Tumor Organoid and Microenvironment Cocultures: Implications for Basic and Translational Cancer Research

Abstract

Organoids are innovative three-dimensional (3D) cellular constructs, offering a unique platform to replicate the architectural and functional complexity of organs and tissues. In oncology, the tumor microenvironment (TME) dictates tumor evolution and therapeutic resistance. Consequently, therapies targeting TME components have emerged as a burgeoning frontier in cancer treatment. However, accurately recapitulating the dynamic, multicellular crosstalk of TME remains a significant hurdle for clinical translation. This review encapsulates the spectrum of current organoid coculture methodologies, ranging from direct coculture and air–liquid interface to advanced microfluidics and 3D bioprinting. These models not only deepen our understanding of the fundamental mechanisms at play in cancer but also evaluate emerging therapeutic modalities, such as antibody–drug conjugates and immunotherapy. By closely mimicking the in vivo tumor milieu, organoid cocultures enhance our ability to predict therapeutic outcomes and pave the way for the development of precision medicine approaches, thereby propelling forward the frontiers of oncology. This review aims to provide a comprehensive overview of organoid coculture models, spanning from construction methodologies to clinical applications. We envision this work serving as a definitive guide for the field, ultimately accelerating the transition from theoretical research to clinical practice.

Keywords

coculture / immunotherapy / organoid / precision medicine / tumor microenvironment

References

[1] C. Jubelin, J. Munoz-Garcia, L. Griscom, et al., “Three-Dimensional In Vitro Culture Models in Oncology Research,” Cell Bioscience 12 (2022): 155.

[2] A. Golebiewska and R. C. Fields, “Advancing Preclinical Cancer Models to Assess Clinically Relevant Outcomes,” BMC Cancer 23 (2023): 230.

[3] T. Sato, R. G. Vries, H. J. Snippert, et al., “Single Lgr5 Stem Cells Build Crypt-Villus Structures In Vitro Without a Mesenchymal Niche,” Nature 459 (2009): 262–265.

[4] T. Sato, D. E. Stange, M. Ferrante, et al., “Long-Term Expansion of Epithelial Organoids from Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium,” Gastroenterology 141 (2011): 1762–1772.

[5] G. Rossi, A. Manfrin, and M. P. Lutolf, “Progress and Potential in Organoid Research,” Nature Reviews Genetics 19 (2018): 671–687.

[6] Q. Yao, S. Cheng, Q. Pan, et al., “Organoids: Development and Applications in Disease Models, Drug Discovery, Precision Medicine, and Regenerative Medicine,” MedComm 5, no. 2024 (2020): e735.

[7] M. van de Wetering, H. E. Francies, J. M. Francis, et al., “Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients,” Cell 161 (2015): 933–945.

[8] M. Fujii, M. Shimokawa, S. Date, et al., “A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis,” Cell Stem Cell 18 (2016): 827–838.

[9] D. Gao, I. Vela, A. Sboner, et al., “Organoid Cultures Derived From Patients With Advanced Prostate Cancer,” Cell 159 (2014): 176–187.

[10] S. H. Lee, W. Hu, J. T. Matulay, et al., “Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer,” Cell 173 (2018): 515–528.e17.

[11] N. Sachs, J. de Ligt, O. Kopper, et al., “A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity,” Cell 172 (2018): 373–386.e10.

[12] H. H. N. Yan, H. C. Siu, S. Law, et al., “A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening,” Cell Stem Cell 23 (2018): 882–897.e11.

[13] T. Seidlitz, S. R. Merker, A. Rothe, et al., “Human Gastric Cancer Modelling Using Organoids,” Gut 68 (2019): 207–217.

[14] L. Broutier, G. Mastrogiovanni, M. M. Verstegen, et al., “Human Primary Liver Cancer-Derived Organoid Cultures for Disease Modeling and Drug Screening,” Nature Medicine 23 (2017): 1424–1435.

[15] O. Kopper, C. J. de Witte, K. Lõhmussaar, et al., “An Organoid Platform for Ovarian Cancer Captures Intra- and Interpatient Heterogeneity,” Nature Medicine 25 (2019): 838–849.

[16] M. Kim, H. Mun, C. O. Sung, et al., “Patient-Derived Lung Cancer Organoids as In Vitro Cancer Models for Therapeutic Screening,” Nature Communications 10 (2019): 3991.

[17] R. Shi, N. Radulovich, C. Ng, et al., “Organoid Cultures as Preclinical Models of Non-Small Cell Lung Cancer,” Clinical Cancer Research 26 (2020): 1162–1174.

[18] Y. Xia, S. Xie, Z. Cai, et al., “The Techniques and Applications of Patient-Derived Head and Neck Cancer Organoids: A Systematic Review,” Holistic Integrative Oncology 4 (2025): 18.

[19] K. Lõhmussaar, R. Oka, J. Espejo Valle-Inclan, et al., “Patient-Derived Organoids Model Cervical Tissue Dynamics and Viral Oncogenesis in Cervical Cancer,” Cell Stem Cell 28 (2021): 1380–1396.

[20] S. Mo, P. Tang, W. Luo, et al., “Patient-Derived Organoids From Colorectal Cancer With Paired Liver Metastasis Reveal Tumor Heterogeneity and Predict Response to Chemotherapy,” Advanced Science (Weinheim) 9 (2022): e2204097.

[21] K. E. de Visser and J. A. Joyce, “The Evolving Tumor Microenvironment: From Cancer Initiation to Metastatic Outgrowth,” Cancer Cell 41 (2023): 374–403.

[22] Y. Xiao and D. Yu, “Tumor Microenvironment as a Therapeutic Target in Cancer,” Pharmacology & Therapeutics 221 (2021): 107753.

[23] B. A. Hassell, G. Goyal, E. Lee, et al., “Human Organ Chip Models Recapitulate Orthotopic Lung Cancer Growth, Therapeutic Responses, and Tumor Dormancy In Vitro,” Cell Reports 21 (2017): 508–516.

