimensional organoid platform for pediatric brain tumor modeling." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i175. http://dx.doi.org/10.1093/neuonc/noac079.651.
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Abstract Brain tumors have become the leading cause of cancer-related death in children. An important hurdle to scientific and clinical progress in the field has been the limited availability of preclinical tumor models. Historically, few pediatric brain tumor cell lines have been established and these often poorly recapitulate the phenotypes of the original tumors. In recent years, the Children’s Brain Tumor Network (CBTN) has accelerated the development of patient-derived cell lines and xenografts, offering these resources to the community through open-source access. While these models are extremely valuable, their development process can be lengthy and result in clonally selected lines which presents a challenge for studying complex tumor biology. To address the need for three-dimensional tissue culture, our group in conjunction with CBTN, utilized organoid culture from fresh tissue specimens obtained directly from surgical resection of various pediatric brain tumor histologies. This resulted in the development and banking of over 30 organoid models, which included ependymoma, high-grade glioma, medulloblastoma, atypical teratoid-rhabdoid tumor, diffuse midline glioma, and low-grade glioma diagnoses. Tissue was processed within an hour post extraction and cultured with universal media composition for each diagnosis. Organoid growth was observed within 2-3 weeks of initiation and continued for up to three months before banking. Banked organoids established growth upon return to culture. Phenotypic analysis revealed organoid cell composition that represented clinical histology. Importantly, organoids returned to culture post-banking demonstrated similar cell composition to those in the original culture, indicating their utility for subsequent preclinical testing. Here we provide a simple and efficient workflow for the generation and characterization of three-dimensional tumor organoids generated from fresh surgical pediatric brain tumor tissue. The platform has the potential to accelerate investigations into tumor biology and empower a diverse array of translational studies for the pediatric brain tumor field.
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Tran, Loan, Raheleh Sheibani Tezerji, Carlos Uziel Perez Malla, Anna Malzer, Ajna Logo, Katarina Misura, Theresia Mair, et al. "Abstract A011: Epigenetic vulnerabilities in patient-derived colorectal cancer organoids." Cancer Research 82, no. 23_Supplement_2 (December 1, 2022): A011. http://dx.doi.org/10.1158/1538-7445.cancepi22-a011.
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Abstract Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide. Here, we established human patient-derived colorectal cancer organoid cultures (PDOs) to decipher the patient-specific DNA methylation profile and drug sensitivity towards 5-aza-2’-deoxycytidine, a DNA methyltransferase inhibitor. Organoid lines (n=15) were generated from adjacent normal mucosa and tumor tissue located in different anatomical sites of the colon and rectum. The derived PDOs were characterized on a histopathological level and reproduced the grade and differentiation capacity of their parental tumors. Additional genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. However, the stability of the DNA methylation landscape of human cancer organoids remains largely unknown. In genome-wide methylation analysis of long-term organoid cultures (up to 6 months in culture), we observed surprisingly stable methylation signatures that recapitulates the patient’s profile whilst demonstrating the vast intertumoral heterogeneity among patients. Strikingly, we identified a tumor-specific methylation signature that consisted of 39 CpG sites, which were unmethylated in normal epithelial cells but highly (91-96%) methylated in all tumor cells analyzed. This signature allows for the estimation of the percentage of tumor content in resected tissues and might represent potent biomarkers for early CRC diagnostics. Notably, it has been demonstrated that PDOs have the potential to predict clinical outcome and response to chemo- and radiation therapy in patients. Herein, drug screening with 5-aza-2’-deoxycytidine revealed heterogeneous responses and a clustering into drug sensitive, median and resistant organoid lines. ATAC- and RNA-seq analyses identified pathways rendering drug sensitivity and resistance. Our findings highlight the utility of PDOs as an advanced model system to study the role of the epigenome, especially DNA methylation, and its impact on tumor burden and vulnerability towards epigenetic modifiers. Citation Format: Loan Tran, Raheleh Sheibani Tezerji, Carlos Uziel Perez Malla, Anna Malzer, Ajna Logo, Katarina Misura, Theresia Mair, Thomas Dillinger, Madeleine Kuroll, Velina Atanasova, Julijan Kabiljo, Helga Schachner, Katharina Wöran, Judith Stift, Helmut Dolznig, Walter Pulverer, Michael Bergmann, Gerda Egger. Epigenetic vulnerabilities in patient-derived colorectal cancer organoids. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A011.
