Cancer precision medicine has been deeply and widely explored through experimental studies to clinical evaluations to achieve the goal of individualized treatment and improve patient prognosis. However, genotype-based cancer precision medicine has some limitations or deficiencies. Genomic instability during sustained cancer proliferation within the tumor microenvironment (TME) gives rise to intra- and inter-tumoral heterogeneity, contributing to drug resistance, treatment failure, and progression. Cancer development is an evolving, highly regulated, dynamic biochemical process associated with TME. Genetic mutations accumulated in the copying process during DNA replication of cancer cells drive the generation of intratumoral heterogeneity within a tumor or inter-tumoral heterogeneity between tumors, with interactions among cancer cells, immune cells, and fibroblasts leading to TME alterations. Moreover, tumor heterogeneity increases due to TME alterations. Thus, the provoked interaction network within TME hinders the clinical application of precision cancer therapy. Further developing preclinical models to investigate the TME and guide clinical precision therapy is required.

Organoids are three-dimensional (3D) cell clusters in vitro that contain key characteristics of an organ in vivo, including a self-organizing stem cell population that can differentiate into organ-specific cells (1). Organoids recapitulate the structure, function, and genetic signature of the original organ, thus providing a solid foundation for future research on stem cells, regenerative biology, organogenesis, precision medicine, and human pathologies (2). Organoids can be derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) through a process similar to the acquisition of their distinctive organization (1). Self-assembly and differentiation through organoid formation depend on implicated cell signaling pathways mediated by intrinsic components and the extracellular environment, including the extracellular matrix (ECM) and media. Due to a better understanding of ECM biology and technologies for cell culture (3, 4), organoid culture has been progressively implemented. Moreover, organoids have shown exciting potential and have been a popular research focus in tissue engineering and biological research in the past decade.

Intra-tumoral heterogeneity is one of the characteristics of cancers that promotes tumor evolution and makes cancer treatment challenging (5). Given that heterogeneous cancer cells and cancer-associated cells within a tumor contribute unequally to progression with complex intercellular interactions, cancer research findings using 2D cancer cell lines have been challenged. Cancer organoids retain the 3D structure of the TME, providing a physical context for molecular interactions. Recently, a cancer organoid co-culture model was developed and utilized to elucidate cell-to-cell interactions, mechanisms of cancer immune responses, and the mechanism of tumor metastasis. We summarize the history of cancer organoids that show great potential and promise in modeling human diseases. We also summarized the applications of cancer organoids in cancer research on multiple systems in the human body. Various excellent reviews have discussed the applications of cancer organoid model systems in genomic analysis and drug screening. Here, we discuss a novel cancer organoid co-culture model system with the advancement of its applications for investigating cell-cell interactions, immune response within cancers, and underlying mechanisms of cancer evolution and personalized precision medicine. We also provide further insights into novel applications and development directions of the cancer organoid co-culture model system. We hope that this review provides a better perspective to help researchers apply this practical method to cancer research and precision medicine.

An organoid is a stem cell-derived 3D cell culture that obtains the structure, collection of multiple cell types, and functionality of the corresponding tissue. Organoids are promising candidates for biomedical applications with tremendous potential and appealing prospects (6, 7). For the past few decades, numerous studies of stem cells have led to a better understanding of their behavior, with emerging methodologies of controlling stem cells’ self-organization and differentiation, which provides a scientific basis for the further establishment of organoids (7). There are two types of stem cells. One is ESCs and the other is ASCs. ESCs possess developmental totipotency and can differentiate into all cell types. ASCs are undifferentiated stem cells with the capacity to differentiate, maintain homeostasis, and regenerate in a specific organ (8). Since 1981, substantial scientific progress in stem cell research has paved the way for organoid development ( Figure 1 ). Moreover, more restricted ASCs have exhibited the capacity to form organoids in vitro once grown in the appropriate extracellular matrix and provided with specific molecular factors (9). The critical point for organoid formation and growth is whether the culture conditions duplicate the in vivo niche signaling pathways for stem cells, which contributes to sustaining stem cell functions and inducing differentiation (10).

