Mégane Jassin1
Alix Block1,2Laury Désiront1Louise Vrancken1,2
Céline Grégoire2Frédéric Baron1,2
Grégory Ehx1,3Thi Tham Nguyen1
Jo Caers1,2*- 1Laboratory of Hematology, Interdisciplinary Cluster for Applied Genoproteomics Institute (GIGA) Institute, University of Liege, Liege, Belgium
- 2Department of Hematology, University Hospital of Liege, Liege, Belgium
- 3Walloon Excellence in Life Sciences and Biotechnology (WELBIO) Department, Walloon Excellenxe in Life Research Institute, Wavre, Belgium
Chimeric Antigen Receptor T-cell (CAR-T) therapy is a revolutionary immunotherapy involving the genetic modification of T cells to express chimeric receptors targeting specific tumor antigens. Over the past decade, CAR-T therapy has significantly advanced with the development of five generations of CAR-T cells, each introducing modifications to enhance T cell efficacy, persistence, and the ability to overcome immune evasion mechanisms. The manufacturing of CAR-T cells has also evolved, employing techniques such as viral vector transduction or CRISPR-based gene editing, lipid nanoparticle, or transposon mediated approaches, to optimize their function. However, the development of CAR-T therapy for solid tumors faces significant challenges, primarily due to the hostile tumor microenvironment (TME), which traditional two-dimensional (2D) culture systems fail to accurately replicate. This review explores the potential of three-dimensional (3D) culture models, including spheroids and organoids, as tools for studying CAR-T cells in the context of solid tumors. Unlike 2D models, 3D systems offer a more physiologically relevant environment, better mimicking the TME, tumor heterogeneity, and immune interactions which CAR-T cells must encounter. We examine the advantages and limitations of 2D versus 3D models and discuss four key methods for generating spheroids/organoids: direct cell aggregation, scaffold-based, microfluidic, organs-on-chip and bioprinting, and patient-derived organotypic tumor approaches. Moreover, we explore the use of murine models in preclinical CAR-T research, highlighting their role in studying the dynamics of CAR-T cell trafficking, efficacy, and off-target effects. While CAR-T therapy has shown impressive success in some hematological malignancies, there is still a critical need for improved models to study CAR-T efficacy against solid tumors, particularly in relation to the TME. 2D models remain a valuable tool but should be combined with 3D models and in vivo murine studies for more accurate clinical outcome predictions. As we advance toward preclinical and clinical applications, ongoing efforts to develop and refine 3D culture systems are essential for overcoming the unique challenges of CAR-T therapy in solid tumors.
1 Introduction to CAR-T therapy
1.1 CAR-T cells: from concept to clinical result
A pioneering form of immunotherapy has emerged for cancer treatment: Chimeric Antigen Receptor T-cell (CAR-T) therapy. This highly innovative approach involves genetically modifying a patient’s T cells to express chimeric antigen receptors (CAR) on their surface. These receptors are engineered to specifically recognize antigens present on the surface of tumor cells. After modification, CAR-T cells are reinfused into the patient, where they target and eliminate tumor cells. The concept of leveraging T cells to combat cancer was first pioneered by Steven Rosenberg and colleagues in 1988, when they treated metastatic melanoma patients with tumor-infiltrating T lymphocytes (TILs) isolated from the tumor, expanded ex vivo and reintroduced into patients (1). Moreover, lifileucel, a TIL based therapy was approved in 2024 for metastatic melanoma treatment (2). By creating the first generation of CAR-T cells termed « T-bodies », Zelig Eshhar and his colleagues made significant progress in the field in 1993 (3). CAR-T cells demonstrated impressive outcomes in treating hematological malignancies within the past ten years. In 2017, the U.S. Food and Drug Administration (FDA) approved Kymriah (tisagenlecleucel), the first CAR-T treatment for refractory B-cell acute lymphoblastic leukemia (ALL), (4). It was followed by other CAR-T therapies for lymphomas such as TECARTUS (brexucabtagene autoleulcel), (5) for refractory mantle cell lymphoma and ALL, YESCARTA (axicabtagene ciloleucel), (6) for refractory large B-cell (LBCL) and follicular lymphomas, BREYANZI (lisocabtagene maraleucel, 7) for refractory LBCL and chronic lymphocytic leukemia (CLL), and for refractory multiple myeloma (MM) such as ABCMA (idecabtagene vicleucel), (8) and CARVYKTI (ciltacabtagene autoleulcel, 9). Although ongoing efforts to develop CAR-T therapies for solid tumors, and overcome numerous obstacles related to the hostile tumor microenvironment (TME), further progress is needed before these therapies can reach clinical approval.