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Abdullah, Kalil, Joseph Buehler, Cylaina Bird, MIlan Savani, Alex Sternisha, Michael Levitt, Kimmo Hatanpaa, Timothy Richardson, and Samuel McBrayer. "TMOD-06. CREATION OF PATIENT-DERIVED LOWER GRADE GLIOMA ORGANOID MODELS FOR PERSONALIZED TREATMENT RESPONSE ASSESSMENT." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi216—vi217. http://dx.doi.org/10.1093/neuonc/noab196.868.
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Abstract Creating in vitro models of lower grade glioma represents a major challenge in neuro-oncology research. There are few such models that are tractable and widely used, which has hindered understanding of the biology of these tumors. Recently, substantial progress has been made in generating patient-derived in vitro organoid models of high grade glioma, but modeling lower grade disease remains difficult. Based on our experience creating neurosphere cultures of lower grade astrocytomas from genetically engineered mice, we hypothesized that modifying patient-derived organoid generation protocols to incorporate physiological oxygen levels would allow establishment and propagation of lower grade glioma organoids. In this study, we show that this approach supports efficient organoid model generation from primary glioma specimens across all histological subtypes and tumor grades (WHO Grade I-IV, n = 20). These organoid models retain key characteristics of their respective parental tumors, including IDH mutations and other genetic alterations, metabolite profiles, intratumoral heterogeneity, cellular composition, and cytoarchitectural features. Importantly, lower grade glioma organoids can be cultured for months and reanimated after biobanking. Our high success rate ( >90%) in establishing organoid models from primary lower grade glioma tissue samples further highlighted opportunities for treatment response assessments. To perform longitudinal measurements of therapy-induced changes in glioma organoid viability, we designed a novel, non-invasive imaging assay (termed rapid apex imaging) to determine real-time treatment response in low and high grade gliomas. We evaluated longitudinal responses of glioblastoma and IDH1 R132H-positive Grade II astrocytoma organoids to temozolomide and olaparib with and without radiation treatment. We quantified topological changes in organoid structure by building a bioinformatics tool to translate imaging data into a cellularity metric as a biomarker of organoid response. Our work unveils an effective new method to create in vitro, personalized models of lower grade glioma that supports elucidation of treatment sensitivity profiles.
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Su, Chang, Kelly A. Olsen, Catherine E. Bond, and Vicki L. J. Whitehall. "The Efficacy of Using Patient-Derived Organoids to Predict Treatment Response in Colorectal Cancer." Cancers 15, no. 3 (January 28, 2023): 805. http://dx.doi.org/10.3390/cancers15030805.
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Colorectal cancer is an important cause of morbidity and mortality worldwide. The current treatment landscape includes chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. A key challenge to improving patient outcomes is the significant inter-patient heterogeneity in treatment response. Tumour organoids derived from the patients’ tumours via surgically resected or endoscopically biopsied tissue, have emerged as promising models for personalised medicine. This review synthesises the findings, to date, of studies which have explored the efficacy of ex vivo organoid sensitivity testing for predicting treatment response. Most studies have focused on predicting the response to standard-of-care radiotherapy and chemotherapy options. There is strong evidence to support organoid sensitivity testing of ionising radiation, 5-fluorouracil, and irinotecan, and to a lesser extent, oxaliplatin and TAS-102. Fewer studies have used organoids to identify patients who are likely to benefit from novel treatment options that otherwise remain in clinical trials. This review also summarises recent advancements in organoid culture to include non-epithelial components of the tumour microenvironment, to allow testing of immunotherapy and certain targeted therapy options. Overall, further prospective trials will support the implementation of organoid-based personalised medicine for colorectal cancer patients in the future.
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Bock, Nathalie, Farzaneh Forouz, Luke Hipwood, Julien Clegg, Penny Jeffery, Madeline Gough, Tirsa van Wyngaard, et al. "GelMA, Click-Chemistry Gelatin and Bioprinted Polyethylene Glycol-Based Hydrogels as 3D Ex Vivo Drug Testing Platforms for Patient-Derived Breast Cancer Organoids." Pharmaceutics 15, no. 1 (January 12, 2023): 261. http://dx.doi.org/10.3390/pharmaceutics15010261.
