2.1.1 Dual co-culture models

First, dual co-culture models allow for the investigation of specific cell–cell interactions. For example, PDTOs can assess toxicity when co-cultured with T-cells. One study co-cultured rectal cancer organoids and TILs and found that cytotoxicity was enhanced to a greater extent when TILs were embedded in the matrix than when they were added to the medium [32]. Another study reported a method for expanding and characterizing tumor-reactive T-cells using organoid culture [21, 33]. They generated tumor organoids, exposed them to interferon-gamma (IFN-γ) for 24 h, and then co-cultured them with autologous peripheral blood lymphocytes. This study demonstrated the feasibility of continuous production of patient-derived T-cell products and, surprisingly, that these tumor-specific T-cells recognized and eliminated autologous tumor organoids but not normal ones [21, 33], indicating great promise for future immunotherapies.

Furthermore, many researchers have explored and optimized co-culture methods and medium components for investigation of the killing of tumor organoids by natural killer (NK) cells [31] and T-cells [34]. For example, colon cancer organoids were embedded in Matrigel, cultured on a thin layer of Matrigel, or submerged in suspension before adding NK cells to investigate the best co-culture method. Microscopic examination revealed that effector cells migrated on the surface of the Matrigel but could not penetrate the dense extracellular matrix (ECM). After 8 h, culture on thin Matrigel increased migration of NK cells and stabilized effector-target cell interactions when compared with culture in suspension, leading to significant chimeric antigen receptor (CAR)-induced lysis [31]. This was consistent with the poor infiltration capacity of NK cells in vivo [35], which may have been attributable to the high levels of ECM and fibrous tissue surrounding the tumor. In contrast, T-cells added to the medium surrounding cholangiocarcinoma (CCA) organoids immersed in the basement membrane extract (BME) dome for 7 days destroyed the organoids without direct contact [34]. This killing may be mediated by soluble factors produced by T-cells with patient-specific specificity, revealing that T-cells can act as a killer independent of ECM, unlike NK cells [31]. As with NK cells, co-culture in a 10% BME suspension preserves 3D organoid morphology and allows direct interaction of PBMCs with organoids [34]. Furthermore, the study optimized medium conditions for PDTOs and T-cell survival and function, emphasizing that nicotinamide-free organoid medium should be used and 10% human serum should be added to a 10% BME suspension [34]. These optimization strategies have paved the way for accelerated translational research.

2.1.2 Triple and multiple co-culture models

Researchers have also established a triple co-culture scheme of tumor organoids with T-cells and other immune cells to assess toxicity. Chakrabarti and colleagues [36, 37] isolated dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) from mouse bone marrow and thymus, respectively, and cultured them with tumor organoids. They found that Hedgehog signaling induced expression of programmed cell death ligand 1 (PD-L1) and proliferation of tumor cells in gastric cancer. When pulsed DCs and CTLs were co-cultured with organoids in the presence of an anti-PD-L1 antibody, immune cells migrated to the organoids, and there was significant apoptosis only in tumor organoids. The research team then applied this co-culture model in patients [38, 39] and demonstrated that PDTO/immune cell co-cultures may be used to study the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) within the gastric TME, providing a more comprehensive model for investigating the complex roles of other cells in the TME.

A recent study described a platform that integrates patient-specific mature lymph node antigen-presenting cells into organoids without cell sorting to generate adaptive immunity [40-42]. When matched peripheral blood T-cells from patients were exposed to co-cultures, they became activated and developed memory for various heterogeneous tumor neoantigens. These trained T-cells efficiently destroyed tumor cells in naive tumor organoids from the same patient [42]. The researchers screened immune-enhanced and nonenhanced PDTOs with checkpoint inhibitors and found increased destruction of immune-enhancing organoids. When the immune-enhanced PDTO response was compared with the patient's clinical response to immunotherapy, there was a correlation in 85% of cases [40]. This platform uses immune-enriched tumor organoids to test the efficacy of immunotherapies and represents a major advance in patient-specific immune organoid culture, providing a reliable and efficient model for the development of personalized immunotherapy [40-42].