1.2 CAR-T structure
Over the years, CAR designs have rapidly improved, resulting in the development of five distinct generations (Figure 1). Each generation has added unique characteristics to enhance the efficacy and persistence of CAR-T cells (10, 11). These developments have significantly improved the intrinsic CAR-T cell activity and enhanced the ability to overcome immune evasion of cancer cells and challenges occurring within the hostile TME. While preclinical and early data are promising (12, 13), none of these constructs have yet received FDA approval, excepted CAR-Ts from the second generation. The first-generation CAR construct consists of an extracellular antigen-recognition domain linked to the intracellular CD3ζ signaling domain. In the second-generation construct, a co-stimulatory domain (CD28 or 4-1BB) is added alongside CD3ζ to enhance T cell activation and proliferation. The third-generation CAR construct is similar to the second one but incorporates more than one co-stimulatory domain to enhance CAR T cell activity and persistence. The fourth-generation CAR construct, also called T-cells redirected for universal cytokine killing (TRUCK), is designed to secrete cytokines upon activation to modulate the TME, attract innate immune cells, and improve antitumor efficacy (14). The fifth-generation CAR construct combines a STAT3-binding motif between the co-stimulatory domain and the CD3ζ signaling domain with a truncated cytoplasmic region of the IL-2 receptor β-chain. Without requiring external cytokine support, this design allows for antigen-dependent activation of the JAK-STAT pathway to enhance T cell proliferation and persistence (15).
Figure 1. Schematic representation of the five distinct generations of CAR. The first-generation CAR construct consists of an extracellular antigen-recognition domain linked to the intracellular CD3ζ signaling domain. The second-generation contains a co-stimulatory domain (CD28 or 4-1BB) added alongside CD3ζ. The third-generation CAR incorporates more than one co-stimulatory domain. The fourth-generation secretes cytokines upon activation. The fifth-generation CAR incorporates a truncated cytoplasmic domain of the IL-2 receptor β-chain, coupled with a STAT3-binding motif between the co-stimulatory domain and the CD3ζ signaling domain to activate the JAK-STAT. This figure was adapted and generated on Biorender.
1.3 CAR-T manufacturing
In addition to design improvement, CAR-T manufacturing has been enriched by several innovations such as CRISPR-Cas9 gene editing to remove immune checkpoint inhibitors such as LAG-3, PD-1 or TIM-3 and enhance resistance to exhaustion and antitumor efficacy (16–18), adenoviral transduction providing efficient gene expression without genome integration and low risk of mutagenesis but offering a short-lived expression (19), retroviral transduction with stable gene integration for a long-term expression but limited to dividing cells with a risk of insertional mutagenesis (19, 20), and lentiviral transduction with stable gene integration for a long-term expression on a broader cell targeting range with lower risk of insertional mutagenesis but with high cost and manufacturing complexity (19). There are also mRNA electroporation, lipidic nanoparticles and transposon-mediated approaches (Figure 2) (21, 22). Moreover, CAR-T cells can be manufactured either ex vivo by extracting cells from the patient, engineering them, and reinfusing back or in vivo by directly injecting the CAR construct into the patient’s body (23). While in vivo CAR-T therapies represent a promising alternative to conventional ex vivo approaches, the first clinical trials have recently begun, and their efficacy and safety remain to be fully established. This is in contrast with second-generation CAR-T therapies produced ex vivo, some of which have already been approved by the FDA. Despite these advances, challenges remain such as immunosuppressive TME, tumor infiltration, antigen downregulation, and CAR-T cell expansion and persistence issues still arise in preclinical and clinical trials. Although results from in vitro characterization could differ from those obtained in in vivo mouse models and do not always predict outcomes in human clinical trials. Therefore, three-dimensional (3D) co-culture systems appear as attractive intermediate systems between in vitro and in vivo models to provide more physiologically relevant models for rapid assessment of CAR-T efficacy.
Figure 2. CAR-T manufacturing processes. The first method to generate CAR-T cells is the CRISPR-Cas9 genome editing followed by viral transduction or the direct adenoviral, retroviral or lentiviral transduction of the CAR sequence (I). The second method includes the CAR construct delivered using lipid nanoparticles (II). The third method involves mRNA electroporation transposon-mediated approaches to integrate the CAR sequence into T cells (III). This figure was generated on Biorender.