[24] I. J. Fidler, “The Pathogenesis of Cancer Metastasis: The 'Seed and Soil' Hypothesis Revisited,” Nature Reviews Cancer 3 (2003): 453–458.

[25] M. J. Pittet, O. Michielin, and D. Migliorini, “Clinical Relevance of Tumour-Associated Macrophages,” Nature Reviews Clinical Oncology 19 (2022): 402–421.

[26] N. Niu, X. Shen, Z. Wang, et al., “Tumor Cell-Intrinsic Epigenetic Dysregulation Shapes Cancer-Associated Fibroblasts Heterogeneity to Metabolically Support Pancreatic Cancer,” Cancer Cell 42 (2024): 869–884.e9.

[27] G. Caligiuri and D. A. Tuveson, “Activated Fibroblasts in Cancer: Perspectives and Challenges,” Cancer Cell 41 (2023): 434–449.

[28] R. Xu, A. Boudreau, and M. J. Bissell, “Tissue Architecture and Function: Dynamic Reciprocity via Extra- and Intra-Cellular Matrices,” Cancer and Metastasis Reviews 28 (2009): 167–176.

[29] R. Xu, X. Zhou, S. Wang, et al., “Tumor Organoid Models in Precision Medicine and Investigating Cancer-Stromal Interactions,” Pharmacology & Therapeutics 218 (2021): 107668.

[30] Y. Liu, W. Wu, C. Cai, et al., “Patient-Derived Xenograft Models in Cancer Therapy: Technologies and Applications,” Signal Transduction and Targeted Therapy 8 (2023): 160.

[31] D. Tuveson and H. Clevers, “Cancer Modeling Meets Human Organoid Technology,” Science 364 (2019): 952–955.

[32] J. T. Neal, X. Li, J. Zhu, et al., “Organoid Modeling of the Tumor Immune Microenvironment,” Cell 175 (2018): 1972–1988.e16.

[33] G. Vlachogiannis, S. Hedayat, A. Vatsiou, et al., “Patient-Derived Organoids Model Treatment Response of Metastatic Gastrointestinal Cancers,” Science 359 (2018): 920–926.

[34] G. Zhou, R. Lieshout, G. S. van Tienderen, et al., “Modelling Immune Cytotoxicity for Cholangiocarcinoma With Tumour-Derived Organoids and Effector T Cells,” British Journal of Cancer 127 (2022): 649–660.

[35] K. K. Dijkstra, C. M. Cattaneo, F. Weeber, et al., “Generation of Tumor-Reactive T Cells by Co-Culture of Peripheral Blood Lymphocytes and Tumor Organoids,” Cell 174 (2018): 1586–1598.e12.

[36] L. Huang, Y. Rong, X. Tang, et al., “Engineered Exosomes as an In Situ Dc-Primed Vaccine to Boost Antitumor Immunity in Breast Cancer,” Molecular Cancer 21 (2022): 45.

[37] C. M. Cattaneo, K. K. Dijkstra, L. F. Fanchi, et al., “Tumor Organoid-T-Cell Coculture Systems,” Nature Protocols 15 (2020): 15–39.

[38] H. Dang, T. J. Harryvan, C. Y. Liao, et al., “Cancer-Associated Fibroblasts Are Key Determinants of Cancer Cell Invasion in the Earliest Stage of Colorectal Cancer,” Cellular and Molecular Gastroenterology and Hepatology 16 (2023): 107–131.

[39] V. Koh, J. Chakrabarti, M. Torvund, et al., “Hedgehog Transcriptional Effector Gli Mediates Mtor-Induced Pd-L1 Expression in Gastric Cancer Organoids,” Cancer Letters 518 (2021): 59–71.

[40] S. Parte, A. B. Kaur, R. K. Nimmakayala, et al., “Cancer-Associated Fibroblast Induces Acinar-to-Ductal Cell Transdifferentiation and Pancreatic Cancer Initiation via Lama5/Itga4 Axis,” Gastroenterology 166 (2024): 842–858.

[41] X. Luo, E. L. S. Fong, C. Zhu, et al., “Hydrogel-Based Colorectal Cancer Organoid Co-Culture Models,” Acta Biomaterialia 132 (2021): 461–472.

[42] Q. Sui, D. Liu, W. Jiang, et al., “Dickkopf 1 Impairs the Tumor Response to Pd-1 Blockade by Inactivating Cd8+ T Cells in Deficient Mismatch Repair Colorectal Cancer,” Journal for ImmunoTherapy of Cancer 9 (2021): e001498.

[43] E. Strating, M. P. Verhagen, E. Wensink, et al., “Co-Cultures of Colon Cancer Cells and Cancer-Associated Fibroblasts Recapitulate the Aggressive Features of Mesenchymal-Like Colon Cancer,” Frontiers in Immunology 14 (2023): 1053920.

[44] T. Seino, S. Kawasaki, M. Shimokawa, et al., “Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence During Disease Progression,” Cell Stem Cell 22 (2018): 454–467.

[45] W. Ye, C. Luo, C. Li, et al., “Organoids to Study Immune Functions, Immunological Diseases and Immunotherapy,” Cancer Letters 477 (2020): 31–40.

[46] S. Jiang, T. Deng, H. Cheng, et al., “Macrophage-Organoid Co-Culture Model for Identifying Treatment Strategies Against Macrophage-Related Gemcitabine Resistance,” Journal of Experimental & Clinical Cancer Research 42 (2023): 199.

[47] D. Öhlund, A. Handly-Santana, G. Biffi, et al., “Distinct Populations of Inflammatory Fibroblasts and Myofibroblasts in Pancreatic Cancer,” Journal of Experimental Medicine 214 (2017): 579–596.

[48] V. S. Atanasova, C. de Jesus Cardona, V. Hejret, et al., “Mimicking Tumor Cell Heterogeneity of Colorectal Cancer in a Patient-Derived Organoid-Fibroblast Model,” Cellular and Molecular Gastroenterology and Hepatology 15 (2023): 1391–1419.