In summary, choosing the appropriate method for immuno-organoid co-culture depends on downstream assays. Short-term organoid immune cell co-culture is appropriate for therapies that function within hours. Longer co-cultures are required for therapies that produce results over time to support tumor and immune cell viability, and the time to add immune components needs to be optimized. The composition of patient-specific immune cells is complex and cannot be easily replicated. The source of exogenous immune cells can be patient-specific PBMCs or allogeneic immune cells. Donor T-cells that recognize major histocompatibility complex (MHC) molecules on organoid cells as nonself pose a challenge in allogeneic cultures, resulting in high background killing compared with autologous systems and impairing assay specificity. Autologous co-culture or human leukocyte antigen (HLA)-matched immune cells are required if T-cell antigen presentation is to be detected.

2.2.1 Patient-derived tumor fragments

The PDTF platform characterizes early antiprogrammed cell death protein 1 (PD-1)-induced immune changes in tumors by embedding small tumor fragments in a collagen and Matrigel ECM to prevent efflux of lymphocytes and treating them ex vivo with anti-PD-1 for 48 h [44, 45]. The treated fragments are then assayed for induction of cytokines, chemokines, and markers of T-cell activation, allowing for creation of an unbiased immunological score that distinguishes PDTF responders from nonresponders. This score can be retrospectively correlated with clinical outcomes in patients who receive anti-PD-1 treatment. A recent report showed 100% concordance between ex vivo PDTF immune responses and clinical outcomes [22]. This method has been used to create PDTFs from various types of cancer, including melanoma, nonsmall cell lung cancer, and breast, ovarian, and renal cell carcinoma. However, this method has some caveats, including the absence of radiographic confirmation in some cases and the possibility of heterogeneous PDTF immune responses by different tumor lesions in the same patient [22].

While PDTFs hold promise for investigating tumor biology and treatment responses, it is important to acknowledge their inherent limitations. First, challenges arise in acquiring and processing samples, with factors such as tumor location, size, and morphology potentially complicating the collection and handling of patient tumor tissue samples, thereby affecting the establishment of PDTFs. Second, there is concern regarding cell heterogeneity, because PDTFs may not fully capture the diverse nature of the patient's tumor tissue owing to possible loss of specific tumor subsets during the culture process. In conclusion, although PDTFs have advantages, researchers must carefully consider these limitations. Thoughtful experimental design and data interpretation are essential when utilizing such models, which should align with the objectives of the study.

2.2.2 Air-liquid interface culture

To fully harness the potential of organoids in tumor immune research, it is essential to investigate the feasibility of constructing an immune TME within organoids. Finnberg et al. [46] developed ALI cultures from surgical tumor specimens obtained from patients with colorectal cancer (CRC) or lung cancer. ALI culture involves mechanically dissociated tissue placed atop a type I collagen matrix within a culture well. Medium is introduced from an adjacent well through a permeable membrane, and the upper surface of the matrix interacts directly with air, facilitating diffusion of oxygen [47-49]; this likely contributes to the ability of ALI cultures to grow substantial multicellular organoids while preserving their inherent tissue architecture. Stromal cells inherently sustain organoid growth within ALI culture, eliminating the need for supplementary growth factors through the production of essential endogenous agents [47]. Finnberg et al. [46] integrated peripheral and tumor-derived immune cells into the in vitro tumor cultures to assess their ability to mimic the immunosuppressive TME. While CD45⁺ cells were readily detectable in 3D ALI cultures, a significant reduction in CD3⁺ cells was observed. Moreover, only growth of CAFs was recorded in squamous 3D ALI nonsmall cell lung cancer cultures. This is certainly an interesting development in the field of tumor immune research.

A subsequent report further optimized ALI culture [23]. ALI PDTOs can grow from various tumor sites and preserve the tumor epithelium and its matrix microenvironment in vitro for at least 30 days [23, 50]. Notably, a decrease in interstitial myofibroblasts was observed in 70% of renal and colon tumor cultures. Immune components of cells were maintained for up to 30 days in organoid cultures. The heterogeneity of T-cell receptors (TCRs) found in primary tumors was also preserved. The authors used these organoids to model immune checkpoint blockade, leading to the proliferation and activation of tumor antigen-specific T-cells and subsequent tumor killing. PDTOs offer opportunities for in vitro modeling of human immunotherapy [23]. In contrast with the tumor epithelium that can be continuously passaged and cryopreserved, the immune component of ALI PDTOs declines over time, which does not last longer than approximately 2 months despite supplementation with interleukin (IL)-2 [23].