2 Advantages and limitations of two-dimensional co-culture systems in CAR-T research
In CAR-T cell research two-dimensional (2D) co-culture systems have served as essential and fundamental tools for preliminary mechanistic investigations and rapid testing of CAR constructs. These systems consist of mixing CAR-T cells directly with tumor cells without any extracellular matrix support, allowing direct cell-cell interactions in a simplified flat 2D environment. While convenient and cost-effective, 2D models remain limited in recapitulating physiological conditions of a complex TME, as they lack crucial immunosuppressive factors, stromal components, and spatial architecture. Thus, results derived from these co-culture systems may be less predictive of in vivo responses, limiting their clinical relevance (24). To address these limitations, in vivo models such as NOD scid gamma (NSG) and Patient-Derived Xenograft (PDX) provide a TME that better mimics disease conditions such as hypoxia, nutrient gradients and stromal interactions. However, these models still lack host immune interactions due to the use of immunodeficient mice, thereby limiting their ability to fully replicate the immune dynamics observed in patients. Additionally, in vivo models remain expensive, time-consuming, and limited by ethical barriers with 3R regulations (Replacement, Reduction and Refinement). Given these challenges, the use of more complex models such as a 3D co-culture and organ-on-chip are in high demand and represent attractive approaches. 3D culture systems incorporate extracellular matrix components, tumor spheroids or organoids, providing a more realistic representation of tumor architecture and cell-cell interactions (25). Organ-on-chip technologies integrate microfluidic systems to recreate tissue-like environments, enabling dynamic perfusion and immune-tumor interactions. These 3D co-culture systems can potentially offer more predictive insights for advancing CAR-T research with conventional 2D and in vivo systems (26).
2.1 Advantages of 2D models
There are some advantages of using 2D co-culture systems (Table 1), such as the ease of use and reproducibility, because these systems are straightforward to establish and follow standardized protocols. Another advantage is the real-time observation because the single-layer arrangement of cells enables direct assessment of CAR-T activity, including cell adhesion, tumor cell lysis and proliferation, through techniques like microscopy and flow cytometry. The absence of a TME enables the targeted study of key parameters such as cytokine production, cytotoxicity, and CAR-T cell proliferation without interference from external factors. Moreover, the 2D cultures can easily integrate a variety of analytical techniques, including ELISA, flow cytometry, lactate dehydrogenase (LDH), or calcium flux assays, facilitating a comprehensive evaluation of CAR-T function. Finally, 2D models remain accessible for widespread use because of their lower cost and minimal specialized equipment required compared to in vivo or preclinical research.
Table 1. Advantages and limitations of 2D culture assay.
2.2 Limitations of 2D models
Nevertheless, 2D co-culture systems have several limitations (Table 1), such as their inability to recreate a realistic TME. This is due to their failure to replicate oxygen and nutrient gradients as well as the complex interactions between tumor cells and extracellular matrix (ECM). Importantly, the 2D co-culture systems lack the ability to simulate immunosuppressive elements of the TME, such as cancer-associated fibroblasts (CAFs), M2 macrophages, and regulatory T cells (Tregs), which are critical obstacles to solid tumor therapy. The oversimplification can lead to an overestimation of CAR-T efficacy (27). Tumor cells in 2D models use cell lines, naturally expressing the target antigen, which may cause a loss of their native tumor phenotype and lead to false-positive results regarding CAR-T efficacy (28). Moreover, 2D culture seems not suitable for some primary cells, such as primary CLL and MM cells for hematological malignancies or primary pancreatic ductal adenocarcinoma (PDAC) which undergo rapid apoptosis or senescence in vitro, and the use of cell lines does not fully recapitulate the biology of the disease. However, cultivating primary cells in 3D models might provide the right microenvironment to support cancer cell survival, making it possible to study CAR-T cell activity for a longer time than in 2D cocultures. Furthermore, in a physiological context, CAR-T cells must overcome physical and biochemical barriers before interacting with tumors cells. This complexity is not adequately represented in 2D models, where CAR-T cells directly interact with tumor cells (29). Thus, 2D systems do not capture dynamics like CAR-T cell migration, penetration into dense tumor tissues, or exhaustion in hypoxic, poorly vascularized environments, and fail to include essential stromal and vascular components, which significantly influence tumor growth, immune evasion, and CAR-T cell functionality (28). Because of their simplicity to use, low cost and analytical compatibility, 2D culture systems remain indispensable for initial CAR-T cell research. However, their inability at accurately replicating the complexities of the TME highlights the necessity to switch to more physiologically relevant models. To fill these gaps, researchers increasingly depend on 3D co-culture systems to provide a transitional step between basic in vitro studies and in vivo validation. This allows for a more accurate evaluation of CAR-T behavior and efficacy in realistic tumor environments (Figure 3).
