Journal articles on the topic 'Primary Human Tumor Cultures'
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Girinsky, T., J. M. Cosset, N. Chavaudra, F. Geara, R. Lubin, H. Bounik, E. Janot, et al. "Primary Cultures from Human Tumor Biopsies." International Journal of Radiation Biology 56, no. 5 (January 1989): 845–46. http://dx.doi.org/10.1080/09553008914552181.
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Zaffaroni, N., R. Villa, L. Oriandi, and R. Silvestrini. "Radiosensitivity testing of human tumor primary cultures." European Journal of Cancer 29 (January 1993): S217. http://dx.doi.org/10.1016/0959-8049(93)91839-d.
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Westphal, Manfred, Hildergard Nausch, and Hans-Dietrich Herrmann. "Cyst Fluids of Malignant Human Brain Tumors Contain Substances That Stimulate the Growth of Cultured Human Gliomas of Various Histological Type." Neurosurgery 25, no. 2 (August 1, 1989): 196–201. http://dx.doi.org/10.1227/00006123-198908000-00007.
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Abstract The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.
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Dougherty, Mark, Eric Taylor, and Marlan Hansen. "TMET-34. RADIATION METABOLOMICS IN PRIMARY HUMAN MENINGIOMA AND SCHWANNOMA: EARLY EXPERIENCE AND INITIAL RESULTS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii269. http://dx.doi.org/10.1093/neuonc/noac209.1039.
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Abstract Introduction Meningiomas and schwannomas account for 45% of primary CNS tumors. Yet when surgery and radiation fail, no further treatments exist. Metabolomics has been used to discover new cancer therapies; however, to date few have used metabolomics to study meningiomas and schwannomas. Here we present initial results and lessons learned from this novel endeavor. METHODS Primary tumors were obtained from patients during surgery and immediately taken for culturing or xenograft implantation. Upon reaching >90% confluence, cultures were treated with 0gy, 3gy, 10gy, or 20gy gamma radiation, then flash frozen 6 or 72 hours post-treatment. Xenograft tumors were implanted in nude mice. MRI 4 weeks post-implantation confirmed tumor viability. Mice were then given 10gy, 20gy, or sham radiation treatment. Xenografts were harvested 72 hours post-treatment. Metabolites were measured with a ThermoISQ gas chromatography-mass spectrometer. RESULTS Eleven meningiomas and nine schwannomas were successfully cultured. Unsupervised hierarchical clustering of cultures demonstrated greater influence from tumor of origin than from radiation. Univariate analysis of schwannoma xenografts demonstrated elevated ornithine following radiation (fold change 1.62; P = 0.008). However, principal component analysis did not show significant between-group differentiation. Orthotopic meningioma xenografts did not produce sufficient tissue for metabolomics; however, subsequent subcutaneous implants have been successful (data forthcoming). CONCLUSION Standard cell cultures did not reveal significant metabolic changes following radiation; it is unclear whether this was due to culture technique or inter-tumor heterogeneity. In radiated schwannoma xenografts, elevated ornithine may implicate related pathways such as ornithine decarboxylase-mediated polyamide synthesis for DNA double-strand break repair. Compared to other ‘-omics’ studies, metabolomics requires more tissue per sample ( >10mg) and is more sensitive to environmental conditions. Thus, large sample sizes are needed to detect significant changes, and xenografts are likely superior to cell culture. Future plans include increased xenograft sample size and stable isotope tracing for pathway analysis.
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Strietz, Juliane, Stella S. Stepputtis, Marie Follo, Peter Bronsert, Elmar Stickeler, and Jochen Maurer. "Human Primary Breast Cancer Stem Cells Are Characterized by Epithelial-Mesenchymal Plasticity." International Journal of Molecular Sciences 22, no. 4 (February 11, 2021): 1808. http://dx.doi.org/10.3390/ijms22041808.
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Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with only limited treatment options available. Recently, cancer stem cells (CSCs) have emerged as the potential drivers of tumor progression due to their ability to both self-renew and give rise to differentiated progeny. The CSC state has been linked to the process of epithelial-mesenchymal transition (EMT) and to the highly flexible state of epithelial-mesenchymal plasticity (EMP). We aimed to establish primary breast cancer stem cell (BCSC) cultures isolated from TNBC specimens. These cells grow as tumor spheres under anchorage-independent culture conditions in vitro and reliably form tumors in mice when transplanted in limiting dilutions in vivo. The BCSC xenograft tumors phenocopy the original patient tumor in architecture and gene expression. Analysis of an EMT-related marker profile revealed the concomitant expression of epithelial and mesenchymal markers suggesting an EMP state for BCSCs of TNBC. Furthermore, BCSCs were susceptible to stimulation with the EMT inducer TGF-β1, resulting in upregulation of mesenchymal genes and enhanced migratory abilities. Overall, primary BCSC cultures are a promising model close to the patient that can be used both in vitro and in vivo to address questions of BCSC biology and evaluate new treatment options for TNBC.
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Muller, D., J. P. Fricker, R. Millon-Collard, J. Abecassis, J. Pusel, M. Eber, and G. Methlin. "Characterization of cell types in human breast tumor primary cultures." Biology of the Cell 61, no. 1-2 (1987): 91–99. http://dx.doi.org/10.1111/j.1768-322x.1987.tb00574.x.
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Arundel, Carla, Susan Bock, William A. Brock, and Philip J. Tofilon. "Radiosensitization of primary human tumor cell cultures by N-methylformamide." International Journal of Radiation Oncology*Biology*Physics 13, no. 5 (May 1987): 753–57. http://dx.doi.org/10.1016/0360-3016(87)90295-1.
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Le Rhun, E., C. von Achenbach, F. Sahm, S. S. Wang, M. C. Neidert, E. Rushing, T. Lawhon, H. Schneider, A. von Deimling, and M. Weller. "OS8.6 Sensitivity of human meningioma cells to the cyclin-dependent kinase inhibitor, TG02." Neuro-Oncology 21, Supplement_3 (August 2019): iii17. http://dx.doi.org/10.1093/neuonc/noz126.056.
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Abstract BACKGROUND Standards of care for meningioma include surgical resection, which may be curative, and radiotherapy as required. Pharmacotherapy plays only a minor role in this disease; however, novel systemic approaches are urgently needed for patients who are no longer candidates for local therapy. MATERIAL AND METHODS We generated primary cultures from surgically removed meningiomas to explore the activity of a novel cyclin-dependent kinase inhibitor, TG02, in meningioma cell cultures. Tumor and cell cultures were characterized by mutation profiling and DNA methylation profiling. DNA methylation data were used to allot each sample to one out of six previously established meningioma methylation classes: benign (ben)-1, 2, 3, intermediate (int)-A, B, and malignant (mal). The activity of TG02 was assessed by standard cell culture assays. RESULTS Cell cultures were derived from nine meningiomas. Four tumors assigned to the methylation class ben-2 showed the same class in the cell culture whereas cell cultures from five non-ben-2 tumors showed a different class, a more malignant class in four of five patients. Cell cultures were uniformly sensitive to the growth inhibitory effects of TG02 in the nanomolar range. Assignment of the cell cultures to a more malignant methylation classifier appeared to be more closely associated with TG02 sensitivity than assignment to a higher WHO grade of the primary tumors. CONCLUSION Primary cell cultures from meningioma facilitate the investigation of the anti-meningioma activity of novel agents. TG02, an orally available cyclin-dependent kinase inhibitor, warrants further exploration in this disease.
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Чернов, А. Н., Е. П. Баранцевич, Э. С. Галимова, and М. М. Галагудза. "Cell cultures of human malignant tumors in development of new anticancer therapies." Nauchno-prakticheskii zhurnal «Patogenez», no. 4() (January 30, 2018): 13–23. http://dx.doi.org/10.25557/gm.2018.4.9744.