As a novel tool and methodology, organoids have been sufficiently developed, widely studied, and adopted in various biomedical research fields (11). Organoids preserve the principal features of organ biology with greater experimental accessibility than that of animal models. Moreover, studies focusing on human embryonic and fetal tissues can be easier to conduct without restricting ethical concerns, such as prenatal development and tissue maintenance. Organoids also provide a platform to simulate the pathological environment for experiments at the organ level that cannot be performed at molecular, cellular, or animal levels. Based on this, precision medicine can be carried out using an organoid-based high-throughput screening and profiling strategy, facilitating preclinical evaluation and treatment guidance.

Organoids have been widely used to investigate neoplastic diseases, aside from being used to establish a normal developmental model. Patient-derived prostate cancer organoids were established for nearly a decade by Chen et al. (12). Recently, various cancer organoids, including colorectal cancer (CRC) (13), breast cancer (14), hepatocellular cancer (15), and non-small cell lung cancer (16), have been developed for drug screening, radiotherapy screening, genome editing, transplantation, and oncogene identification ( Table 1 ). Most cancer organoids were obtained from patient-derived cancer samples and generated under ASCs-organoid conditions, but CRISPR-Cas9 nuclease genome editing system established the minority. Yilmaz et al. established CRC organoids via CRISPR-Cas9-based APC editing with lentivirus transfection using colon organoids derived from transgenic mice (33). Few studies have focused on developing organoids using other biological samples. Gao et al. successfully established prostate cancer organoids by culturing collected circulating tumor cells (12). Moreover, cancer organoids from cells in the urine and bronchoalveolar lavage fluid have already been established, providing a novel approach for cancer organoid establishment (34, 35).

Likewise, cancer organoids contain the cancerous cell composition of the original tumors and possess the corresponding features and genetics. The generation of various cancer organoids requires distinct methods. There are no standardized culture media or procedures for experiments. Optimal tissues are commonly obtained from tumor margins with a minimal necrosis rate. Generally, the entire process is initiated by mechanical and enzymatic digestion of tumor samples into ~1 mm diameter pieces, subsequently seeding the tissue suspension onto Matrigel as a biomimetic scaffold. Matrigel, which mainly contains laminin, entactin, proteoglycans, and collagen IV, primarily contributes to the cellular architecture of organoids (36). Unlike culturing healthy organoids, media with reduced growth factors is preferred for cancer organoid culture to minimize clonal selection and avoid confounding drug treatment effects (37). Growth factors for cancer organoid culture include Wnt3A, R-spondin-1, TGF-β receptor inhibitor, epidermal growth factor, and Noggin, but the combination and concentration of these factors added to the media depend on the specific cancer type. Compared to the initiated culture process, organoid passage is a more simplified but essential process during the culture period. The issue we must be concerned with is the passage number of cancer organoids that can be used for investigations. Upon passage, the genetic material of cancer cells within organoids undergoes mutations or alterations (18, 20, 23). Consequently, the authentication of cancer organoids is particularly important for their authenticity and credibility. Although cancer organoids are preferred and widely used in cancer research, their limitations cannot be neglected. Exogenous growth factors and small molecules added to organoid growth may result in clonal selection, and the components of the culture media may interact with the tested drug, complicating the conclusion. In addition, inappropriate sample collection may significantly impact the successful culturing rate because of less active proliferating cells and increased necrosis.

As advancements have been made in cancer organoid development, cancer organoids have become a widely accepted practical model in cancer research ( Figure 2 ). Cancer organoids are primarily used in drug screening for personalized medicine approaches. The drug response of patient-derived organoids (PDOs) mainly simulates patients’ initial responses to the same agents (28, 38–40). These studies have shown that the genetic changes that drive oncogenic pathways correspond to this therapy. Therefore, drug screening is the primary part of cancer organoid applications in cancer research to screen the most effective drugs and predict their therapeutic effects. A combination of other conditions, including radiation, was added to determine the most effective therapeutic strategy. For example, the FOXM1 inhibitor thiostrepton combined with radiotherapy (4 Gy) remarkably suppresses the proliferation of meningioma organoid models (26). Such a cancer organoid model may be applied in immunoradiotherapy investigations. In addition, the transplantation model is an excellent platform for mimicking human disease.