[49] S. V. Murphy and A. Atala, “3d Bioprinting of Tissues and Organs,” Nature Biotechnology 32 (2014): 773–785.

[50] Z. Gong, L. Huang, X. Tang, et al., “Acoustic Droplet Printing Tumor Organoids for Modeling Bladder Tumor Immune Microenvironment Within a Week,” Advanced Healthcare Materials 10 (2021): e2101312.

[51] S. Flores-Torres, N. M. Dimitriou, L. A. Pardo, et al., “Bioprinted Multicomponent Hydrogel Co-Culture Tumor-Immune Model for Assessing and Simulating Tumor-Infiltrated Lymphocyte Migration and Functional Activation,” ACS Applied Materials & Interfaces 15 (2023): 33250–33262.

[52] M. H. Kim, Y. P. Singh, N. Celik, et al., “High-Throughput Bioprinting of Spheroids for Scalable Tissue Fabrication,” Nature Communications 15 (2024): 10083.

[53] W. Shi, S. Mirza, M. Kuss, et al., “Embedded Bioprinting of Breast Tumor Cells and Organoids Using Low-Concentration Collagen-Based Bioinks,” Advanced Healthcare Materials 12 (2023): e2300905.

[54] S. E. Park, A. Georgescu, and D. Huh, “Organoids-on-a-Chip,” Science 364 (2019): 960–965.

[55] S. N. Bhatia and D. E. Ingber, “Microfluidic Organs-on-Chips,” Nature Biotechnology 32 (2014): 760–772.

[56] J. Zhang, H. Tavakoli, L. Ma, et al., “Immunotherapy Discovery on Tumor Organoid-on-a-Chip Platforms That Recapitulate the Tumor Microenvironment,” Advanced Drug Delivery Reviews 187 (2022): 114365.

[57] J. Deng, E. S. Wang, R. W. Jenkins, et al., “Cdk4/6 Inhibition Augments Antitumor Immunity by Enhancing T-Cell Activation,” Cancer Discovery 8 (2018): 216–233.

[58] Z. Zou, Z. Lin, C. Wu, et al., “Micro-Engineered Organoid-on-a-Chip Based on Mesenchymal Stromal Cells to Predict Immunotherapy Responses of Hcc Patients,” Advanced Science 10 (2023): e2302640.

[59] Y. Zhang, Q. Hu, Y. Pei, et al., “A Patient-Specific Lung Cancer Assembloid Model With Heterogeneous Tumor Microenvironments,” Nature Communications 15 (2024): 3382.

[60] H. G. Yi, Y. H. Jeong, Y. Kim, et al., “A Bioprinted Human-Glioblastoma-on-a-Chip for the Identification of Patient-Specific Responses to Chemoradiotherapy,” Nature Biomedical Engineering 3 (2019): 509–519.

[61] A. C. Daly, G. M. Cunniffe, B. N. Sathy, et al., “3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering,” Advanced Healthcare Materials 5 (2016): 2353–2362.

[62] A. Blaeser, D. F. Duarte Campos, U. Puster, et al., “Controlling Shear Stress in 3D Bioprinting Is a Key Factor to Balance Printing Resolution and Stem Cell Integrity,” Advanced Healthcare Materials 5 (2016): 326–333.

[63] A. Sontheimer-Phelps, B. A. Hassell, and D. E. Ingber, “Modelling Cancer in Microfluidic Human Organs-on-Chips,” Nature Reviews Cancer 19 (2019): 65–81.

[64] G. M. Whitesides, “The Origins and the Future of Microfluidics,” Nature 442 (2006): 368–373.

[65] S. Halldorsson, E. Lucumi, R. Gomez-Sjoberg, et al., “Advantages and Challenges of Microfluidic Cell Culture in Polydimethylsiloxane Devices,” Biosensors & Bioelectronics 63 (2015): 218–231.

[66] D. Hanahan and R. A. Weinberg, “Hallmarks of Cancer: The Next Generation,” Cell 144 (2011): 646–674.

[67] L. Peng, F. Wang, Z. Wang, et al., “Cell-Cell Communication Inference and Analysis in the Tumour Microenvironments From Single-Cell Transcriptomics: Data Resources and Computational Strategies,” Briefings in Bioinformatics 23 (2022): bbac234.

[68] A. Naba, “Mechanisms of Assembly and Remodelling of the Extracellular Matrix,” Nature Reviews Molecular Cell Biology 25 (2024): 865–885.

[69] R. Kalluri, “The Biology and Function of Exosomes in Cancer,” Journal of Clinical Investigation 126 (2016): 1208–1215.

[70] N. N. Pavlova and C. B. Thompson, “The Emerging Hallmarks of Cancer Metabolism,” Cell Metabolism 23 (2016): 27–47.

[71] B. Farhood, M. Najafi, and K. Mortezaee, “Cd8+ Cytotoxic T Lymphocytes in Cancer Immunotherapy: A Review,” Journal of Cellular Physiology 234 (2019): 8509–8521.

[72] A. M. van der Leun, D. S. Thommen, and T. N. Schumacher, “Cd8(+) T Cell States in Human Cancer: Insights From Single-Cell Analysis,” Nature Reviews Cancer 20 (2020): 218–232.

[73] E. J. Wherry and M. Kurachi, “Molecular and Cellular Insights Into T Cell Exhaustion,” Nature Reviews Immunology 15 (2015): 486–499.

[74] M. Hashimoto, A. O. Kamphorst, S. J. Im, et al., “Cd8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions,” Annual Review of Medicine 69 (2018): 301–318.

[75] J. S. Dolina, N. Van Braeckel-Budimir, G. D. Thomas, et al., “Cd8(+) T Cell Exhaustion in Cancer,” Frontiers in Immunology 12 (2021): 715234.

[76] U. Smole, N. Gour, J. Phelan, et al., “Serum Amyloid a Is a Soluble Pattern Recognition Receptor That Drives Type 2 Immunity,” Nature Immunology 21 (2020): 756–765.