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Современный эффективный скрининг новых противоопухолевых химиопрепаратов и биологических препаратов на доклиническом этапе невозможен без применения моделей культур опухолевых клеток. К таким моделям относят первичные культуры клеток и клеточные линии опухолей человека, культивируемые в двумерной (2D) и трехмерной (3D) системах. В обзоре обсуждаются различные аспекты применения моделей клеточных культур неоплазий человека, их актуальность в исследованиях противоопухолевой эффективности препаратов. Current effective preclinical screening of new anticancer chemotherapies and biological medicines requires cancer cell culture models. Such models include primary cell cultures and human tumor cell lines cultured in two-dimensional (2D) and three-dimensional (3D) systems. This review discussed different aspects of using human tumor cell culture models and their relevance for studying efficacy of antitumor drugs.
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Whigham, Amy, Brown Brandee, Avtandyl Kochiashvili, James L. Netterville, Brian B. Burkey, Robert J. Sinard, and Wendell G. Yarbrough. "Short-Term Culture and In-Vivo Modeling of Primary HNSCC." Otolaryngology–Head and Neck Surgery 139, no. 2_suppl (August 2008): P93—P94. http://dx.doi.org/10.1016/j.otohns.2008.05.502.
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Problem Head and neck squamous cell carcinoma (HNSCC) accounts for 4% of annual U.S. cancer deaths. In-vivo models exist using established HNSCC lines, but currently there is no such model that allows consistent growth of HNSCC from primary tumors. Methods Primary HNSCC tissue was obtained from 103 patients at biopsy/resection, disaggregated and seeded onto collagen-coated plates in keratinocyte growth media with 10% FBS, additives and antibiotics. After short-term growth in culture, cells were transferred to denuded rat tracheas and implanted subcutaneously in nude mice. Indirect immunofluorescent staining using antibodies specific to cytokeratin, vimentin and nuclear Ku was performed to determine cell lineage and origin. Results Cultured cells exhibited morphology consistent with epithelial or stromal derivation. 80% of cultures had viable cells present at 10 days and 24% were maintained 30 days or longer. 5 cultures (5%) proliferated after multiple passages and thrived on uncoated plates in the absence of mesenchymal cells. The xenograft model was able to successfully establish tumors in vivo from 59% of primary tumors. Immunostaining for nuclear Ku and cytokeratin confirmed human origin and epithelial cell lineage, respectively. Conclusion The high success rate indicates that selective growth and survival pressures for short-term culture of primary HNSCC may be considerably less than for establishment of cell lines. Additionally, the techniques permit tumor-derived epithelial and mesenchymal cells to be cultured simultaneously. Preliminary data for the in-vivo trachea xenograft model is promising. A luciferase lentiviral system has been created to allow monitoring of tumor growth in vivo with serial live animal imaging. Significance These short-term culture techniques may more accurately characterize both the biological diversity of HNSCC and tumor-stromal cell interactions. Once optimized, the trachea xenograft model can be used to determine the in-vivo response of a heterogeneous group of HNSCC to standard and novel therapies. Support Funds provided by an endowment for the Barry Baker Laboratory for Head and Neck Oncology, the Vanderbilt Ingram Cancer Center Endowed Professorship Fund, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.
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Ihler, Friedrich, Ronja Gratz, Hendrik A. Wolff, Bernhard G. Weiss, Mattis Bertlich, Julia Kitz, Gabriela Salinas, Margret Rave-Fränk, and Martin Canis. "Epithelial-Mesenchymal Transition during Metastasis of HPV-Negative Pharyngeal Squamous Cell Carcinoma." BioMed Research International 2018 (2018): 1–12. http://dx.doi.org/10.1155/2018/7929104.
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In epithelial tumors, a shift towards a mesenchymal phenotype has been associated with increased invasiveness and metastasis. It is assumed that this phenomenon plays a major role in disease progression and ultimately prognosis. This study investigated epithelial-mesenchymal transition (EMT) in human papillomavirus- (HPV-) negative pharyngeal squamous cell carcinoma. Tissue was obtained from one hypopharyngeal primary tumor and a regional lymph node metastasis during surgery with curative intention. A cell culture was established from the primary tumor and mesenchymal growth conditions were emulated. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition), applying gene set expression analysis (GSEA). Immunohistochemical staining and flow cytometry of CD44 and E-cadherin were compared in primary tumor, metastasis, and cell cultures. Primary tumor and metastasis were highly positive for CD44. A loss of E-cadherin occurred in the metastasis. Flow cytometry showed the appearance of a population without E-cadherin in spheroid colonies. In GSEA, the EMT phenotype was enriched in the primary tumor compared to metastasis and cell cultures (FDR < 25%,p< 5%). EMT showed variable expression during metastasis. It may thereby be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active only during the process of metastasis itself. Thereby, the primary tumor as well as the metastasis may exhibit fewer EMT properties.
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Carra, Elisa, Federica Barbieri, Daniela Marubbi, Alessandra Pattarozzi, Roberto E. Favoni, Tullio Florio, and Antonio Daga. "Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures." Cell Cycle 12, no. 3 (February 2013): 491–500. http://dx.doi.org/10.4161/cc.23372.
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Danilova, Anna B., Natalia A. Efremova, Tatiana L. Nekhaeva, Irina A. Baldueva, Mykhail A. Maydin, Anna A. Murashkina, Ekaterina S. Artemyeva, and Anna S. Artemyev. "Evolution of the Solid Human Tumor Cells Properties in Various Experimental Systems in Vitro." Journal of Hematology and Oncology Research 4, no. 2 (January 19, 2022): 9–29. http://dx.doi.org/10.14302/issn.2372-6601.jhor-22-4061.
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Background Human malignant cell models which reflect the structural and physiological complexity of tumor tissue are of great importance for preclinical research in oncology. Spheroids/tumoroids derived from solid tumors are of great interest as cellular models mimicking the first vascular-free growth phase of a tumor node. The fact of the identity between artificially created tumor multicellular aggregates and the real tumor tissue, however, needs to be specified, described and validated in order to see how closely the spheroids are biologically similar to the malignized tissues in vivo compared to the monolayer cell cultures traditionally used. We present here a comparison study of the characteristics of solid tumor cells of different histogenesis (melanomas, soft tissue sarcomas and bone sarcomas, epithelial tumors) cultured in two dimensions (monolayer culture) and three dimensional space (spheroid), namely: spatial organization, multiplication, metabolic activity. Patients and Methods For the creation of 2 D and 3D cell models the cells isolated from the patient's solid tumor fragments obtained intraoperatively were used. 15 samples of skin melanoma, 20 samples of soft tissue and osteogenic sarcomas (STBS), and 9 samples of epithelial tumors (ET). The tumor cells were all cultivated for at least 10 passages. We used phase contrast, confocal microscopy, and immunohistochemistry to investigate spheroids and monolayer cultures. The supernatants of tumor cells grown in 2D and 3D cultures were studied using ELISA and multiplex analysis for the production of a spectrum of chemokines and cytokines supporting the immunosuppression, invasion and metastasis processes. Results Tumor specimens received were predominantly of metastatic origin (75%). In 100% of cases 2D cultures were received, in 88.6% of cases (39 out of 44) we succeeded in obtaining spheroids. There was no direct correlation between the efficiency of tumoroid formation and the tumor's histogenetic origin and the stage of the cancer process (primary tumor, recurrence, metastasis). The median size of spheroids by 4-5 days of cultivation with a starting concentration of 10000 cells per well was 657.14 μm for melanoma (min 400 - max 1000 μm), 571.42 μm (min 400 - max 700 μm), 507.14 μm (min 300 - max 600 μm) for soft tissue sarcomas, 650.0 μm (min 400 - max 900 μm) for osteogenic sarcomas. Immunochemical analysis of Ki-67, GLUT1, and Ecadherin markers was carried out for tumor tissue samples, single-layer tumor cultures, and tumoroids of every patient. The distribution of the stained groups in the spheroids was distinct from the monolayer cultures and more in accordance with the distribution of such in the tissue tumor, the number of Ki-67+ cells was increasing in the spheroids. We detected no dependence of Ki-67+ and GLUT1+ cell localization grade on spheroid size. We identified E-cadherin in tumor tissue and tumoroids of breast carcinoma and one melanoma culture. Monolayer cultures did not express it. The increase in secretory cell activity of the solid tumor cells from 2D to 3D system was observed when CCL2, CCL3, CXCL1, CXCL16, MIF, IL10, MICA (p<0.01) were investigated. Conclusion The presence of patient-specific cells of solid tumors in a 3D environment causes activation of the proliferative and metabolic processes as compared to monolayer cultures, which makes these models approximate the real world clinical picture. The production of chemokines that can attract to the tumor various types of immune system cells, to include their immature versions, as well as production of cytokines and Immunosuppression factors that, when present in the tumor microenvironment in the high concentrations, contribute to the formation of immune cells having suppressive capacities occurs in the 3D cell system. Three-dimensional model of the initial tumor nodule formation stage thus demonstrates the forming process of tumor cells favorable for them microenvironment. Construction of three-dimensional models - spheroids of tumor cells of differing histogenesis demands individual approach and more thorough investigation
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Lachota, Mieszko, Katarzyna Zielniok, Agata Gozdz, Patrycja Szpak, Ilona Kalaszczynska, and Radoslaw Zagozdzon. "Analysis of chemokine network in primary human glioblastoma." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 179.06. http://dx.doi.org/10.4049/jimmunol.208.supp.179.06.