The orthotopic transplantation of cancer organoids has been established in preclinical models. Sequencing analysis is commonly used to identify type-specific differentially expressed genes. RNA sequencing analysis and whole-exome sequencing were performed to identify cancer-related oncogenes. Moreover, as time passes, these techniques are the primary authentication methods for cancer organoids, and targeted sequencing is used to identify target mutations. CRISPR-Cas9 technology is revolutionary in genome editing and has been applied in organoid research. However, the precise integration of exogenous DNA sequences into human organoids is deficient in knock-in approaches. To address this, CRISPR-Cas9-mediated homology-independent organoid transgenesis was established to enable the efficient generation of knock-in human organoids representing different tissues (41), concluding that this technique can be used to achieve fast and efficient gene knock-in in human wild-type organoids.

Cancer organoids are now versatile tools in cancer research with a wide range of potential applications, showing the prospects of this advanced methodology in cancer research. However, there are some shortcomings. Organoids only comprise the epithelial layer without the native microenvironment of the surrounding mesenchyme, immune cells, nervous system, or muscular layer (42). Developing a novel co-culture model system of cancer organoids with other cells or organoids may recapitulate cell-cell interactions. In addition, the culture media should be improved to promote organoid growth and long-term expansion while minimizing the impact of growth factors in the media on the behavior of organoids (43). Cancer organoids have been established based on various human cancers; however, some cancer types, such as sarcomas, are still not involved. Consequently, refining culture approaches for rare heterogeneous cancer organoids and applying this tool for precision medicine should be addressed in future studies.

Co-culture is a method for culturing multiple distinct cell types, directly or indirectly, within the same culture environment (44). The cancer organoid co-culture model can efficiently simulate the environment for interactions between cancer organoids and cells within a tumor. Given that there are no immune cells, the nervous system, or mature TME in cancer organoids, a co-culture model was developed to solve this problem. The cancer organoid co-culture model was established for three main purposes. The first and most common application is to drive organoid formation via direct or indirect interactions between specific cell types within tumors. The second is the generation of specific tumor-targeting cytotoxic immune cells for cancer therapy using cancer organoids. The third is to detect the immune crosstalk between cancer organoids and specific cells, which is commonly implemented as a suspension of one cell population, usually cancer-associated fibroblasts, with the secretion of signaling factors and cytokines to condition the medium for the organoid. Marked advances in cancer organoids have been made by employing co-cultures of cancer organoids with specific cell types ( Table 2 ). Moreover, the cancer organoid co-culture system with a specific type of cell can be used for different research purposes, and the co-culture of cancer organoids with multiple types of cells may accurately mimic tumor conditions.

As a major technological breakthrough, organoids are now well-established and vigorously developed as a vital methodology in biomedical studies. Organoids have been applied in tissue engineering, regenerative medicine, disease modeling, drug screening, and toxicological studies, restoring the 3D structure and primary cell types, but also in translational applications such as the prediction of chemotherapy, radiotherapy resistance before treatment, and gene editing, enabling mutation rectification (76). Although organoids have a wide range of applications in cancer research and clinical practice, the current version is a rough model, and culture procedures for specific cancer types must be constantly standardized and improved. Organoids are found in various organs that have been created, such as the brain, retina, gastrointestinal tract, tongue, thyroid, liver, pancreas, skin, lung, kidney, and heart (7). However, obstacles impede the generation of organoids in some organs, such as bone and soft tissue engineering. Human‐periosteum‐derived cells allow the scalable generation of semiautonomous callus organoids that induce the formation of bone micro-organs upon implantation (77). For most cancers, 3D organoid models have already been established for cancer investigations with barely any technological restrictions. However, no relevant studies have investigated organoids in rare malignant tumors that are equally promising in research on these rare cancers, such as bone and soft tissue sarcomas and neuroendocrine tumors. Bone and soft tissue sarcomas are commonly characterized by chromosomal translocations with a low mutational burden. Because of the advantage of gene editing of cancer organoids, mechanism-based sarcoma organoid models may provide theoretical support for investigations focusing on elucidating sarcomagenesis and other extensive applications.