Figure 3. Comparison of 2D vs. 3D co-culture system. In a 2D co-culture system containing a monolayer of tumor cells and fibroblasts, CAR-T cells can interact directly and this could be observed in real-time while evaluating cytokines production, cytotoxicity and proliferation without external factors. However, this system does not represent the microenvironment and the migration of tumor cells or CAR-T cells cannot be followed according to gradient of oxygen or nutrients. In a 3D co-culture system, CAR-T migration, infiltration, cell-cell-interaction and tumor killing can be monitored between the several layers of cells with or without gradients of oxygen and nutrients. This figure was generated on Biorender.
3 Three-dimensional co-culture system: a bridge between 2D and in vivo studies
The introduction of 3D cell culture systems marked a significant advancement in research, offering a more physiologically relevant model than traditional 2D cultures (Table 2). By enabling spatially organized cell interactions, these 3D systems better mimic the natural tissue microenvironment, which is essential for studying complex processes like cancer progression, immune responses, and tissue regeneration. In the context of solid tumors, 3D co-culture systems provide valuable insights into CAR-T cell interactions with the TME, revealing key challenges such as tumor resistance, immune evasion, and infiltration limitations. This approach enhances the evaluation of therapeutic outcomes and helps refine CAR-T cell strategies for improved efficacy in solid tumors (28, 30). In 1956, Ehrmann and Gey replaced the flat-surface cell culture method with the culture of human cell lines in rat tail-isolated collagen to promote cell aggregations in 3D (31). However, the term « spheroid » emerged in 1970 to describe Rheinwald and Green 3D cell culture using human progenitor cells where cells aggregate as a sphere (32). In 1980’s and 1990’s, they further developed in vitro cultures from neuroblastomas and lung tissues considered to as the first organoids, a self-organizing structure derived from stem cells, mimicking key features of an organ. Indeed, spheroids are related to tissue architecture whereas organoids are linked to organ architecture because cells spontaneously organize themselves to form a structure similar to the organ they derived from (33). Moreover, organoids can be derived from spheroids (34), (Figure 4). Finally, Rheinwald and Green’s further investigated 3D culture and started to cultivate skin organoids from human primary cells (35, 36).
Table 2. Comparison between 2D and 3D co-culture systems for CAR-T research.
Figure 4. Differences between monolayer 2D cultures, spheroids and organoids. Monolayer cultures consist of single cell type or multiple cell types while spheroids and organoids are composed of multiple cell types. Additionally, organoid structure requires at least epithelial or mesenchymal cells. The architecture complexity increases from monolayers (flat structure) to spheroids (tissue-like aggregates) and organoids (organ-like structures). Furthermore, organoids are composed of monolayers or spheroids composed of monolayers. This figure was generated on Biorender.
4 Strategies for culturing spheroids and organoids including CAR-T cells
Three-dimensional culture systems offer more physiologically relevant models for studying CAR-T cells in solid tumors where they face challenges such as immune suppression and poor tissue penetration. Unlike hematological cancers, in solid cancers, CAR-T cells must infiltrate dense extracellular matrices and pass through multiple barriers created by stromal cells and deposited matrix proteins, making in vitro 3D models essential for in vivo studies or preclinical evaluation. This section highlights strategies to generate spheroid and organoid cultures and integrate CAR-T cells to investigate their efficacy in solid tumor therapy (Table 3).
Table 3. Strategies to produce spheroids or organoids while studying CAR-T cells in solid tumors.