Although the ALI model has successfully retained stromal and immune cells for at least a month in various tumors, questions remain about its transformability. Researchers have used surgically dissected samples to isolate the test population without evaluating significant parallel patient treatment controls since researchers have not been comparing their experimental setups with equivalent conditions observed in actual patients undergoing treatments. This leaves gaps in the expected correlation between simulating the TME and patient immunotherapy responses. Furthermore, ALI techniques cannot address the limitation that immune and stromal components in organoid cultures can only be maintained for a short time; this is partly because of the rapid reduction of the stromal cell bank, making long-term studies of epithelial cell-immune cell interactions difficult.

2.2.3 3D suspension culture

A recent study showed that fragments of glioblastoma from patients can be expanded into organoids using a serum, epidermal growth factor/basic fibroblast growth factor, and ECM-free medium on a 120 rpm orbital oscillator at 37°C and 5% CO₂ [20]. These patient-derived glioblastoma organoids accurately reproduce the histological features, cellular heterogeneity, gene expression patterns, and mutation profiles of their original tumors. This culture method enables the rapid and reliable generation and biobanking of glioblastoma organoids, providing an abundant resource for both basic and translational glioblastoma research [51]. Nevertheless, this suspension culture may not faithfully replicate the nutrient, oxygen, and other gradients seen in solid tumors. Furthermore, it frequently lacks the mechanical forces and physical cues inherent in solid tumors, which are factors that can profoundly impact tumor growth, behavior, and drug response. These discrepancies could potentially result in deviations from in vivo conditions.

2.2.4 Microfluidic tumor-on-chip

A limitation of organoid cultures is their lack of reproducibility owing to variations in size, shape, cell count, and geometry. To address this issue, emerging organ-on-chip technologies, particularly ToC, have been developed by combining cell biology with advanced techniques like micromachining and microfluidics, which provide a stable and reproducible platform, to improve consistency and control for enhanced use in high-throughput screening and testing [52]. The ToC platform is created by co-culturing tumor and stromal cells in a continuously perfused chamber within a 3D biomimetic matrix on a microfluidic device [53]. ECM-mimicking culture scaffolds provide structural and functional support to promote cell survival, proliferation, and differentiation [54], allowing ToC models to recapitulate key mechanobiological features of the TME for studying the collective migration and invasion of cancer cells [55]. The microfluidic device can also generate chemokine gradients for studying the chemotaxis of immune cells toward the tumor nest and distal metastasis of tumor cells [53, 56].

The combination of ToC technology, which allows PDTOs to exhibit diverse functionalities and structures corresponding to individual patients and types of cancer, along with the advantages of microfluidic technology, enables precise monitoring and control, enhancing the relevance of cancer immunotherapy screening [57]. For example, Jenkins et al. [58] have described a 3D microfluidic device for the short-term culture of mouse-derived and patient-derived organotypic tumor spheroids with preserved immune cell populations. The sensing device allows for real-time monitoring of tumor-immune interactions, delivery of checkpoint inhibitors and small-molecule drugs through microfluidic channels, and live/dead screening and cytokine analysis. Their study demonstrated ex vivo sensitivity and resistance to PD-1 blocking therapy within a short period (3–6 days), highlighting the potential for rapid evaluation of immune checkpoint blockade-based therapies in a clinical setting [58]. Another study used a microfluidic system to screen small molecules that enhance T-cell activity under inhibition of PD-1 [59]. Treatment with a CDK4/6 inhibitor increased infiltrating T-cell levels and synergized with anti-PD-1 blocking antibodies to enhance antitumor activity [59]. Moreover, by establishing an in situ breast tumor model with varying levels of PD-L1 expression, it was shown that on-chip interrogation of primary tumor responses to PD-1 as early as 10 days post-tumor inoculation could predict in vivo tumor responses to PD-1 at Day 24 [60].