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Abstract Chemokines play an integral role in cancer immunoevasion by modulating leukocyte infiltration. They selectively increase the migration of immunosuppressive cells while impairing the infiltration of cytotoxic lymphocytes with potent antitumor activity. Some cancer-associated chemokines may also enhance tumor progression by acting as growth factors or promoting epithelial-mesenchymal transition. Here, we acquired fresh human glioblastoma (GBM) tissues to define chemokine expression patterns in GBM tumors by integrating bulk and single-cell RNA sequencing of both tumor and healthy brain tissues along with Luminex secretome profiling of GBM primary cell cultures. Extensive mapping of GBM-associated chemokines has enabled us to gain insight into intratumoral chemokine-mediated cell-to-cell communication. Among others, we identified CCL7 and midkine (MDK) to play a crucial role in the formation of the hostile tumor microenvironment (TME) in GBM. We hypothesize that these chemoattractants can facilitate the GBM tumor growth by recruiting M2 macrophages, promoting epithelial-mesenchymal transition, and activating the prosurvival PI3K-Akt pathway. Indeed, high expression of both chemokines was linked to poor overall survival. Additionally, by integrating the data, we further identified the cellular source of the most abundantly secreted chemokines in GBM tumors. Since immune cells require TME infiltration to mediate an appropriate antitumor response, impaired tumor infiltration is one of the critical limiting factors for cancer cell therapy. Our chemokine atlas provides novel targets to enhance adoptive cell therapies by improving the potential of effector cells for tumor homing and anticancer activities in GBM. This research was funded by the National Science Centre, Poland (2020/37/B/NZ6/02191), the Polish Ministry of Science and Higher Education (grant number DI2018 020548), and the National Centre for Research and Development (STRATEGMED3/307326/6/NCBR/2017).
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Papadopoulos, Thomas, Thomas Kirchner, Alexander Marx, and Hans Konrad Müller-Hermelink. "Primary cultures of human thymic epithelial tumors." Virchows Archiv B Cell Pathology Including Molecular Pathology 56, no. 1 (December 1988): 363–70. http://dx.doi.org/10.1007/bf02890038.
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Wegert, Jenny, Lisa Zauter, Silke Appenzeller, Christoph Otto, Sabrina Bausenwein, Christian Vokuhl, Karen Ernestus, Rhoikos Furtwängler, Norbert Graf, and Manfred Gessler. "High-risk blastemal Wilms tumor can be modeled by 3D spheroid cultures in vitro." Oncogene 39, no. 4 (September 27, 2019): 849–61. http://dx.doi.org/10.1038/s41388-019-1027-8.
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Abstract In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) — the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.
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Dolgova, E. V., A. S. Proskurina, E. A. Potter, T. V. Tyrinova, O. S. Taranov, Ya R. Efremov, K. E. Orishchenko, et al. "Evaluation of a strategy for tumor-initiating stem cell eradication in primary human glioblastoma cultures as a model." Vavilov Journal of Genetics and Breeding 22, no. 7 (November 9, 2018): 825–36. http://dx.doi.org/10.18699/vj18.31-o.
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Primary cultures of human glioblastoma were obtained from the surgical material of patients K. (female, 61 years, Ds: relapse of glioblastoma) and Zh. (female, 60 years, Ds: relapse of glioblastoma). The effectiveness of a new therapeutic approach aimed at destroying the cancer cell community was evaluated on the primary cell lines of human glioblastoma culture by employing a new strategy of tumor-initiating stem cell synchronization and a domestic strategy of their eradication "3+1". The key elements of the strategy were the following indicator results: (1) evaluation of the presence of tumor-initiating stem cells in a population of cells from analyzed cultures by their ability to internalize double-stranded labeled DNA (TAMRA+ cells); (2) determination of the reference time points of the repair cycle of DNA interstrand cross-links induced by cross-linking cytostatic mitomycin C; (3) evaluation of cell cycle synchronization; (4) determination of the time (day after therapy initiation) when TAMRA+ cells were synchronously present in phase G1/S of the cell cycle, sensitive to the therapy; and (5) establishment of the TAMRA+ (tumor-initiating stem cells) eradication schedule. The cultures were treated with cross-linking cytostatic mitomycin C and a compositional DNA preparation. After the treatments, cell division slows down, and the cultures degrade. The K cell line completely degraded within 30 days of observation. The cell number of the Zh culture fell to nearly one-third of the starting value by day 15 of observation. On day 15, this indicator constituted 1/7.45 for mitomycin C and 1/10.28 for mitomycin C + DNA with reference to the control. The main target of the mitomycin C + DNA regimen was TAMRA+ tumor-initiating stem cells of the glioblastoma cell populations. The action of mitomycin C alone or in the combination with DNA demonstrated effective elimination of TAMRA+ tumor-initiating stem cells and the whole primary cultures of human glioblastomas.
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Nie, Jing, Guang-long Huang, Sheng-Ze Deng, Yun Bao, Ya-Wei Liu, Zhan-Peng Feng, Chao-Hu Wang, Ming Chen, Song-Tao Qi, and Jun Pan. "The purine receptor P2X7R regulates the release of pro-inflammatory cytokines in human craniopharyngioma." Endocrine-Related Cancer 24, no. 6 (June 2017): 287–96. http://dx.doi.org/10.1530/erc-16-0338.
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Craniopharyngiomas (CPs) are usually benign, non-metastasizing embryonic malformations originating from the sellar area. They are, however, locally invasive and generate adherent interfaces with the surrounding brain parenchyma. Previous studies have shown the tumor microenvironment is characterized by a local abundance of adenosine triphosphate (ATP), infiltration of leukocytes and elevated levels of pro-inflammatory cytokines that are thought to be responsible, at least in part, for the local invasion. Here, we examine whether ATP, via the P2X7R, participates in the regulation of cytokine expression in CPs. The expression of P2X7R and pro-inflammatory cytokines were measured at the RNA and protein levels both in tumor samples and in primary cultured tumor cells. Furthermore, cytokine modulation was measured after manipulating P2X7R in cultured tumor cells by siRNA-mediated knockdown, as well as pharmacologically by using selective agonists and antagonists. The following results were observed. A number of cytokines, in particular IL-6, IL-8 and MCP-1, were elevated in patient plasma, tumor tissue and cultured tumor cells. P2X7R was expressed in tumor tissue as well as in cultured tumor cells. RNA expression as measured in 48 resected tumors was positively correlated with the RNA levels of IL-6, IL-8 and MCP-1 in tumors. Furthermore, knockdown of P2X7R in primary tumor cultures reduced, and stimulation of P2XR7 by a specific agonist enhanced the expression of these cytokines. This latter stimulation involved a Ca2+-dependent mechanism and could be counteracted by the addition of an antagonist. In conclusion, the results suggest that P2X7R may promote IL-6, IL-8 and MCP-1 production and secretion and contribute to the invasion and adhesion of CPs to the surrounding tissue.