4.1 Cell aggregation
The first method for generating spheroids is direct formation by letting cells aggregate spontaneously together in a flat-bottom plate such as the liquid overlay technique, or in a U-bottom plate such as the hanging drop spheroid (22, 37–41). In these techniques, cell aggregation can be facilitated using low or ultra-low attachment plates coated to prevent cell adherence to the plastic surface. This coating promotes cells to interact with each other, forming small spherical structures (42–51). Additionally, 1% of agarose can be added to the plate bottom to maintain the stemness of cells when necessary, as seen in organoid formation using stem-cell-derived beta cells using plate shaking techniques (37, 38, 52),. Alternatively, direct spheroid formation can be fostered by adding specific molecules and factors to mimic the TME and promote cell growth and aggregation. For instance, for brain cancer and glioblastoma models, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor (IGF) are used along with neurobasal medium supplemented with B27 or N2 to replicate brain tumor signaling and support neural cell growth (Figure 5; 51, 53),. For CAR-T cell studies, CAR-T cells are subsequently introduced into the established 3D models using different strategies, depending on the research goals. For instance, to study CAR-T cell-mediated cytotoxicity, CAR-T cells can be added directly with tumor cells for co-culture into the spheroid during the initial aggregation phase. Alternatively, to investigate infiltration dynamics, CAR-T cells can be introduced after spheroid formation.
Figure 5. Schematic representation of direct spheroid formation. First method is the liquid overlay technique where cells aggregate together spontaneously to form a spheroid after being seeded. Second method is the hanging drop technique using a U-bottom well to facilitate natural aggregation. Third method is the same as the first one while cells are seeded by growth factors. Fourth method is the use of ultra-low or -low attachment plate with a specific coatting to avoid cell adherence to plastic. This figure was generated on Biorender.
4.2 Using matrix and scaffold
The second method for producing spheroids or organoids is to use a matrix as a scaffold (Figure 6). There are several matrices available such as matrigel (54–63), hydrogel (64–73), and collagen (74–76) from rat or mouse tails. In this model, cells are cultured in the matrix as in 3D space instead of monolayer. Another kind of matrix is the ECM derived from tissues and organs. ECM can provide a more natural microenvironment compared to synthetic hydrogels, such as polyethylene glycol (PEG) or poly-lactic-co-glycolic acid (PLGA), which facilitate the creation of more physiologically and biochemically relevant models for spheroid and organoid formation (54, 60, 66, 69), (73), (75),,– (77). Another way is to produce the spheroid using a low-attachment plate before transferring it into a gel bubble to let it grow in 3D (55, 56),. CAR-T cells can be directly added into pre-formed spheroids or organoids in the matrix or into the culture medium surrounding the spheroid/organoid (65, 67),. To investigate the early interactions with tumor cell aggregates, or CAR-T activation and tumor killing (72), CAR-T cells can be co-cultured before or during the formation of the spheroids/organoids. On the other hand, to evaluate CAR-T infiltration, migration as well as killing tumor cells, CAR-T cells can also be added after the spheroid/organoid formation as a well-established tumor structure. This method mimics the conditions of treating an existing tumor rather than targeting cells in the early stages of aggregation.
Figure 6. Schematic representation of using matrix coupled with microfluidic, organ-on-chip and bioprinting. Microfluidic device is composed of different channels, one to culture the spheroid or organoid with CAR-T cells while other channels can bring oxygen, nutrients, chemotaxis molecules or growth factors or can mimick blood vessels. Channels can be coupled with filters to remove debris and dead cells. This figure was generated on Biorender.
4.3 Advanced techniques: microfluidic, organs-on-chip, or bioprinting technologies
The third method for creating spheroids and organoids involves microfluidic, organs-on-chip, or bioprinting technologies (Figure 6). Several devices and chips have been engineered to deliver oxygen or nutrients, regulate and study chemotaxis (78, 79), mimic blood vessels and vascularization (80), remove dead cells and deliver CAR-T cells using microdroplets (81) or microhydrogels (45, 82, 83),. In these approaches, spheroids, organoids, or biopsy-derived cells are cultured on a matrix and connected to microfluidic systems (78, 84). Similar to microfluidic systems, organ-on-chip provides precise regulation of biochemical and physical microenvironments, enabling accurate modeling of tumor microenvironment complexity and CAR-T cell behavior. Additionally, organ-on-chip systems simulate blood flow to allow the investigation of CAR-T cell infiltration and interactions with endothelial barriers, which is critical for studying solid tumors (82, 84–86). Using multiple channels, these devices allow simultaneous testing on various conditions, such as cytokine gradients or different CAR construct injections (86). Most recently, bioprinting has emerged as a cutting-edge technology offering precise tissue construction (87). Thus, these technologies facilitate the 3D dynamic studies of CAR-T cell migration, proliferation, and cytotoxicity under well-controlled parameters, giving kinetic results that static cultures cannot achieve.