[77] X. Wang, S. Wen, X. Du, et al., “Saa Suppresses Alpha-Pd-1 Induced Anti-Tumor Immunity by Driving T(H)2 Polarization in Lung Adenocarcinoma,” Cell Death & Disease 14 (2023): 718.

[78] K. A. Ahmed, M. A. Munegowda, Y. Xie, et al., “Intercellular Trogocytosis Plays an Important Role in Modulation of Immune Responses,” Cellular & Molecular Immunology 5 (2008): 261–269.

[79] J. H. Shin, J. Jeong, S. E. Maher, et al., “Colon Cancer Cells Acquire Immune Regulatory Molecules from Tumor-Infiltrating Lymphocytes by Trogocytosis,” Proceedings of the National Academy of Sciences 118 (2021): e2110241118.

[80] H. Cheroutre, F. Lambolez, and D. Mucida, “The Light and Dark Sides of Intestinal Intraepithelial Lymphocytes,” Nature Reviews Immunology 11 (2011): 445–456.

[81] R. Morikawa, Y. Nemoto, Y. Yonemoto, et al., “Intraepithelial Lymphocytes Suppress Intestinal Tumor Growth by Cell-to-Cell Contact via Cd103/E-Cadherin Signal,” Cellular and Molecular Gastroenterology and Hepatology 11 (2021): 1483–1503.

[82] C. M. Laumont and B. H. Nelson, “B Cells in the Tumor Microenvironment: Multi-Faceted Organizers, Regulators, and Effectors of Anti-Tumor Immunity,” Cancer Cell 41 (2023): 466–489.

[83] C. Sautes-Fridman, F. Petitprez, J. Calderaro, et al., “Tertiary Lymphoid Structures in the Era of Cancer Immunotherapy,” Nature Reviews Cancer 19 (2019): 307–325.

[84] R. Cabrita, M. Lauss, A. Sanna, et al., “Tertiary Lymphoid Structures Improve Immunotherapy and Survival in Melanoma,” Nature 577 (2020): 561–565.

[85] B. A. Helmink, S. M. Reddy, J. Gao, et al., “B Cells and Tertiary Lymphoid Structures Promote Immunotherapy Response,” Nature 577 (2020): 549–555.

[86] L. E. Wagar, A. Salahudeen, C. M. Constantz, et al., “Modeling Human Adaptive Immune Responses With Tonsil Organoids,” Nature Medicine 27 (2021): 125–135.

[87] Y. Chen, Y. Song, W. Du, et al., “Tumor-Associated Macrophages: An Accomplice in Solid Tumor Progression,” Journal of Biomedical Science 26 (2019): 78.

[88] S. Aras and M. R. Zaidi, “Tameless Traitors: Macrophages in Cancer Progression and Metastasis,” British Journal of Cancer 117 (2017): 1583–1591.

[89] N. Linde, C. M. Gutschalk, C. Hoffmann, et al., “Integrating Macrophages Into Organotypic Co-Cultures: A 3D In Vitro Model to Study Tumor-Associated Macrophages,” PLoS ONE 7 (2012): e40058.

[90] F. Veglia, E. Sanseviero, and D. I. Gabrilovich, “Myeloid-Derived Suppressor Cells in the Era of Increasing Myeloid Cell Diversity,” Nature Reviews Immunology 21 (2021): 485–498.

[91] E. Schouppe, E. Van Overmeire, D. Laoui, et al., “Modulation of Cd8+ T-Cell Activation Events by Monocytic and Granulocytic Myeloid-Derived Suppressor Cells,” Immunobiology 218 (2013): 1385–1391.

[92] L. Holokai, J. Chakrabarti, J. Lundy, et al., “Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma,” Cancers 12 (2020): 3816.

[93] X. Lu, J. W. Horner, E. Paul, et al., “Effective Combinatorial Immunotherapy for Castration-Resistant Prostate Cancer,” Nature 543 (2017): 728–732.

[94] A. Gardner and B. Ruffell, “Dendritic Cells and Cancer Immunity,” Trends in Immunology 37 (2016): 855–865.

[95] Y. Wang, Y. Xiang, V. W. Xin, et al., “Dendritic Cell Biology and Its Role in Tumor Immunotherapy,” Journal of Hematology & Oncology 13 (2020): 107.

[96] A. Leblanc-Hotte, C. Audiger, G. Chabot-Roy, et al., “Immature and Mature Bone Marrow-Derived Dendritic Cells Exhibit Distinct Intracellular Mechanical Properties,” Scientific Reports 13 (2023): 1967.

[97] B. Subtil, K. K. Iyer, D. Poel, et al., “Dendritic Cell Phenotype and Function in a 3D Co-Culture Model of Patient-Derived Metastatic Colorectal Cancer Organoids,” Frontiers in Immunology 14 (2023): 1105244.

[98] P. Gandellini, F. Andriani, G. Merlino, et al., “Complexity in the Tumour Microenvironment: Cancer Associated Fibroblast Gene Expression Patterns Identify Both Common and Unique Features of Tumour-Stroma Crosstalk Across Cancer Types,” Seminars in Cancer Biology 35 (2015): 96–106.

[99] C. Fan, W. Zhu, Y. Chen, et al., “Cancer-Associated Fibroblasts: Origin, Classification, Tumorigenicity, and Targeting for Cancer Therapy,” MedComm 6, no. 2025 (2020): e70415.

[100] K. E. Sung, X. Su, E. Berthier, et al., “Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models,” PLoS ONE 8 (2013): e76373.

[101] G. Biffi, T. E. Oni, B. Spielman, et al., “IL1-Induced Jak/Stat Signaling Is Antagonized by Tgfbeta to Shape Caf Heterogeneity in Pancreatic Ductal Adenocarcinoma,” Cancer Discovery 9 (2019): 282–301.