Tumor cell lines in mice and patient-derived xenografts have long been used as cancer research models and have made significant contributions. However, various shortcomings hinder using these experimental models in clinical applications. Cell lines generally contain only one type of cell without co-cultured immune cells, stromal cells, TME, or organ-specific capability, losing the genetic heterogeneity of the origin tumor after multiple passages and clonal selection. In addition, there is a lack of immune response between the original tumor and the immune environment in immunodeficient mice. Human-derived cancer cells have evolved, potentially impacting chemotherapy by reshaping the genomic landscape. Such xenograft models are highly time- and resource-intensive. Cancer organoids may also overcome the aforementioned restrictions. Genetic modification of the insertions of oncogenic mutations in stem cells leads to the generation of genetically modified organoids. Unlike patient-derived xenografts, cancer organoids are easier to obtain and establish biobanks for restoration and can be used for high-throughput drug screening. Cancer organoids based on specific cancers and even on a specific individual used in high-throughput screening are expected to become powerful tools for precision therapy. Furthermore, screening can be performed using biobanks to identify new drugs and explore new indications.

Cancer organoids have a major shortcoming: fewer immune cells and specific types of cancer-associated stromal cell organoids. A cancer organoid co-culture model system appears to address this issue ( Figure 3 ). This approach may be considered closer to what occurs within the TME (48). With regard to the co-culture of cancer-associated cells, CAFs account for most cancer organoid co-culture studies. CAFs, as essential complements of TME, have been shown to facilitate cancer progression and promote treatment resistance. CAFs contribute to treatment resistance, mainly through impaired drug delivery and biochemical signaling (61).

Given the existence of direct or indirect biochemical crosstalk between cancer cells and CAFs, the roles of CAFs in the immune response, drug resistance, and cancer proliferation must be demonstrated in specific cancer types. Although other cell types, such as BMSCs, were observed to be radiation protective through their well-known regenerative functions after ionizing radiation (65), such co-culture models with additional conditions are encouraged in cancer research for comprehensive treatment of cancer owing to the characteristics of the subtype. Co-culture of cancer organoids and immune cells can not only establish a model system to interrogate cancer sensitivity to immunotherapy for individuals at any period during treatment but also provide a clinically practical method for the creation of patient-specific T cell products for adoptive T cell transfer through expanding circulating tumor-reactive T cells by co-culture (47). NK cells, T cells, and dendritic cells were co-cultured with cancer organoids to investigate immune responses in cancer. This novel model was considered an efficient method for rapidly evaluating the effect of immune checkpoint inhibitors on activating cytotoxic lymphocytes and increasing infiltration in the context of T-cell infiltrates (46). Single-cell T-cell receptor sequencing (scTCR-seq) is another novel single-cell approach that can identify paired α- and β-TCR subunits that determine the specificity of infiltrating T cells. Association analysis of scTCR-seq with T-cell phenotypes (activation, memory, and exhaustion) and antigen specificity determination may provide more in-depth insights into cancer immunotherapy based on this novel tool.

The advanced technology of co-culturing CAR-engineered lymphocytes and cancer organoids is superior in personalized medicine owing to the maintenance of heterogeneity and the TME. CAR-engineered lymphocytes combined with organoid applications in drug tests, genome editing, and high-throughput screening will be our future research direction. However, because the therapeutic effect of CAR-T cell immunotherapy in solid tumors has not been as effective as in blood cancers owing to poor trafficking, limited persistence, limited infiltration, and T cell inhibitory activity in the TME, there has been little research on this area to date (78–80). Consequently, it has been proposed that checkpoint blockade inhibitors combined with CAR-engineered cells are a promising treatment approach for solid tumors (81–85), with a platform of organoids providing recapitulation of the environment. Recently, CAR-engineered cell therapy has been expanded to novel cell types, and the expression of CARs in NK cells has been considered a more successful variant. Cancer organoid-based co-culture systems may provide a platform for evaluating the clinical therapeutic effects of adoptive cell therapies. Moreover, genetic mutations in altered surface antigens in specific cancer cells must be identified to provide primary evidence for this methodology to achieve higher targeting and therapeutic efficiency. To broaden its applications, further research is needed to establish various co-culture model systems for organoid cancer research.

JY: Conceptualization, Investigation, Writing- Original draft preparation, SY, XL: Conceptualization, Supervision, Writing- Review and Editing. All authors contributed to the article and approved the submitted version.