4.4 Patient-derived organotypic tumor or tumor slice culture assays
The fourth method is patient-derived organotypic tumor or tumor slice culture assays (Figure 7). This technique is related to ex vivo studies, where human tumors are extracted from the patient and cultured on a matrix (21, 88–94). Depending on the size of the tumor and the microenvironment components extracted, the tumor can be cultured whole or sliced into thin sections (93). These cultures can also be connected to a microfluidic system to supply medium, and nutrients or to facilitate CAR-T cell injection (95). Alternatively, CAR-T cells can be introduced using similar methods to those described in the second method of organoid manufacturing.
Figure 7. Schematic representation of patient-derived organotypic tumor preparation. Solid tumors are extracted from human patient organ biopsies and cultured on a matrix. Extracted tumor can be sliced depending on their size and can be cultured as organoids or spheroids before injecting CAR-T cells. This figure was generated on Biorender.
4.5 Methods to study CAR-T cells in a 3D co-culture system
As with 2D analysis, several methods can be used to study CAR-T cell behavior in a 3D co-culture system. Indeed, confocal microscopy and live-cell imaging are used to track CAR-T activity such as the infiltration into the spheroid/organoid, the spatial distribution of tumor and CAR-T cells, CAR-T migration, and cytotoxicity. To track CAR-T and tumor cells in the spheroid/organoid, cells are labelled with a fluorescent molecule or a reporter system. Flow cytometry can also be used after the dissociation of the co-culture system into single-cell suspensions to further analyze CAR-T cell activity, cytotoxicity, persistence, expansion and proliferation, differentiation phenotype, and exhaustion. Coupled with flow cytometry, ELISAs can be employed to analyze release cytokine production for CAR-T cells such as TFN-α, IFNγ, and IL-2. Viability assays using LDH release assays or live-dead staining can be used to quantify tumor cell damage or death before and after CAR-T treatment. Finally, compared to 2D co-culture systems, histological analysis can be applied to fixed spheroids/organoids using immunohistochemistry (IHC) or immunofluorescence to detect tumor killing or apoptosis, CAR-T cell infiltration and phenotype (97).
5 From mouse to clinic: murine models driving preclinical progress
In vivo research provides key insights into the physiological complexities of cancer treatments and acts as an essential link between in vitro studies and human clinical trials. For CAR-T cell research, murine models are the most commonly used, enabling researchers to assess the efficacy, safety, and pharmacokinetics of treatments in a living organism, and dynamics, including their migration, proliferation, and cytotoxic effects in the context of a systemic environment where cancer may be disseminated. CAR-T cell interactions that are missing in 3D culture systems are typically represented in these models, including interactions with the broader biological system, such as blood vessels, and distant tissues to evaluate potential off-target effects and toxicity in non-tumor tissues. In vivo studies allow researchers to investigate systemic interactions such as immune responses, biodistribution, and off-target effects on healthy tissues in the context of a fully functional organism. However, studying immune-mediated processes is limited by the use of immunodeficient mice. Moreover, in vivo experiments remain expensive, time-consuming, resource-intensive, and subject to ethical restrictions. Despite these challenges, in vivo models offer a global insight of therapeutic effects in a living dynamic biological organism. Depending on different cancer models, tumor cells can be injected subcutaneously using matrigel or not [e.g. melanoma (98), breast cancer (99), prostate cancer (100), rhabdomyosarcoma (101), mesothelin (102)], intravenously in the blood-stream [e.g. hematological malignancies (103–105)], intraperitoneally [e.g. ovarian cancer (106)] or orthotopically in the organ of origin [e.g. breast cancer (99), glioblastoma (91), lung cancer (107), glioma (108)]. Furthermore, murine models are also used to evaluate procedures commonly used in CAR-T therapies such as isolation of T cells, their genetical engineering to express the CAR, and their expansion before reinfusion. Moreover, CAR-T cells can be administered either directly into the tumor to study local effects or intravenously to observe their migration, tumor-homing behavior, and systemic activity. Compared to preclinical or clinical trials on human patients, there are some limitations of murine models such as the use of murine cells, or xenografted human cells injected in immunocompromised mice lacking some immune cells, and the issue of lack of interaction between human and murine cells or microenvironment or cytokines. Nevertheless, despite these limitations, murine models remain an invaluable tool for preclinical research, providing essential insights into CAR-T cells potential efficacy and safety before advancing to human clinical trials.