[102] O. Badran, I. Cohen, and G. Bar-Sela, “Cancer-Associated Fibroblasts in Solid Tumors and Sarcomas: Heterogeneity, Function, and Therapeutic Implications,” Cells 14 (2025): 1398.

[103] K. Feldmann, C. Maurer, K. Peschke, et al., “Mesenchymal Plasticity Regulated by Prrx1 Drives Aggressive Pancreatic Cancer Biology,” Gastroenterology 160, no. 2021 (1943): 346–361.e24.

[104] S. Schwörer, F. V. Cimino, M. Ros, et al., “Hypoxia Potentiates the Inflammatory Fibroblast Phenotype Promoted by Pancreatic Cancer Cell–Derived Cytokines,” Cancer Research 83 (2023): 1596–1610.

[105] T. Zhang, Y. Ren, P. Yang, et al., “Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma,” Cell Death & Disease 13 (2022): 897.

[106] J. Liu, P. Li, L. Wang, et al., “Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance,” Cellular and Molecular Gastroenterology and Hepatology 11 (2021): 407–431.

[107] T. Shinkawa, K. Ohuchida, Y. Mochida, et al., “Subtypes in Pancreatic Ductal Adenocarcinoma Based on Niche Factor Dependency Show Distinct Drug Treatment Responses,” Journal of Experimental & Clinical Cancer Research 41 (2022): 89.

[108] C. Thery, “Exosomes: Secreted Vesicles and Intercellular Communications,” F1000 Biology Reports 3 (2011): 15.

[109] S. Kunou, K. Shimada, M. Takai, et al., “Exosomes Secreted From Cancer-Associated Fibroblasts Elicit Anti-Pyrimidine Drug Resistance Through Modulation of Its Transporter in Malignant Lymphoma,” Oncogene 40 (2021): 3989–4003.

[110] S. H. Kang, S. Y. Oh, K.-Y. Lee, et al., “Differential Effect of Cancer-Associated Fibroblast-Derived Extracellular Vesicles on Cisplatin Resistance in Oral Squamous Cell Carcinomavia Mir-876-3p,” Theranostics 14 (2024): 460–479.

[111] H. Zhao, E. Jiang, and Z. Shang, “3D Co-Culture of Cancer-Associated Fibroblast with Oral Cancer Organoids,” Journal of Dental Research 100 (2021): 201–208.

[112] W. Xiao, M. Pahlavanneshan, C. Y. Eun, et al., “Matrix Stiffness Mediates Pancreatic Cancer Chemoresistance Through Induction of Exosome Hypersecretion in a Cancer Associated Fibroblasts-Tumor Organoid Biomimetic Model,” Matrix Biology Plus 14 (2022): 100111.

[113] L. Hutchinson and R. Kirk, “High Drug Attrition Rates—Where Are We Going Wrong?,” Nature Reviews Clinical Oncology 8 (2011): 189–190.

[114] S. Schuth, S. Le Blanc, T. G. Krieger, et al., “Patient-Specific Modeling of Stroma-Mediated Chemoresistance of Pancreatic Cancer Using a Three-Dimensional Organoid-Fibroblast Co-Culture System,” Journal of Experimental & Clinical Cancer Research 41 (2022): 312.

[115] H. F. Farin, M. H. Mosa, B. Ndreshkjana, et al., “Colorectal Cancer Organoid-Stroma Biobank Allows Subtype-Specific Assessment of Individualized Therapy Responses,” Cancer Discovery 13 (2023): 2192–2211.

[116] Y. Zhang, Q. Fu, W. Sun, et al., “Mechanical Forces in the Tumor Microenvironment: Roles, Pathways, and Therapeutic Approaches,” Journal of Translational Medicine 23 (2025): 313.

[117] P. Lu, V. M. Weaver, and Z. Werb, “The Extracellular Matrix: A Dynamic Niche in Cancer Progression,” Journal of Cell Biology 196 (2012): 395–406.

[118] A. Marchini and F. Gelain, “Synthetic Scaffolds for 3D Cell Cultures and Organoids: Applications in Regenerative Medicine,” Critical Reviews in Biotechnology 42 (2022): 468–486.

[119] J. Wang, Z. Sui, W. Huang, et al., “Biomimetic Hydrogels With Mesoscale Collagen Architecture for Patient-Derived Tumor Organoids Culture,” Bioactive Materials 38 (2024): 384–398.

[120] C. E. Nason-Tomaszewski, E. E. Thomas, D. L. Matera, et al., “Extracellular Matrix-Templating Fibrous Hydrogels Promote Ovarian Tissue Remodeling and Oocyte Growth,” Bioactive Materials 32 (2024): 292–303.

[121] S. S. Ng, K. Saeb-Parsy, S. J. I. Blackford, et al., “Human Ips Derived Progenitors Bioengineered Into Liver Organoids Using an Inverted Colloidal Crystal Poly (Ethylene Glycol) Scaffold,” Biomaterials 182 (2018): 299–311.

[122] B. R. Dye, R. L. Youngblood, R. S. Oakes, et al., “Human Lung Organoids Develop Into Adult Airway-Like Structures Directed by Physico-Chemical Biomaterial Properties,” Biomaterials 234 (2020): 119757.

[123] A. Marchini, A. Raspa, R. Pugliese, et al., “Multifunctionalized Hydrogels Foster HNSC Maturation in 3D Cultures and Neural Regeneration in Spinal Cord Injuries,” Proceedings of the National Academy of Sciences 116 (2019): 7483–7492.

[124] R. Xu, X. Zhou, S. Wang, et al., “Tumor Organoid Models in Precision Medicine and Investigating Cancer-Stromal Interactions,” Pharmacology & Therapeutics 218 (2021): 107668.

[125] M. B. Schaaf, A. D. Garg, and P. Agostinis, “Defining the Role of the Tumor Vasculature in Antitumor Immunity and Immunotherapy,” Cell Death & Disease 9 (2018): 115.

[126] J. Folkman, “Tumor Angiogenesis: Therapeutic Implications,” New England Journal of Medicine 285 (1971): 1182–1186.