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3D organoid model technologies have led to the development of innovative tools for cancer precision medicine. Yet, the gold standard culture system (Matrigel®) lacks the ability for extensive biophysical manipulation needed to model various cancer microenvironments and has inherent batch-to-batch variability. Tunable hydrogel matrices provide enhanced capability for drug testing in breast cancer (BCa), by better mimicking key physicochemical characteristics of this disease’s extracellular matrix. Here, we encapsulated patient-derived breast cancer cells in bioprinted polyethylene glycol-derived hydrogels (PEG), functionalized with adhesion peptides (RGD, GFOGER and DYIGSR) and gelatin-derived hydrogels (gelatin methacryloyl; GelMA and thiolated-gelatin crosslinked with PEG-4MAL; GelSH). Within ranges of BCa stiffnesses (1–6 kPa), GelMA, GelSH and PEG-based hydrogels successfully supported the growth and organoid formation of HR+,−/HER2+,− primary cancer cells for at least 2–3 weeks, with superior organoid formation within the GelSH biomaterial (up to 268% growth after 15 days). BCa organoids responded to doxorubicin, EP31670 and paclitaxel treatments with increased IC50 concentrations on organoids compared to 2D cultures, and highest IC50 for organoids in GelSH. Cell viability after doxorubicin treatment (1 µM) remained >2-fold higher in the 3D gels compared to 2D and doxorubicin/paclitaxel (both 5 µM) were ~2.75–3-fold less potent in GelSH compared to PEG hydrogels. The data demonstrate the potential of hydrogel matrices as easy-to-use and effective preclinical tools for therapy assessment in patient-derived breast cancer organoids.
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Wang, Zhaohui, Emilio Cortes-Sanchez, Chieh-Hsiang Yang, Daniel Nelson, Daniel Delubac, Shiaowen David Hsu, Alana Welm, and Xiling Shen. "Micro-organospheres: An automated patient-derived model platform for rapid drug screening of breast cancer." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e12628-e12628. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e12628.
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e12628 Background: Patient-derived breast cancer (BC) organoids are valuable preclinical models to study patient drug responses, demonstrating good correlations with patients’ clinical outcomes. However, establishment and expansion of such organoids from patient tumors for drug screening is currently a time-consuming and labor-intensive process. A more rapid and high-throughput method will enable broader utility in diagnostics and drug development. Methods: An automated, rapid and scalable microfluidic platform was used to process and develop BC micro-organospheres. Drug sensitivities studies on BC micro-organospheres were performed on day 3 and day 6 using 10-FDA approved drugs, including palbociclib, adriamycin, 5-FU, gemcitabine, methotrexate, everolimus, paclitaxel, docetaxel, ixabepilone, and vinblastine. The responses of micro-organospheres and organoids to the drugs were assessed by CellTiter 3D Glo assay on day 6 after the drug treatment. The growth and establishment of the micro-organospheres by imaging. The drug sensitivity and resistance of the micro-organospheres were analyzed by calculating the percentage cell viability and normalized growth rate inhibition (GRI) and compared to organoids. Results: We successfully established micro-organospheres from eight patient-derived BC organoids with a 100% success rate. The micro-organospheres preserved similar cell morphologies to the bulk organoids. 7/8 micro-organosphere models had similar drug response patterns to organoids between day 3 and day 6 as evident by the GRI heatmap. Specifically, we treated matching micro-organospheres and conventional organoids from two patients with 10 frontline BC chemotherapy drugs, and both showed similar response patterns with GRI heatmap. For the other 6 patient-derived models, the responses of micro-organospheres to docetaxel and everolimus also matched the historical drug responses of in bulk organoid culture with similar GRI heatmap. Conclusions: We have now shown the feasibility of establishing micro-organospheres as a rapid, scalable, and cost-effective platform to study patient-derived BC drug response. This technology has the potential to be used for both diagnostics to guide patient treatment and as a screening platform for new BC drug discovery.
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Kim, Tae Il. "Clinical applications and optimization of patient-derived organoids in intestinal diseases." Organoid 2 (August 25, 2022): e22. http://dx.doi.org/10.51335/organoid.2022.2.e22.
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Since the first successful establishment of organoids from adult intestinal stem cells, organoid technology has rapidly developed. With advances in normal organoid technology, intestinal disorders, such as colorectal tumors and inflammatory bowel disease, have been major target diseases for patient-derived organoid (PDO) development. PDO biobanking for colorectal cancer has subsequently been developed, and some reports have shown the possibility of using PDO models to predict anticancer drug responses. However, to apply these models to real-world practice, we need more long-term clinical follow-up data from further large-scale PDO biobanks, as well as advanced technology for more rapid and efficient PDO establishment. In addition, in the field of regenerative medicine, the implantation of healthy intestinal PDOs to refractory tissue defects could be a new treatment strategy to accelerate the healing and repair of mucosal defects. This PDO technology could also be applied to inflammatory bowel diseases and serve as a very useful model for drug development via high-throughput screening of useful candidate drugs.