In addition to recapitulating the TME, microfluidic devices can encapsulate an important aspect of the immune environment, namely, vascularization. Organoids in culture reach a finite size beyond which oxygen, nutrient, and metabolite exchange can no longer rely solely on diffusion. This leads to the formation of a central hypoxic core [61, 62]. Hypoxic cores can be observed in organoids or spheres larger than 100–200 μm in size, and necrosis occurs in organoids larger than 500 μm in diameter [63]. Therefore, achieving the vascularization of organoids remains a major challenge in terms of maintaining their complexity and scale. Vascular co-culture can be added in vitro through layer-by-layer deposition of endothelial cells or by selectively removing material to form tubular voids seeded with endothelial cells connected to the perfusion network. More complex organoids containing vascular-like structures can also be created [54, 64]. Alternatively, angiogenesis can be induced and guided by hypoxia gradients in vascular endothelial growth factor using a separate microfluidic chip for organoid-endothelial cell co-cultures [54]. Organoid and vascular co-cultures have been extensively investigated across various cancer types in academic research. For example, a microfluidic model of breast cancer has been developed to assess the effect of TME on the response to treatment and extravasation of cancer cells in breast cancer [65]. Mannino et al. [66] created a microfluidic model of diffuse large B-cell lymphoma with endothelialized blood vessels and separate lymph node lumens to mimic tumor-immune interactions as well as the vasculature. Roy's laboratory has also developed an on-chip lung cancer model containing a microvasculature and fibroblasts [67].

In summary, ToC models enhance the reproducibility of studies and recapitulate key mechanobiological features in the TME, including the vasculature, thereby improving the relevance of cancer immunotherapy screening. Nevertheless, ToC has limitations that require recognition and consideration. These include high technical complexity, significant costs, challenges in establishing large-scale tumor models owing to limited dimensions, lack of standardized protocols leading to poor reproducibility and comparability, and difficulties with long-term cultivation, which restrict comprehensive investigation of prolonged effects and dynamic changes.

3.2.1 Immune checkpoint inhibitor response in organoids

Checkpoint inhibitors are a well-studied class of immunotherapy [86]. These agents block proteins on the surface of cancer cells, helping them to evade the immune system, and have been widely investigated in PDTOs. For example, Scognamiglio et al. [90] found that PD-1/CD8-positive lymphocytes from PD-L1-positive organoids show promise as tools for immune checkpoint inhibition. However, considering that immune checkpoint inhibitors (ICIs) are not always effective, researchers have developed combination therapies to overcome immune resistance. These approaches involve targeting specific molecules within pathways and directly modulating immune cells to induce antitumor effects. Researchers have attempted to enhance the efficacy of ICIs by inhibiting pathways targeting specific molecules. For example, Della Corte et al. [91] confirmed that MEK inhibitors regulate the immune microenvironment by downregulating expression of PD-L1, enhancing expression of MHC-I on tumor cells, and increasing the production of several cytokines, including IFN-γ, IL-6, IL-1β, and tumor necrosis factor-alpha, which recruit immune cells to the tumor site, triggering a broader antitumor immune response. Another approach involves targeting Dickkopf 1 (DKK1), which is associated with tumor progression [92]. Sui et al. [93] found that DKK1 suppresses CD8⁺ T-cell antitumor immunity through the GSK3β/E2F1/T-bet axis. Neutralizing DKK1 may restore sensitivity to PD-1 blockade in DNA mismatch repair-deficient CRC [93]. Although half of PD-L1-positive gastric tumors co-express HER2, the interaction between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined [94]. Chakrabarti et al. [38] used autologous gastric cancer organoids co-cultured with CTLs and MDSCs to study this interaction. They found that inhibiting expression of HER2 decreased PD-L1 levels in organoids, increased proliferation of CTLs, and enhanced killing of tumor cells, suggesting that co-expression of HER2 and PD-L1 may contribute to tumor immune escape [38]. Koikawa et al. [95] similarly discovered that Pin1 promoted proliferation of fibroblasts and an immunosuppressive TME when indirectly co-cultured with PDAC PDOs. Furthermore, Pin1 induced lysosomal degradation of both PD-L1 and the gemcitabine transporter equilibrative nucleoside transporter 1, which helps cancer cells take in the chemotherapy drug gemcitabine and activates multiple cancer pathways. Targeting Pin1 with clinically available drugs led to complete elimination or sustained remission of invasive PDAC when performed in combination with anti-PD-1 and gemcitabine.