[127] P. Leone, E. Malerba, N. Susca, et al., “Endothelial Cells in Tumor Microenvironment: Insights and Perspectives,” Frontiers in Immunology 15 (2024): 1367875.

[128] N. Bayat, R. Izadpanah, S. Ebrahimi-Barough, et al., “The Anti-Angiogenic Effect of Atorvastatin in Glioblastoma Spheroids Tumor Cultured in Fibrin Gel: In 3D In Vitro Model,” Asian Pacific Journal of Cancer Prevention: APJCP 19 (2018): 2553–2560.

[129] Y. Wang, K. Takeishi, Z. Li, et al., “Microenvironment of a Tumor-Organoid System Enhances Hepatocellular Carcinoma Malignancy-Related Hallmarks,” Organogenesis 13 (2017): 83–94.

[130] V. O. Oria and J. T. Erler, “Tumor Angiocrine Signaling: Novel Targeting Opportunity in Cancer,” Cells 12 (2023): 2510.

[131] J. T. C. Lim, L. G. Kwang, N. C. W. Ho, et al., “Hepatocellular Carcinoma Organoid Co-Cultures Mimic Angiocrine Crosstalk to Generate Inflammatory Tumor Microenvironment,” Biomaterials 284 (2022): 121527.

[132] B. Dirat, L. Bochet, M. Dabek, et al., “Cancer-Associated Adipocytes Exhibit an Activated Phenotype and Contribute to Breast Cancer Invasion,” Cancer Research 71 (2011): 2455–2465.

[133] N. K. Pallegar and S. L. Christian, “Adipocytes in the Tumour Microenvironment,” Advances in Experimental Medicine and Biology 1234 (2020): 1–13.

[134] K. M. Nieman, H. A. Kenny, C. V. Penicka, et al., “Adipocytes Promote Ovarian Cancer Metastasis and Provide Energy for Rapid Tumor Growth,” Nature Medicine 17 (2011): 1498–1503.

[135] Y. A. Wen, X. Xing, J. W. Harris, et al., “Adipocytes Activate Mitochondrial Fatty Acid Oxidation and Autophagy to Promote Tumor Growth in Colon Cancer,” Cell Death & Disease 8 (2017): e2593.

[136] D. R. Mertz, E. Parigoris, J. Sentosa, et al., “Triple-Negative Breast Cancer Cells Invade Adipocyte/Preadipocyte-Encapsulating Geometrically Inverted Mammary Organoids,” Integrative Biology (Cambridge) 15 (2023): zyad004.

[137] C. Liebig, G. Ayala, J. A. Wilks, et al., “Perineural Invasion in Cancer: A Review of the Literature,” Cancer 115 (2009): 3379–3391.

[138] R. Mancusi and M. Monje, “The Neuroscience of Cancer,” Nature 618 (2023): 467–479.

[139] S. H. Jiang, L. P. Hu, X. Wang, et al., “Neurotransmitters: Emerging Targets in Cancer,” Oncogene 39 (2020): 503–515.

[140] B. K. C. Chan, C. Zhang, C. H. Poon, et al., “A Combined Enteric Neuron-Gastric Tumor Organoid Reveals Metabolic Vulnerabilities in Gastric Cancer,” Cell Stem Cell 32 (2025): 1595–1613 e10.

[141] D. Nejman, I. Livyatan, G. Fuks, et al., “The Human Tumor Microbiome Is Composed of Tumor Type-Specific Intracellular Bacteria,” Science 368 (2020): 973–980.

[142] A. B. Dohlman, J. Klug, M. Mesko, et al., “A Pan-Cancer Mycobiome Analysis Reveals Fungal Involvement in Gastrointestinal and Lung Tumors,” Cell 185 (2022): 3807–3822 e12.

[143] L. T. Geller, M. Barzily-Rokni, T. Danino, et al., “Potential Role of Intratumor Bacteria in Mediating Tumor Resistance to the Chemotherapeutic Drug Gemcitabine,” Science 357 (2017): 1156–1160.

[144] B. Routy, E. Le Chatelier, L. Derosa, et al., “Gut Microbiome Influences Efficacy of Pd-1-Based Immunotherapy Against Epithelial Tumors,” Science 359 (2018): 91–97.

[145] J. Puschhof, C. Pleguezuelos-Manzano, A. Martinez-Silgado, et al., “Intestinal Organoid Cocultures With Microbes,” Nature Protocols 16 (2021): 4633–4649.

[146] C. Pleguezuelos-Manzano, J. Puschhof, A. Rosendahl Huber, et al., “Mutational Signature in Colorectal Cancer Caused by Genotoxic Pks(+) E. coli,” Nature 580 (2020): 269–273.

[147] A. Fu, B. Yao, T. Dong, et al., “Tumor-Resident Intracellular Microbiota Promotes Metastatic Colonization in Breast Cancer,” Cell 185 (2022): 1356–1372 e26.

[148] A. Ribas and J. D. Wolchok, “Cancer Immunotherapy Using Checkpoint Blockade,” Science 359 (2018): 1350–1355.

[149] X. He, Y. Jiang, L. Zhang, et al., “Patient-Derived Organoids as a Platform for Drug Screening in Metastatic Colorectal Cancer,” Frontiers in Bioengineering and Biotechnology 11 (2023): 1190637.

[150] J. Mason, J. Cumming, A. U. Eriksson, et al., “High Throughput Screen in a Co-Culture Model to Uncover Therapeutic Strategies to Potentiate the Cancer-Inhibiting Properties of the Tumor-Stroma in Pancreatic Cancer,” bioRxiv, 2022, 2022.03.11.483991.

[151] Z. Zhou, K. Van der Jeught, Y. Fang, et al., “An Organoid-Based Screen for Epigenetic Inhibitors That Stimulate Antigen Presentation and Potentiate T-Cell-Mediated Cytotoxicity,” Nature Biomedical Engineering 5 (2021): 1320–1335.