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Ali-Osman, F., J. Caughlin, M. Berger, A. Spence, and Alexander Spence. "PRIMARY CULTURES OF HUMAN GLIAL TUMOR CELLS AND THEIR RELATIONSHIP TO HISTOPATHOLOGY." Journal of Neuropathology and Experimental Neurology 46, no. 3 (May 1987): 371. http://dx.doi.org/10.1097/00005072-198705000-00126.
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Liverani, Chiara, Chiara Spadazzi, Alberto Bongiovanni, Federica Pieri, Flavia Foca, Chiara Calabrese, Giorgio Ercolani, et al. "HRAS expression predicts Lenvatinib responsiveness in human primary gastroenteropancreatic neuroendocrine tumor cells." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e16211-e16211. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e16211.
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e16211 Background: Neuroendocrine neoplasms (NENs) are a rare group of heterogeneous tumors, with clinical presentation ranging from indolent and small lesions to very aggressive, metastatic and therapy refractory forms. Despite a high number of trials, few drugs have been introduced in the clinical practice and therapeutic options for systemic intervention are limited. The recent TALENT trial demonstrated the efficacy of Lenvatinib, a multi-tyrosine kinase inhibitor (MKI), in the treatment of patients with advanced well differentiated gastroenteropancreatic neuroendocrine tumors (GEP-NET) progressing from targeted therapies or somatostatin analogues with no other treatment indication. Lenvatinib might represent a novel therapeutic opportunity for these rare tumor patients. However, the treatment approach with MKI lacks clinically validated tissue or blood biomarkers to guide patient selection and improve efficacy. Methods: Here we established 11 human primary cultures from patients with GEP-NEN of different grades and sites of origin and assessed the antitumor activity of Lenvatinib compared with standard treatment agents. All primary cultures were molecularly characterized to identify possible predictors of response. Results: Lenvatinib exerted a significant inhibition of cell growth in primary GEP-NET cells, with median survival inhibitions similar or higher than those of standard treatments. Of the 11 primary cultures analyzed in our case series, 6 were classified as responder with survival inhibition higher than 15%, and 5 as non-responder with survival inhibition lower than 15%. A high expression of HRAS in the tumor tissues compared to the matched healthy tissues significantly correlated with responsiveness of primary cells to Lenvatinib. All 5 non-responder patients showed low expression of HRAS, while of the 6 responder patients, 5 showed a high HRAS expression and only 1 low. None of the other evaluated clinical variables (grade, Ki67, site of origin and syndromic disease) correlated with response. Conclusions: In conclusion, we believe that the evaluation of HRAS expression and mutation might be of great interest in GEP-NET patients receiving Lenvatinib to improve patient selection.
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Failli, Alessandra, Rita Consolini, Annalisa Legitimo, Roberto Spisni, Maura Castagna, Antonella Romanini, Gaetano Crimaldi, and Paolo Miccoli. "The Challenge of Culturing Human Colorectal Tumor Cells: Establishment of a Cell Culture Model by the Comparison of Different Methodological Approaches." Tumori Journal 95, no. 3 (May 2009): 343–47. http://dx.doi.org/10.1177/030089160909500312.
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Background Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents. Methods This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry. Results We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells. Conclusion In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.
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Bex, Axel, Aurelie Guislain, Hester van Boven, and Christian U. Blank. "Effect of sunitinib pretreatment on yield from tumor-infiltrating lymphocyte (TIL) cultures in patients with renal cell carcinoma." Journal of Clinical Oncology 30, no. 5_suppl (February 10, 2012): 419. http://dx.doi.org/10.1200/jco.2012.30.5_suppl.419.
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419 Background: Targeted therapies achieve response rates up to 50% in metastatic RCC, but durable complete remissions are very rare. Therefore additional therapeutic options are warranted. Adoptive transfer (ACT) of tumor infiltrating lymphocytes (TIL) has revealed response rates of up to 72% in melanoma patients and may lead to consolidation of responses upon sunitinib in RCC. Recent data suggest that sunitinib influences lymphocyte infiltration and regulatory T cells (Treg) into tumor, dendritic cells (DC), and myeloid derived suppressor cells (MDSC) in animal models, but impaired proliferation and function of human T cells and was unable to improve human DC activity in vitro. Methods: We compared TIL grown from primary tumors pretreated with 2 courses sunitinib prior to cytoreductive nephrectomy to TIL generated from treatment-naïve RCC. Patients took part in a phase II trial of presurgical sunitinib registered under EudraCT 2006-006491-38 and gave written informed consent. From 6 pretreated primary tumors and 6 untreated controls sterile tumor material was obtained and digested with collagenase, hyaluronidase and DNase to obtain tumor single cell suspension. This TIL/tumor suspension was cultured for up to 25 days in 6000IU/ml interleukin 2 (IL-2) and rapidly expanded by anti-CD3+IL-2 stimulation. Results: TIL harvested after coculture with tumor cells yielded 36+/−SD fold expansion from non-pretreated tumors versus a 128+/−SD fold expansion from tumors from patients that had been pretreated with sunitinib (p=0.015). Phenotypic analysis of culture product indicated a higher content of natural killer (NK) cells from treatment naïve TIL cultures, while sunitinib pretreatment skewed towards NK T cells. Interferon-gamma-production was not different on a per cell level.Rapid expansion after culture using anti-CD3/IL-2 induced 100-200 fold expansion towards a content of more than 75% T cells with no significant differences between both groups. Conclusions: Sunitinib in vivo pretreatment improves TIL grow during initial culture and does not impair function or rapid expansion, allowing higher final yield of TIL for ACT studies in RCC.
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Guigni, Blas A., Jos van der Velden, C. Matthew Kinsey, James A. Carson, and Michael J. Toth. "Effects of conditioned media from murine lung cancer cells and human tumor cells on cultured myotubes." American Journal of Physiology-Endocrinology and Metabolism 318, no. 1 (January 1, 2020): E22—E32. http://dx.doi.org/10.1152/ajpendo.00310.2019.
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Factors secreted from tumors/tumor cells are hypothesized to cause skeletal muscle wasting in cancer patients. We examined whether cancer cells secrete factors to promote atrophy by evaluating the effects of conditioned media (CM) from murine lung cancer cells and primary cultures of human lung tumor cells on cultured myotubes. We evaluated murine Lewis lung carcinoma (LLC) and KRASG12D cells, and primary cell lines derived from tumor biopsies from patients with lung cancer (hTCM; n = 6). In all experiments, serum content was matched across treatment groups. We hypothesized that CM from murine and human tumor cells would reduce myotube myosin content, decrease mitochondrial content, and increase mitochondrial reactive oxygen species (ROS) production. Treatment of myotubes differentiated for 7 days with CM from LLC and KRASG12D cells did not alter any of these variables. Effects of murine tumor cell CM were observed when myotubes differentiated for 4 days were treated with tumor cell CM and compared with undiluted differentiation media. However, these effects were not apparent if tumor cell CM treatments were compared with control cell CM or dilution controls. Finally, CM from human lung tumor primary cell lines did not modify myosin content or mitochondrial content or ROS production compared with either undiluted differentiated media, control cell CM, or dilution controls. Our results do not support the hypothesis that factors released from cultured lung cancer/tumor cells promote myotube wasting or mitochondrial abnormalities, but we cannot dismiss the possibility that these cells could secrete such factors in vivo within the native tumor microenvironment.