Recently, researchers have started work on enhancing the response to ICIs by modulating immune cells. For example, one study performed a differential transcriptome analysis of tumors with variable potential for immune escape and co-cultured tumor organoids with PBMCs91. Functional tests showed that organoids treated with ICIs and dexamethasone recruited more CD3⁺ and CD8⁺ T-cells and expressed higher levels of cleaved caspase 3 and cleaved caspase 8, suggesting that T-cells enhanced immune surveillance and highlighting the potential of dexamethasone to improve the response to ICIs [96]. Likewise, the inhibition of immunosuppression of polymorphonuclear MDSCs by the tyrosine kinase inhibitor cabozantinib sensitized gastric cancer organoids to nivolumab and mubritinib in co-cultures [39, 97].

3.2.2 Adoptive cell transfer therapy in organoids

Tumors are usually infiltrated by T lymphocytes that recognize self or mutated antigens but are usually inactive, although they often show signs of prior clonal expansion [98]. To activate TILs, Yin et al. [99] prepared nanoparticles containing innate immune stimulators that were able to induce strong activation of endogenous T-cells in PDTOs. However, this activation has often been limited, leading to the development of immune cell therapies for adoptive cell transfer therapy, including therapeutic T-cells, NK cells, DCs, and macrophages [100]. In adoptive cell transfer therapy, circulating lymphocytes or TILs are collected, selected or modified, expanded, and activated ex vivo before they are used in the patient [101].

PDTOs can be used to generate and expand tumor-specific T-cells [21, 33]. In one study, PDTOs were used to achieve antigen-specific stimulation of T-cells in the PBMC fraction [21]. CTLs expanded in co-culture and efficiently destroyed PDTOs without T-cell-mediated cytotoxicity against healthy lung organoids generated from the same patients. This study showed that cancer organoids can produce antigens that induce proliferation and stimulation of T-cells, and more importantly, be used to evaluate the tumor-specific killing efficiency of T-cells. Another study investigated circulating tumor-targeted TCRs using autologous pancreatic tumor organoids [102]. Autologous PDTOs were stimulated with T-cells obtained from patients' PBMCs for 2 weeks to generate organoid-primed T-cells. TCR sequencing revealed significant clonal expansion of T-cells with a restricted subset of TCRs. Cloning and transferring these TCRs to heterologous T-cells conferred tumor cell recognition and cytotoxic properties in a patient-specific manner. This approach facilitates the creation of tumor-specific T-cells capable of recognizing and selectively eliminating cancer cells based on individual patient characteristics. This advance holds significant promise in the realm of cancer immunotherapy, providing a cornerstone for adoptive cell transfer therapy and the potential to enhance the efficacy of treatment for cancer at the individual level.

Organoid platforms are effective in generating CAR T-cells. Seet et al. [103] created artificial thymic organoids that produce human-engineered T-cells with cancer recognition receptors and without off-target effects, which are critical for established adoptive T-cell therapies. This system is reproducible and scalable, potentially accelerating engineered development of T-cell therapy [104]. Another study reported that the pluripotent stem cell (PSC) thymic organoid (ATO) system efficiently generates functional mature T-cells from human PSCs [105]. The introduction of MHC-I-restricted TCRs in PSCs generates naive antigen-specific CD8αβ⁺ T-cells that lack endogenous TCR expression and show antitumor efficacy in vitro and in vivo. A recent study found that ATO 3D organoid cultures supported the differentiation of human CAR⁺ induced pluripotent stem cells (iPSCs) into high-functioning CAR T-cells [106, 107]. Expanded iPSC CD19-CAR T-cells, which refers to T-cells derived from iPSCs that have been engineered to express a CAR specific to CD19, a protein found on the surface of certain immune cells and cancer cells, show antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion comparable with that of conventional CD19-CAR T-cells and maintain homogeneous expression of TCRs derived from initial clones [106].