[152] S. Bagchi, R. Yuan, and E. G. Engleman, “Immune Checkpoint Inhibitors for the Treatment of Cancer: Clinical Impact and Mechanisms of Response and Resistance,” Annual Review of Pathology 16 (2021): 223–249.

[153] C. Robert, “A Decade of Immune-Checkpoint Inhibitors in Cancer Therapy,” Nature Communications 11 (2020): 3801.

[154] K. Pang, Z.-D. Shi, L.-Y. Wei, et al., “Research Progress of Therapeutic Effects and Drug Resistance of Immunotherapy Based on Pd-1/Pd-L1 Blockade,” Drug Resistance Updates 66 (2023): 100907.

[155] L. Sun, X. Kang, H. Ju, et al., “A Human Mucosal Melanoma Organoid Platform for Modeling Tumor Heterogeneity and Exploring Immunotherapy Combination Options,” Science Advances 9 (2023): eadg6686.

[156] C. Wan, M. P. Keany, H. Dong, et al., “Enhanced Efficacy of Simultaneous Pd-1 and Pd-L1 Immune Checkpoint Blockade in High-Grade Serous Ovarian Cancer,” Cancer Research 81 (2021): 158–173.

[157] C. H. June, R. S. O'Connor, O. U. Kawalekar, et al., “Car T Cell Immunotherapy for Human Cancer,” Science 359 (2018): 1361–1365.

[158] U. Köhl, S. Arsenieva, A. Holzinger, et al., “Car T Cells in Trials: Recent Achievements and Challenges That Remain in the Production of Modified T Cells for Clinical Applications,” Human Gene Therapy 29 (2018): 559–568.

[159] Z. Wang, H. P. McWilliams-Koeppen, H. Reza, et al., “3D-Organoid Culture Supports Differentiation of Human Car+ Ipscs into Highly Functional Car T Cells,” Cell Stem Cell 29 (2022): 515–527.

[160] M. Themeli, C. C. Kloss, G. Ciriello, et al., “Generation of Tumor-Targeted Human T Lymphocytes From Induced Pluripotent Stem Cells for Cancer Therapy,” Nature Biotechnology 31 (2013): 928–933.

[161] F. Zou, J. Tan, T. Liu, et al., “The Cd39(+) Hbv Surface Protein-Targeted Car-T and Personalized Tumor-Reactive Cd8(+) T Cells Exhibit Potent Anti-Hcc Activity,” Molecular Therapy 29 (2021): 1794–1807.

[162] E. Liu, D. Marin, P. Banerjee, et al., “Use of Car-Transduced Natural Killer Cells in Cd19-Positive Lymphoid Tumors,” New England Journal of Medicine 382 (2020): 545–553.

[163] K. Rezvani and R. H. Rouce, “The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer,” Frontiers in Immunology 6 (2015): 578.

[164] T. E. Schnalzger, M. H. de Groot, C. Zhang, et al., “3D Model for Car-Mediated Cytotoxicity Using Patient-Derived Colorectal Cancer Organoids,” EMBO Journal 38 (2019): e100928.

[165] K. Palucka and J. Banchereau, “Dendritic-Cell-Based Therapeutic Cancer Vaccines,” Immunity 39 (2013): 38–48.

[166] U. Sahin and O. Tureci, “Personalized Vaccines for Cancer Immunotherapy,” Science 359 (2018): 1355–1360.

[167] A. Harari, M. Graciotti, M. Bassani-Sternberg, et al., “Antitumour Dendritic Cell Vaccination in a Priming and Boosting Approach,” Nature Reviews Drug Discovery 19 (2020): 635–652.

[168] L. Shang, X. Jiang, X. Zhao, et al., “Mitochondrial DNA-Boosted Dendritic Cell-Based Nanovaccination Triggers Antitumor Immunity in Lung and Pancreatic Cancers,” Cell Reports Medicine 5 (2024): 101648.

[169] C. Klein, U. Brinkmann, J. M. Reichert, et al., “The Present and Future of Bispecific Antibodies for Cancer Therapy,” Nature Reviews Drug Discovery 23 (2024): 301–319.

[170] Y. Gu and Q. Zhao, “Clinical Progresses and Challenges of Bispecific Antibodies for the Treatment of Solid Tumors,” Molecular Diagnosis and Therapy 28 (2024): 669–702.

[171] S. Dees, R. Ganesan, S. Singh, et al., “Bispecific Antibodies for Triple Negative Breast Cancer,” Trends in Cancer 7 (2021): 162–173.

[172] M. de Miguel, P. Umana, A. L. Gomes De Morais, et al., “T-Cell–Engaging Therapy for Solid Tumors,” Clinical Cancer Research 27 (2021): 1595–1603.

[173] A. Teijeira, I. Migueliz, S. Garasa, et al., “Three-Dimensional Colon Cancer Organoids Model the Response to Cea-Cd3 T-Cell Engagers,” Theranostics 12 (2022): 1373–1387.

[174] V. Anstett, E. Heinzelmann, F. Piraino, et al., “In Vitro Evaluation of the Safety and Efficacy of Cibisatamab Using Adult Stem Cell-Derived Organoids and Colorectal Cancer Spheroids,” Cancers (Basel) 17 (2025): 291.

[175] Z. Xue, Z. Wang, D. Liu, et al., “T-Cell Activation Enhances Anti-Her2-Mediated Antibody-Dependent Cellular Cytotoxicity in Gastric Cancer,” Immunologic Research 73 (2025): 88.

[176] S. Mensurado, R. Blanco-Domínguez, and B. Silva-Santos, “The Emerging Roles of Γδ T Cells in Cancer Immunotherapy,” Nature Reviews Clinical Oncology 20 (2023): 178–191.

[177] J. Dong, D. Holthaus, C. Peters, et al., “γδ T Cell-Mediated Cytotoxicity Against Patient-Derived Healthy and Cancer Cervical Organoids,” Frontiers in Immunology 14 (2023): 1281646.