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Bogush, T. A., S. A. Kaliuzhny, M. R. Chetyrkina, M. A. Yastrebova, A. M. Scherbakov, I. A. Mamichev, and А. А. Kamensky. "VIMENTIN EXPRESSION IN HUMAN CELL LINES OF EPITHELIAL TUMORS." Advances in molecular oncology 5, no. 2 (July 17, 2018): 24–30. http://dx.doi.org/10.17650/2313-805x-2018-5-2-24-30.
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Background.Cell cultures used as a models in studies of epithelial tumors, are obtained not only from solid tumors, but also from extracellular fluids. It is known that dissemination of ovarian cancer in the peritoneum and further growth of tumor cells in ascetic liquid is accompanied with the activation of epithelial-mesenchymal transition, and, therefore, cell cultures derived from extracellular fluids can have a distinct molecular phenotype from primary tumors.Objective:evaluation the “persistence” of epithelial phenotype in breast and ovarian cancer cell cultures.Materials and methods.The cells obtained from pleural fluid (MCF-7, T-47D), colostrum (HBL-100), solid tumors (BT-474, HCC1937) of patients with breast cancer and ascitic fluid (SCOV- 3) of patients with ovarian cancer. The expression of cytokeratins and vimentin was evaluated using a quantitative immunofluorescence method associated with flow cytometry.Results.Vimentin expression in cells derived from extracellular fluids was not changed (line HBL-100), slightly decreased (SCOV-3 cells), or even was lost (MCF-7 and T-47D cells). HCC1937 cells obtained from solid tumor with expected low expression of vimentin acquired a molecular phenotype with a high expression of this mesenchymal marker. In breast cancer cells BT-474 derived from solid tumor a “persistence” of epithelial phenotype was discovered.Conclusion.Quantitative assessment of the de novo expression of mesenchymal protein vimentin showed that the tumor phenotype within the organizm is not always realized in cells adapted to growth in culture, and is not always «strictly» epithelial, and this evidence must be considered with different kinds of molecular studies of epithelial cells in vitro.
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Zarco, Natanael, Emily Norton, Montserrat Lara-Velazquez, Anna Carrano, Alfredo Quinones-Hinojosa, and Hugo Guerrero-Cazares. "TMIC-56. ROLE OF HUMAN BRAIN TUMOR STEM CELLS-DERIVED EXTRACELLULAR VESICLES ON THE PHENOTYPIC TRANSDIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS." Neuro-Oncology 21, Supplement_6 (November 2019): vi260. http://dx.doi.org/10.1093/neuonc/noz175.1090.
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Abstract Glioblastoma (GBM) is the most aggressive of all the brain tumors with a median patient survival less than 15 months. Despite of surgical resection, radiotherapy, and chemotherapy, recurrence rate is almost 100%. A great percentage of GBM tumors (~60%) infiltrate and contact the ventricular-subventricular zone (V-SVZ). Interestingly, these tumors are the most aggressive, and invariably lead to higher distal recurrence rates, shorter time to tumor progression, and lower overall survival of the patient. The reason for this role of V-SVZ-proximity on the outcome of GBM patients is unknown. We suggest that a potential explanation is the interaction of GBM with the V-SVZ. This region is the largest neurogenic niche in the adult brain where neural stem cells (NSCs) give rise to newborn neuroblasts that migrate toward the olfactory bulb. In GBM there is a cell subpopulation called brain tumor stem cells (BTSCs) with NSCs-like characteristics, but with added potential for tumor initiation, recurrence and invasiveness. Tumor microenvironment plays an important role in migration and invasion process. In the present work, we used the total exosome isolation kit to purify Extracellular Vesicles (EVs) from human primary cultures of BTSCs. We determined that BTSCs-derived EVs contain specific information that is transfer to primary cultures of human Neural Progenitors Cells (NPCs) modulating their proliferation rate, cell viability, and migration. In addition, we identify that NPCs taken up BTSCs-derived EVs and significantly increase the expression levels of stemness-related genes such as Nestin, Nanog, and Sox2, suggesting that a phenotypic transdifferentiation is being carry out. These results support our hypothesis that GBM modulate the tumor microenvironment close to the V-SVZ by releasing EVs that target cellular components in this region and promote their phenotypic transformation, highlighting that NPCs biology changes in the context of tumor environment.
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Shamova, T. V., A. O. Sitkovskaya, E. E. Rostorguev, N. S. Kuznetsova, and S. E. Kavitsky. "Preparation of primary glial tumor cell lines." Perm Medical Journal 37, no. 5 (January 7, 2021): 79–89. http://dx.doi.org/10.17816/pmj37579-89.
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Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method.
Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed.
Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes.
At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days.
Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.
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Shamova, T. V., A. O. Sitkovskaya, E. E. Rostorguev, N. S. Kuznetsova, and S. E. Kavitsky. "Preparation of primary glial tumor cell lines." Perm Medical Journal 37, no. 5 (January 7, 2021): 79–89. http://dx.doi.org/10.17816/pmj37579-89.
Full textAbstract:
Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method.
Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed.
Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes.
At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days.
Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.
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Lenggenhager, Daniela, Manoj Amrutkar, Petra Sántha, Monica Aasrum, Johannes-Matthias Löhr, Ivar P. Gladhaug, and Caroline S. Verbeke. "Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells." Cells 8, no. 1 (January 5, 2019): 23. http://dx.doi.org/10.3390/cells8010023.
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Activated pancreatic stellate cells (PSCs) play a central role in the tumor stroma of pancreatic ductal adenocarcinoma (PDAC). Given the limited availability of patient-derived PSCs from PDAC, immortalized PSC cell lines of murine and human origin have been established; however, it is not elucidated whether differences in species, organ disease status, donor age, and immortalization alter the PSC phenotype and behavior compared to that of patient-derived primary PSC cultures. Therefore, a panel of commonly used PSC cultures was examined for important phenotypical and functional features: three primary cultures from human PDAC, one primary from normal human pancreas, and three immortalized (one from human, two from murine pancreas). Growth rate was considerably lower in primary PSCs from human PDAC. Basal collagen synthesis varied between the PSC cultures, and TGF-β stimulation increased collagen synthesis only in non-immortalized cultures. Differences in secretome composition were observed along with a divergence in the DNA synthesis, migration, and response to gemcitabine of PDAC cell lines that were grown in conditioned medium from the various PSC cultures. The findings reveal considerable differences in features and functions that are key to PSCs and in the interactions with PDAC. These observations may be relevant to researchers when selecting the most appropriate PSC culture for their experiments.
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Mergler, Stefan, Adriana Drost, Wolf-Otto Bechstein, Peter Neuhaus, and Bertram Wiedenmann. "Differential Ca2+ channel activity in human primary neuroendocrine tumor cell cultures from fore- and midgut tumors." Gastroenterology 124, no. 4 (April 2003): A135. http://dx.doi.org/10.1016/s0016-5085(03)80669-8.
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GRIMM, DANIELA, JOHANN BAUER, FERDINAND HOFSTÄDTER, GÜNTER A. J. RIEGGER, and ECKHARD P. KROMER. "Characteristics of Multicellular Spheroids Formed by Primary Cultures of Human Thyroid Tumor Cells." Thyroid 7, no. 6 (December 1997): 859–65. http://dx.doi.org/10.1089/thy.1997.7.859.
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Peters, L. J., F. Baker, and W. A. Brock. "Comparison of radiosensitivity of primary human tumor cell cultures for different histological types." International Journal of Radiation Oncology*Biology*Physics 12 (November 1986): 191–92. http://dx.doi.org/10.1016/0360-3016(86)90696-6.