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Khare, Sonal, Chi-Sing Ho, Madhavi Kannan, Brian Larsen, Brandon Mapes, Jenna Shaxted, Jagadish Venkataraman, and Ameen Salahudeen. "62 Applying machine vision to empower preclinical development of cell engager and adoptive cell therapeutics in patient-derived organoid models of solid tumors." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A70. http://dx.doi.org/10.1136/jitc-2021-sitc2021.062.
Full textAbstract:
BackgroundCell engager and adoptive cell therapeutics have emerged as efficacious and durable treatments in patients with B-cell malignancies. Though many analogous strategies are under development in solid tumors, none have received approval. Preclinical development of these therapies requires cell labeling of immortalized cell lines and/or primary expanded T cells to distinguish target and effector cells. However, cell engager and adoptive cell therapies have had limited evidence of reproducibility in primary patient-derived models such as tumor organoid cultures thus far. Here, we build upon our tumor organoid platform1 to measure organoid specific responses to these therapies. Utilizing machine vision coupled with time-lapse-microscopy, we obtain multiparameter kinetic readouts of patient-derived tumor organoid cell killing and allogeneic MHC-matched primary peripheral blood mononuclear cells (PBMCs).MethodsThe patient-derived tumor organoids were co-cultured with PBMCs in the presence of engagers/activators and vital dyes and incubated for 96 hrs. Cell death was measured by quantifying the caspase 3/7 vital dye pixel intensities at different time points using high throughput imaging. As a first step, a fully convolutional neural network was trained to segment out organoids from brightfield images comprised of organoids, immune cells and potential background artifacts. This segmentation mask was then transferred over to registered caspase 3/7 images to quantify tumor cell specific phenotypes in a rapid and automated manner.ResultsThe time-lapse imaging assay allowed for both the tracking of the organoid growth over time as well as the quantification of the kinetics of engagers/activators in comparison to controls resulting in accurate and precise technical reproducibility. Further, this assay allowed for the co-localization of the organoids and the immune cells over time, thus, enabling a spatiotemporal summary of dose dependent efficacy of candidate therapeutics.ConclusionsWe demonstrate the scalability and throughput of a machine vision tumor organoid immune co-culture platform across multiple unique patient-derived tumor organoid lines bearing a target of interest, enabling future discovery of biomarkers of therapeutic response and resistance.ReferenceLarsen B, Kannan M, Langer LF, Khan AA, Salahudeen AA, A pan-cancer organoid platform for precision medicine. Cell Reports 2021; 36:109429
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Frappart, Pierre-Olivier, Karolin Walter, Johann Gout, Alica K. Beutel, Mareen Morawe, Frank Arnold, Markus Breunig, et al. "Pancreatic cancer-derived organoids – a disease modeling tool to predict drug response." United European Gastroenterology Journal 8, no. 5 (February 19, 2020): 594–606. http://dx.doi.org/10.1177/2050640620905183.
Full textAbstract:
Background Organotypic cultures derived from pancreatic ductal adenocarcinoma (PDAC) termed pancreatic ductal cancer organoids (PDOs) recapitulate the primary cancer and can be derived from primary or metastatic biopsies. Although isolation and culture of patient-derived pancreatic organoids were established several years ago, pros and cons for individualized medicine have not been comprehensively investigated to date. Methods We conducted a feasibility study, systematically comparing head-to-head patient-derived xenograft tumor (PDX) and PDX-derived organoids by rigorous immunohistochemical and molecular characterization. Subsequently, a drug testing platform was set up and validated in vivo. Patient-derived organoids were investigated as well. Results First, PDOs faithfully recapitulated the morphology and marker protein expression patterns of the PDXs. Second, quantitative proteomes from the PDX as well as from corresponding organoid cultures showed high concordance. Third, genomic alterations, as assessed by array-based comparative genomic hybridization, revealed similar results in both groups. Fourth, we established a small-scale pharmacotyping platform adjusted to operate in parallel considering potential obstacles such as culture conditions, timing, drug dosing, and interpretation of the results. In vitro predictions were successfully validated in an in vivo xenograft trial. Translational proof-of-concept is exemplified in a patient with PDAC receiving palliative chemotherapy. Conclusion Small-scale drug screening in organoids appears to be a feasible, robust and easy-to-handle disease modeling method to allow response predictions in parallel to daily clinical routine. Therefore, our fast and cost-efficient assay is a reasonable approach in a predictive clinical setting.