[178] G. Delaney, S. Jacob, C. Featherstone, et al., “The Role of Radiotherapy in Cancer Treatment: Estimating Optimal Utilization From a Review of Evidence-Based Clinical Guidelines,” Cancer 104 (2005): 1129–1137.

[179] S. P. Jackson and J. Bartek, “The DNA-Damage Response in Human Biology and Disease,” Nature 461 (2009): 1071–1078.

[180] Y. Yao, X. Xu, L. Yang, et al., “Patient-Derived Organoids Predict Chemoradiation Responses of Locally Advanced Rectal Cancer,” Cell Stem Cell 26 (2020): 17–26.

[181] P. Mu, S. Mo, X. He, et al., “Unveiling Radiobiological Traits and Therapeutic Responses of Braf(V600e)-Mutant Colorectal Cancer via Patient-Derived Organoids,” Journal of Experimental & Clinical Cancer Research 44 (2025): 92.

[182] C. Yakkala, A. Denys, L. Kandalaft, et al., “Cryoablation and Immunotherapy of Cancer,” Current Opinion in Biotechnology 65 (2020): 60–64.

[183] J. G. Baust, A. A. Gage, T. E. Bjerklund Johansen, et al., “Mechanisms of Cryoablation: Clinical Consequences on Malignant Tumors,” Cryobiology 68 (2014): 1–11.

[184] Z. Mou, Y. Chen, Z. Zhang, et al., “Cryoablation Inhibits the Recurrence and Progression of Bladder Cancer by Enhancing Tumour-Specific Immunity,” Clinical and Translational Medicine 13 (2023): e1255.

[185] J. Z. Drago, S. Modi, and S. Chandarlapaty, “Unlocking the Potential of Antibody-Drug Conjugates for Cancer Therapy,” Nature Reviews Clinical Oncology 18 (2021): 327–344.

[186] S. Modi, W. Jacot, T. Yamashita, et al., “Trastuzumab Deruxtecan in Previously Treated Her2-Low Advanced Breast Cancer,” New England Journal of Medicine 387 (2022): 9–20.

[187] Y. Nakazawa, M. Miyano, S. Tsukamoto, et al., “Delivery of a Bet Protein Degrader via a Ceacam6-Targeted Antibody-Drug Conjugate Inhibits Tumour Growth in Pancreatic Cancer Models,” Nature Communications 15 (2024): 2192.

[188] S. Cui, X. Luo, G. Fan, et al., “A Novel Fgfr3-Targeting Antibody-Drug Conjugate Induces Tumor Cell Apoptosis Through the Cgas-Sting Pathway in Bladder Cancer,” Advanced Science (Weinheim) 13 (2026): e09933.

[189] M. Zanoni, F. Piccinini, C. Arienti, et al., “3D Tumor Spheroid Models for In Vitro Therapeutic Screening: A Systematic Approach to Enhance the Biological Relevance of Data Obtained,” Scientific Reports 6 (2016): 19103.

[190] X. Li, G. Fu, L. Zhang, et al., “Assay Establishment and Validation of a High-Throughput Organoid-Based Drug Screening Platform,” Stem Cell Research Therapy 13 (2022): 219.

[191] M. Ramos Zapatero, A. Tong, J. W. Opzoomer, et al., “Trellis Tree-Based Analysis Reveals Stromal Regulation of Patient-Derived Organoid Drug Responses,” Cell 186 (2023): 5606–5619.e24.

[192] S. Guinn, B. Kinny-Köster, J. A. Tandurella, et al., “Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated With Epithelial–Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma,” Cancer Research 84 (2024): 1517–1533.

[193] X. Huang, L. M. Maxson, T. Nguyen, et al., “Organoid Tracker: A SAM2-Powered Platform for Zero-Shot Cyst Analysis in Human Kidney Organoid Videos,” 2025, arXiv:2509.11063.

[194] E. G. Gracey and J. N. Lampe, “Novel Emerging Cell and Organoid Systems for the Study of Drug Metabolism and Toxicity in Humans,” Drug Metabolism and Disposition 53 (2025): 100188.

[195] L. Papamichail, L. S. Koch, D. Veerman, et al., “Organoids-on-a-Chip: Microfluidic Technology Enables Culture of Organoids With Enhanced Tissue Function and Potential for Disease Modeling,” Frontiers in Bioengineering and Biotechnology 13 (2025): 1515340.

[196] E. A. Aisenbrey and W. L. Murphy, “Synthetic Alternatives to Matrigel,” Nature Reviews Materials 5 (2020): 539–551.

[197] T. Kassis, V. Hernandez-Gordillo, R. Langer, et al., “Orgaquant: Human Intestinal Organoid Localization and Quantification Using Deep Convolutional Neural Networks,” Scientific Reports 9 (2019): 12479.

[198] J. Kim, B. K. Koo, and J. A. Knoblich, “Human Organoids: Model Systems for Human Biology and Medicine,” Nature Reviews Molecular Cell Biology 21 (2020): 571–584.

[199] P. Mu, S. Zhou, T. Lv, et al., “Newly Developed 3D In Vitro Models to Study Tumor–Immune Interaction,” Journal of Experimental & Clinical Cancer Research 42 (2023): 81.

[200] D. Huh, G. A. Hamilton, and D. E. Ingber, “From 3D Cell Culture to Organs-on-Chips,” Trends in Cell Biology 21 (2011): 745–754.

[201] B. Grigoryan, S. J. Paulsen, D. C. Corbett, et al., “Multivascular Networks and Functional Intravascular Topologies Within Biocompatible Hydrogels,” Science 364 (2019): 458–464.

[202] M. Wadman, “FDA No Longer Has to Require Animal Testing for New Drugs,” Science 379 (2023): 127–128.

[203] S. N. Ooft, F. Weeber, K. K. Dijkstra, et al., “Patient-Derived Organoids Can Predict Response to Chemotherapy in Metastatic Colorectal Cancer Patients,” Science Translational Medicine 11 (2019): eaay2574.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.