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Cuny, Thomas, Peter M. van Koetsveld, Grégoire Mondielli, Fadime Dogan, Wouter W. de Herder, Anne Barlier, and Leo J. Hofland. "Reciprocal Interactions between Fibroblast and Pancreatic Neuroendocrine Tumor Cells: Putative Impact of the Tumor Microenvironment." Cancers 14, no. 14 (July 18, 2022): 3481. http://dx.doi.org/10.3390/cancers14143481.
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Introduction: Pancreatic neuroendocrine neoplasms (PNENs) present with a fibrotic stroma that constitutes the tumor microenvironment (TME). The role played by stromal fibroblasts in the growth of PNENs and their sensitivity to the mTOR inhibitor RAD001 has not yet been established. Methods: We investigated reciprocal interactions between (1) human PNEN cell lines (BON-1/QGP-1) or primary cultures of human ileal neuroendocrine neoplasm (iNEN) or PNEN and (2) human fibroblast cell lines (HPF/HFL-1). Proliferation was assessed in transwell (tw) co-culture or in the presence of serum-free conditioned media (cm), with and without RAD001. Colony formation and migration of BON-1/QGP-1 were evaluated upon incubation with HPFcm. Results: Proliferation of BON-1 and QGP-1 increased in the presence of HFL-1cm, HPFcm, HFL-1tw and HPFtw (BON-1: +46–70% and QGP-1: +42–55%, p < 0.001 vs. controls) and HPFcm significantly increased the number of BON-1 or QGP-1 colonies (p < 0.05). This stimulatory effect was reversed in the presence of RAD001. Likewise, proliferation of human iNEN and PNEN primary cultures increased in the presence of HFL-1 or HPF. Reciprocally, BON-1cm and BONtw stimulated the proliferation of HPF (+90 ± 61% and +55 ± 47%, respectively, p < 0.001 vs. controls), an effect less pronounced with QGP-1cm or QGPtw (+19 to +27%, p < 0.05 vs. controls). Finally, a higher migration potential for BON-1 and QGP-1 was found in the presence of HPFcm (p < 0.001 vs. controls). Conclusions: Fibroblasts in the TME of PNENs represent a target of interest, the stimulatory effect of which over PNENs is mitigated by the mTOR inhibitor everolimus.
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Steele, Nina, Valerie Irizarry-Negron, Veerin Sirihorachai, Samantha Kemp, Eileen Carpenter, Christopher Halbrook, Costas Lyssiotis, et al. "3373 Modulation of Hedgehog Signaling Alters Immune Infiltration in Pancreatic Cancer." Journal of Clinical and Translational Science 3, s1 (March 2019): 16. http://dx.doi.org/10.1017/cts.2019.40.
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OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.
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Laurie, Nikia, Adithi Mohan, Justina McEvoy, Damon Reed, Jiakun Zhang, Brett Schweers, Itsuki Ajioka, et al. "Changes in Retinoblastoma Cell Adhesion Associated with Optic Nerve Invasion." Molecular and Cellular Biology 29, no. 23 (September 28, 2009): 6268–82. http://dx.doi.org/10.1128/mcb.00374-09.
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ABSTRACT In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.
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Khochenkova, Yu A., I. G. Dyrda, Yu S. Machkova, E. Sh Solomko, T. A. Sidorova, D. A. Khochenkov, and E. A. Avilova. "New approaches in 3D modeling of in vitro growth of primary cultures of malignant gliomas." Advances in molecular oncology 6, no. 4 (December 15, 2019): 69–74. http://dx.doi.org/10.17650/2313-805x-2019-6-4-69-74.
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Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.
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Kiwit, Jurgen Carl Walther, Anja Hertel, and Alexander E. Matuschek. "Reversal of chemoresistance in malignant gliomas by calcium antagonists: correlation with the expression of multidrug-resistant p-glycoprotein." Journal of Neurosurgery 81, no. 4 (October 1994): 587–94. http://dx.doi.org/10.3171/jns.1994.81.4.0587.
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✓ Resistance to multiple drugs is often observed in malignant gliomas. The authors used a microtiter tetrazolium test to analyze primary in vitro chemoresistance and chemosensitivity of 15 early cultures of human malignant glioma exposed to 50 µg/ml (1,4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), 50 µg/ml cisplatin, 1 µg/ml vincristine, or combinations of these chemotherapeutic agents. Primary chemoresistance was observed in 87% of tumors for ACNU, in 87% for cisplatin, and in 83% for vincristine. All tumors were examined for expression of multidrug-resistant p-glycoprotein, a transport protein of 170,000 D, by means of immunohistochemical staining with the JSB-1 antibody on paraffinized tumor sections. Eight of 15 specimens (53%) showed positive staining for the monoclonal antibody. Primary chemoresistance was overcome by addition of the calcium antagonists verapamil or nimodipine to the cultures if the original tumor expressed p-glycoprotein (p < 0.01 for verapamil, p < 0.05 for nimodipine). In tumors not expressing p-glycoprotein, addition of calcium antagonists to the cell cultures did not influence primary chemoresistance. It is concluded from these data that addition of calcium antagonists to the adjuvant chemotherapy of malignant gliomas might overcome primary chemoresistance in tumors expressing the multidrugresistant phenotype.
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Khalid, M. Humayun, Shobu Shibata, Koichi Furukawa, Amal Nadel, Matthew D. Ammerman, and Anthony J. Caputy. "Role of estrogen receptor—related antigen in initiating the growth of human glioma cells." Journal of Neurosurgery 100, no. 5 (May 2004): 923–30. http://dx.doi.org/10.3171/jns.2004.100.5.0923.
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Object. The expression of estrogen receptor—related antigen (ER-D5) has been demonstrated in many tumors, including those of the brain, but the actual role of ER-D5 in cell growth is unknown. The authors evaluated the role of ER-D5 in the growth of gliomas in vitro. Methods. Human glioblastoma multiforme (GBM) cell lines A172, T98G, U87MG, and U118MG; rat C6 glioma and 9L gliosarcoma; AS human astrocytoma; GBM in primary culture and tumor tissues; and normal brain tissues were examined for ER-D5 by using immunohistochemical, Western immunoblot, flow cytometry, and enzyme-linked immunosorbent assays. The ER-D5 was detected in all tumor cell types of human origin, but not in rat cell lines and normal brain; the expression of ER-D5 was not related to cell cycle phase. Kinetic analysis of ER-D5 expression in cultured cell lines revealed that an enhanced and sharp accumulation of ER-D5 occurred during the first 24 hours of culture, followed by a sharp fall in the next 24 hours. Gradual decreases of ER-D5 during the subsequent days were demonstrated in all human cell lines, and in primary cultures of GBM. This accumulation pattern of ER-D5 was confirmed on Western blot analysis. The ER-D5 was also detected in cells cultured in serum-free medium. Culture cells were treated with D5 antibody against ER-D5 for 48 hours and the effects were evaluated using a monotetrazolium colorimetric assay; the result revealed that growth of cultured cells was inhibited in a dose-dependent manner, and that addition of a single median inhibitory concentration dose resulted in complete growth inhibition and arrest of cell growth at the G0/G1 phase at 96 hours posttreatment. Conclusions. These findings indicated that synthesis and accumulation of ER-D5 is an essential event in the very early phase of in vitro growth of human gliomas.
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Yan, Xuefei, Hongjuan Zhang, Jun Zhou, Jia Zheng, Shuang Zhu, Rui Zhang, Mingfa Zang, et al. "237 In vitro potency assays for immune checkpoint blockade using human primary cells, murine huGEMM immune cells and patient-derived tumor organoids." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A254. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0237.