Importantly, organoids are increasingly used in CAR-related evaluations. A recent assay tested CAR NK cell-mediated cytotoxicity against cancer organoids [31]. The investigators engineered CAR NK-92 cells targeting epidermal growth factor receptor variant III, a neoantigen expressed by solid cancers [108]. They established robust assays for continuous cell-resolved analysis at the individual organoid level to monitor effector recruitment and cytotoxicity using luciferase-based endpoint measurements or in vivo microscopy [31]. Another study demonstrated that CAR T-cell-derived tumor necrosis factor is a potent antitumor effector, synergistic with Smac mimetics [109]. CAR T-cell therapy has also been evaluated in glioblastoma and bladder cancer organoids [20, 51, 110]. Glioblastoma organoids preserve the original TME, but their large volume hinders killing by CAR T-cells because they are generated from tumor tissue fragments in culture medium [111]. In contrast, bladder cancer organoids are produced from single cells and are more efficiently destroyed by CAR T-cells [110]. The blood-brain barrier hinders the distribution of antitumor drugs and immune cells, particularly in patients with nonsmall cell lung cancer and brain metastases. Similarly, Li et al. [112] demonstrated that B7-H3.CAR T-cells exhibit antitumor activity against lung cancer organoids in vitro. Co-expression of the CCR2B receptor significantly improves the ability of these cells to cross the blood-brain barrier and enhances their antitumor activity against tumor lesions in the brain [112]. Therefore, PDTOs represent a promising preclinical approach for the evaluation of patient-specific responses to CAR therapy.

3.2.3 Extending the therapeutic reach of immunotherapy in organoids

In addition to ICIs and adoptive cell therapy, several new immunotherapy regimens have shown promise. These include immunomodulatory monoclonal antibodies, bispecific antibodies, and oncolytic virus therapy.

Co-culture organoids can be used to evaluate the efficacy and mode of action of immunomodulatory monoclonal antibodies. For example, a co-culture model using exogenous immune cells and CCR PDOs identified that the NKG2D-MICA/B pathway was involved in the destruction of tumor cells [80]. After the anti-MICA/B treatment, there was an increase in expression of the inhibitory receptor NKG2A expressed by CD8⁺ T and NK cells. Thus, the combination of anti-MICA/B and anti-NKG2A has a synergistic effect.

Bispecific antibodies, which can bind two different antigens simultaneously, can enhance recognition or bring cytotoxic cells and target tumors into proximity, resulting in a cytotoxic effect in a tumor [113]. One study assessed sensitivity to the bispecific antibody cibisatamab in multidrug-resistant metastatic CRC PDOs co-cultured with allogeneic CD8⁺ T-cells [114]. Cibisatamab binds to carcinoembryonic antigen (CEA) on cancer cells and CD3 on T-cells, triggering the destruction of cancer cells expressing moderate to high levels of CEA by T-cells [114]. Live confocal microscopy was used to examine redirected killing in colon cancer, facilitating an ongoing clinical trial of cibisatamab [115]. Another study established co-cultures of high-grade serous ovarian cancer organoids with autologous immune cells treated with a bispecific anti-PD-1/PD-L1 antibody. Transcriptional analysis suggested that the increased efficacy of immunotherapy in ovarian cancer is driven by changes of state in small subsets of NK and CD8⁺ T-cells [116].

Oncolytic viruses are a newly identified class of immunotherapeutic agents with strong potential as immunostimulators [117-121]. One study showed that PDOs can be used to screen for sensitivity to oncolytic adenovirus alone or in combination with chemotherapy [122]. Another study found that the Zika virus has oncolytic activity in glioblastoma stem cells [123]. Using glioblastoma-brain cortical organoids, Zhu et al. [124] demonstrated that the Zika virus preferentially infects and destroys glioblastoma stem cells in a SOX2-dependent manner. Meanwhile, Hamdan et al. [125] designed an oncolytic adenovirus secreting a cross-hybrid Fc fusion peptide targeting PD-L1. When used in a co-culture model of renal cell carcinoma PDOs and PBMCs, the oncolytic virus triggered the effector mechanisms of IgG and IgA1, activating neutrophils and resulting in increased tumor organoid destruction. In conclusion, PDTOs are a potentially valuable tool for the evaluation of the killing efficacy of oncolytic viruses in clinical settings and for studying relevant mechanisms [126].