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BackgroundThe demand of evaluating potency of immune checkpoint modulators is steadily growing for immune-oncology drug development.MethodsWe aimed to establish a platform to assess the effects of immune checkpoint blockade using human primary immune cells, humanized murine primary immune cells, and co-cultures of tumor cells or patient-derived tumor organoids with immune cells.ResultsFirst, we validated the potency of immune checkpoint blockade, such as anti-PD-1 antibodies, using mixed lymphocyte reaction (MLR) assay and T cell activation assay by in vitro stimulation. Secondly, we introduced tumor cell lines into co-culture system with immune cells and validate the potency assay by measuring cytokine production and tumor cell killing by allogenic T cells. Thirdly we used huGEMM mouse-derived immune cells to replace human primary immune cells in potency assays. HuGEMM mice express engineered human immune checkpoint targets on immune cells and they can serve as an excellent resource of primary immune cells to test the drug candidates targeting human checkpoints in vitro. Last, we developed a patient-derived tumor organoid co-culture system with immune cells. We profiled the expression of immune inhibitory molecules on the tumor organoids and assessed the potency of immune checkpoint inhibitors.ConclusionsIn summary, we have established an extensive in vitro platform to evaluate the potency of the next generation of immune checkpoint inhibitors.
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Sébire, G., D. Emilie, C. Wallon, C. Héry, O. Devergne, J. F. Delfraissy, P. Galanaud, and M. Tardieu. "In vitro production of IL-6, IL-1 beta, and tumor necrosis factor-alpha by human embryonic microglial and neural cells." Journal of Immunology 150, no. 4 (February 15, 1993): 1517–23. http://dx.doi.org/10.4049/jimmunol.150.4.1517.
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Abstract The abilities of the different cells from human central nervous system (CNS) to produce IL-6, IL-1 beta, and TNF-alpha were tested in vitro using either cultures enriched in human embryonic microglial cells or primary cultures of human embryonic CNS cells. High amounts of IL-6, low amounts of IL-1 beta but no TNF-alpha were detected in supernatants of microglial cells, kept either in FCS-free conditions or in FCS-containing medium. Moreover, IL-6 mRNA was also present in 45 to 55% of microglial cells cultured in the presence of FCS as visualized by in situ hybridization, whereas IL-1 beta mRNA remained undetectable. After prestimulation of microglial cells with LPS or IL-1 alpha, the percentage of cells labeled with an antisense IL-6 mRNA probe increased to 70% and hybridization with an antisense IL-1 beta mRNA probe became detectable. In contrast to this dyscoordinate production of cytokines by microglial cells, human monocytes, freshly isolated from blood and kept in the same culture conditions, produced high levels of the three cytokines tested. In primary cultures of human embryonic CNS cells, IL-6, IL-1 beta, and TNF-alpha were produced mostly or only by microglial cells because no IL-1 beta mRNA or IL-6 mRNA were detected in astrocytes, even after prestimulation with LPS or IL-1 alpha. Finally, IL-1 was the main inducer of IL-6 production because IL-1 alpha, but not LPS, induced a significant increase in IL-6 synthesis in cultures kept in FCS-free medium. However, in presence of FCS, LPS appeared to initiate a cascade reaction involving the production of IL-1 by microglial cells, acting as an autocrine loop to trigger IL-6 synthesis.
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Shuba, I. M., V. V. Lylo, I. S. Karpova, O. Y. Glavatskyi, and O. I. Kornelyuk. "The primary culture of malignant glioma cells as a model for the study of anti-tumor activity of substances." Visnik ukrains'kogo tovaristva genetikiv i selekcioneriv 17, no. 2 (January 20, 2020): 196–203. http://dx.doi.org/10.7124/visnyk.utgis.17.2.1221.
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Aim. The aim of our work was to optimize the scheme of obtaining primary cell culture of malignant gliomas, which can be a model for a personalized approach in the selection of chemotherapeutic exposure tactics. Methods. The standard glioma cell line U-251MG and cells obtained as a result of mechanical disaggregation of Gr III–IV tumor fragments to single isolated cells were used. Results. A comparative analysis of the results of cultivation of the standard glioma cell line U-251MG and the primary cell culture of malignant gliomas. An optimized scheme for obtaining primary cultures of human malignant glioma cells isolated from glial tumor fragments obtained during surgery is proposed. Conclusions. Today, more and more preferred methods of individual determination of chemosensitivity over the appointment of standard chemotherapy regimens and it is the primary culture of tumor cells, from our point of view, can be used to test the response to the effect of chemotherapy.Keywords: malignant glioma cells, primary culture, standard cell line.
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Peehl, D. M. "Primary cell cultures as models of prostate cancer development." Endocrine-Related Cancer 12, no. 1 (March 2005): 19–47. http://dx.doi.org/10.1677/erc.1.00795.
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This review focuses on primary cultures of human prostatic epithelial cells and their applications as models of normal and malignant biological behavior. Current abilities to culture cells from normal tissues, from premalignant dysplastic lesions (prostatic intraepithelial neoplasia), from primary adenocarcinomas, and from metastases are described. Evidence for representation of the interrelated cells of the normal prostatic epithelium — stem cells, basal epithelial cells, secretory epithelial cells, transit amplifying cells and neuroendocrine cells — in primary cultures is presented. Comparisons between normal and cancer-derived primary cultures are made regarding biological activities relevant to carcinogenesis, such as proliferation, apoptosis, differentiation, senescence, adhesion, migration, invasion, steroid hormone metabolism, other metabolic pathways and angiogenesis. Analyses of tumor suppressor activity, differential gene expression and cytogenetics in primary cultures have revealed changes relevant to prostate cancer progression. Preclinical studies with primary cultures have provided information useful for designing new strategies for chemoprevention, chemotherapy, cytotoxin therapy, radiation therapy, gene therapy and imaging. While the behavior of normal primary cultures is often used as a basis for comparison with established, immortal prostate cancer cell lines, the most informative studies are performed with donor-matched pairs of normal and malignant primary cultures, grown under identical conditions. Challenges that remain to be addressed if the full potential of primary cultures as a model system is to be realized include isolation, culture and characterization of stem cells, improved methodology to induce or maintain a fully differentiated, androgen-responsive phenotype, and identification of cell surface antigens or other markers with which to purify pure populations of live cancer or premalignant cells apart from non-malignant epithelial cells prior to culture.
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Lake, Andrew, Michael Warren, Sonia Das, Christopher Wells, Maria Scrivens, Ernest Smith, Vito Palombella, Bijan Etemad-Gilbertson, Pamela Holland, and Austin Dulak. "726 SRF114 is a fully human, CCR8 selective IgG1 antibody that induces destruction of tumor Tregs through ADCC." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A769. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0726.
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BackgroundT regulatory cells (Tregs) are potent suppressors of immune activation in the periphery and tumor microenvironment (TME). Tumor-infiltrating Tregs have also been associated with resistance to cancer therapies. Loss of peripheral Tregs can lead to widespread autoimmunity and tissue destruction; therefore, specifically depleting tumor Tregs is an attractive therapeutic approach to locally activate the immune system. CCR8 expression is highly restricted to tumor Tregs across multiple cancer types, supporting the notion that CCR8 targeting may induce tumor-specific Treg depletion while sparing peripheral Tregs. Moreover, depletion of CCR8+ Tregs leads to significant tumor growth inhibition with correlative tumor Treg depletion in established CT-26 tumors. These data provide rationale for targeting CCR8 to deplete tumor Tregs. Here, we describe the development of SRF114, a fully human IgG1 anti-CCR8 antibody that induces tumor Treg destruction through antibody-dependent cellular cytotoxicity (ADCC).MethodsVirus panning against the N-terminal region of CCR8 and subsequent affinity maturation process led to discovery of SRF114, a fully human monoclonal antibody that is specific to CCR8. To evaluate SRF114 specificity, binding was profiled on recombinant CCR8 N-terminus, CCR8+ and CCR8- cell lines, and primary cell cultures. An extracellular protein target cell microarray was used to further validate specificity. SRF114 functional assays included the Promega CD16 (V/F variants) ADCC signaling assay, PBMC/293T-hCCR8+ cell co-culture experiments, and natural killer (NK)-activation assays targeting Raji-CCR8+ cell lines. To confirm tumor Treg binding and depletion, NK allogenic co-culture experiments were performed with SRF114 using isolated tumor infiltrating lymphocyte cultures from freshly resected tumors.ResultsA tumor Treg-restricted pattern of CCR8 expression was confirmed using publicly available datasets and profiling of CCR8 expression on Tregs from fresh tumor tissues. SRF114 binds to CCR8-expressing 293T cells with pM affinity and not to parental cells. SRF114 does not bind any cell populations in PBMCs from healthy donors and has no other protein targets assessed by cell microarray. In dose-dependent ADCC assays, SRF114 induces cell killing with pM EC50 values, which is further enhanced by removing the fucose groups from the Fc-domain. Finally, SRF114 specifically binds to human tumor Tregs and induces killing of Tregs in NK co-culture experiments.ConclusionsThe fully human anti-CCR8 antibody SRF114 specifically binds to and targets CCR8+tumor Tregs for depletion, likely through ADCC. Through this mechanism, SRF114 treatment may alter the TME to support immune destruction of human tumors.
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Herrmann, Dieter B. J., and Herbert A. Neumann. "Cytotoxic activity of the thioether phospholipid analogue BM 41.440 in primary human tumor cultures." Lipids 23, no. 1 (January 1988): 76. http://dx.doi.org/10.1007/bf02535310.
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Herrmann, Dieter B. J., and Herbert A. Neumann. "Cytotoxic activity of the thioether phospholipid analogue BM 41.440 in primary human tumor cultures." Lipids 22, no. 11 (November 1987): 955–57. http://dx.doi.org/10.1007/bf02535563.
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Schwab, Tracy S., Tylitha Stewart, Jeff Lehr, Kenneth J. Pienta, Johng S. Rhim, and Jill A. Macoska. "Phenotypic characterization of immortalized normal and primary tumor-derived human prostate epithelial cell cultures." Prostate 44, no. 2 (2000): 164–71. http://dx.doi.org/10.1002/1097-0045(20000701)44:2<164::aid-pros9>3.0.co;2-4.
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van Koetsveld, Peter M., Sara G. Creemers, Fadime Dogan, Gaston J. H. Franssen, Wouter W. de Herder, Richard A. Feelders, and Leo J. Hofland. "The Efficacy of Mitotane in Human Primary Adrenocortical Carcinoma Cultures." Journal of Clinical Endocrinology & Metabolism 105, no. 2 (October 5, 2019): 407–17. http://dx.doi.org/10.1210/clinem/dgz001.
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Abstract Context Patients with adrenocortical carcinoma (ACC) often fail mitotane treatment and deal with severe toxicity, marking the relevance of predictive parameters for treatment outcome. Objective Determine the effects of mitotane in primary ACC cultures, and correlate sensitivity with patient and tumor characteristics. Methods In 32 primary ACC cultures, the effects of mitotane on cell growth and cortisol production were determined. RRM1, SOAT1, and CYP2W1 expression were assessed using reverse transcription-polymerase chain reaction and immunohistochemistry. Results The median percentage cell amount inhibition in primary ACC cultures at 50 µM mitotane was 57%. Seven patients were classified as nonresponders, 14 as partial responders, and 11 as responders. The mean median effective concentration (EC50) value of mitotane for inhibition of cell amount in responders was 14.2 µM (95% CI, 11.3–17.9), in partial responders 41.6 µM (95% CI, 33.5–51.8), and could not be calculated in nonresponders. The percentage cortisol-producing ACC was 14%, 43%, and 73% for nonresponders, partial responders, and responders (P = 0.068). Mitotane inhibited cortisol production with a mean EC50 of 1.4 µM (95% CI, 0.9–2.1), which was considerably lower than the EC50 on cell growth. RRM1, SOAT1, and CYP2W1 expression levels were not predictive for mitotane sensitivity in vitro. Conclusion Direct antitumor effects of mitotane on human primary ACC cultures are highly variable between patients, reflecting heterogeneous responses in patients. Cortisol was inhibited at lower concentrations, compared with its effect on cell amount. Cortisol secretion by ACC might be associated with enhanced mitotane sensitivity due to increased direct antitumor effects of mitotane.
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Kern, P. A. "Recombinant human tumor necrosis factor does not inhibit lipoprotein lipase in primary cultures of isolated human adipocytes." Journal of Lipid Research 29, no. 7 (July 1988): 909–14. http://dx.doi.org/10.1016/s0022-2275(20)38483-2.
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Suarez-Cuervo, C., N. L. Bryant, R. D. Lopez, G. Y. Gillespie, and L. S. Lamb. "Ex vivo activation and expansion of γδ T cells for immunotherapy of glioblastoma." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 2553. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.2553.
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2553 Background: Activated γδ T cells will efficiently kill established glioblastoma (GBM) cell lines in vitro. However, their potential for cell therapy has been limited by their relatively small numbers in the peripheral blood and their sensitivity to activation induced cell death (AICD) during ex vivo expansion. Our laboratory developed a method whereby γδ T cells, when stimulated by IFN-γ, IL- 12, and anti-CD2, acquire resistance to AICD and can be expanded in culture with anti-CD3 and IL-2. The γδ T cells expanded by this method are highly cytotoxic to GBM. We have now sought to adapt the laboratory method for human use. Methods: The laboratory method and selection procedure were modified to employ pharmaceutical-grade reagents. Peripheral blood mononuclear cells (1.0 × 106/ml) were stimulated overnight with media containing cGMP grade human serum, IFN-γ, IL-12, and anti-CD2. Media containing human serum, IL-2 and anti-CD3 was then added (1 v/v) and refreshed 1 v/v 3× weekly. The cultures were harvested after two weeks followed by depletion of CD4+ and CD8+ T cells, thereby enriching γδ T cells. Cytotoxicity of expanded of γδ T cells was evaluated against GBM primary tumor cultures, established GBM cell lines, and cultured astrocytes using a commercial flow cytometric method. Results: After two weeks in culture, γδ T cells expanded up to 1000 fold (usually 200–400 fold), consistent with results from our previous research-scale method. Positive selection yielded a γδ T cell product with >80% purity and >70% recovery. The cell products exhibited incremental cytotoxicity against several primary GBM cultures and established GBM cell lines. Astrocytoma cells were not killed. Conclusions: Apoptosis-resistant γδ T cells can be expanded and selected using clinically approvable reagents and in numbers sufficient for immunotherapy of malignant brain tumors. Initial data show that expanded γδ T cells retain cytotoxicity against the GBM primary cultures and spare normal astrocytes. No significant financial relationships to disclose.
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Rathje, Florian, Stefan Klingler, and Fritz Aberger. "Organoids for Modeling (Colorectal) Cancer in a Dish." Cancers 14, no. 21 (November 3, 2022): 5416. http://dx.doi.org/10.3390/cancers14215416.
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Functional studies of primary cancer have been limited to animal models for a long time making it difficult to study aspects specific to human cancer biology. The development of organoid technology enabled us to culture human healthy and tumor cells as three-dimensional self-organizing structures in vitro for a prolonged time. Organoid cultures conserve the heterogeneity of the originating epithelium regarding cell types and tumor clonality. Therefore, organoids are considered an invaluable tool to study and genetically dissect various aspects of human cancer biology. In this review, we describe the applications, advantages, and limitations of organoids as human cancer models with the main emphasis on colorectal cancer.
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Fankhauser, Maria, Nicole Bechmann, Michael Lauseker, Judith Goncalves, Judith Favier, Barbara Klink, Doreen William, et al. "Synergistic Highly Potent Targeted Drug Combinations in Different Pheochromocytoma Models Including Human Tumor Cultures." Endocrinology 160, no. 11 (July 19, 2019): 2600–2617. http://dx.doi.org/10.1210/en.2019-00410.
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Abstract There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb−/− cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb−/− cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb−/− cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.