6 Conclusion
CAR-T cell therapy has revolutionized the cancer immunotherapy landscape. However, despite its remarkable successes in clinics in lymphoid malignancies, even if there are still some major challenges in myeloid malignancies, there are still challenges remaining as for solid tumors, including consistent antigen expression across all tumor cells, antigen loss or mutation, or other resistance mechanisms to immune cells. This review focused on the need to use robust 3D in vitro culture systems to study CAR-T therapy in solid tumors or in bone marrow microenvironment to develop CAR-T cell therapy for hematological malignancies such as acute myeloid leukemia.
2D co-culture systems provide useful initial insights into CAR-T cell biology, such as cytotoxicity, proliferation and cytokine production. The advantages regarding simplicity, low cost and real-time observation make 2D co-culture an indispensable tool for preliminary studies. However, their limitations in recapitulating the TME, including immunosuppressive elements, hypoxia, and extracellular matrix barriers reduce their predictive value for in vivo assays or clinical efficacy (109, 110). Thus, 3D culture systems have emerged as essential intermediate models to mimic some of the structural and biochemical complexities of tumors, enabling more accurate and rapid assessments of CAR-T cell infiltration, migration, killing capacity and exhaustion. Moreover, 3D models might be a suitable strategy to evaluate advanced designed CAR constructs; such as TRUCKs from the fourth generation, designed to secrete cytokines upon activation to modulate the TME, or CAR from the fifth generation. Several strategies to generate 3D spheroids and organoids have been developed. Direct cell aggregation techniques using low- or ultra-low-attachment plates are straightforward without any matrix. Matrix-based approaches incorporate extracellular matrix components and can be combined with microfluidic and organ-on-chip technologies, which allow precise control over dynamic parameters such as nutrient gradients and blood flow. Moreover, the use of patient-derived organotypic tumor models, incorporating components of the TME, provides a more accurate and physiologically relevant recapitulation of the TME for studying therapeutic responses.
The transition from 3D culture systems to in vivo murine models remain a critical step for preclinical validation. Murine models better capture the complexity of the disease model, tumor microenvironment, and tumor-immune cell dynamics compared to in vitro studies. However, the commonly used immunodeficient mice lack host immune cells and, therefore do not recapitulate tumor-immune interaction in absence of co-transplantation of PBMCs. Improvements and a combination of humanized mouse models and organ-on-chip technologies mimicking vascularized tumors may overcome differences between in vitro studies and clinical trials. However, despite all these advances, tumor immune escape mechanisms, antigen heterogeneity and the immunosuppressive TME remain limited in these models for assessment of CAR-T cell efficacy.
In conclusion, CAR-T cell therapy represents a pioneering approach to treat hematological malignancies, yet its success in solid tumors in clinical trials will require continued advancements in cell engineering, manufacturing, and modeling systems. Combining 2D and 3D culture systems with in vivo studies allows researchers to overview and evaluate CAR-T functionality. Ultimately, integrating these models with emerging technologies (microfluidic, bioprinting, organ-on-chip, etc.) might lead to more effective and durable CAR-T cell therapies for solid malignancies.
Author contributions
MJ: Conceptualization, Data curation, Investigation, Writing – original draft. AB: Conceptualization, Data curation, Writing – review & editing. LD: Data curation, Investigation, Writing – review & editing. LV: Writing – review & editing. CG: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. FB: Supervision, Writing – review & editing. GE: Supervision, Writing – review & editing, Methodology, Validation. TN: Methodology, Supervision, Writing – review & editing. JC: Conceptualization, Supervision, Validation, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. The laboratory of Haematology is supported by the Foundation Against Cancer, the Intergroup Francophone du Myélome, the Fonds National de la Recherche Scientifique, (FNRS, Belgium), Télévie-FNRS and the Fonds Spéciaux de la Recherche (University of Liège).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision
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The author(s) declare that no Generative AI was used in the creation of this manuscript.
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Glossary
CAR-T: Chimeric Antigen Receptor T-cell
TME: Tumor Microenvironment
2D: Two-Dimensional
3D: Three-Dimensional
CAR: Chimeric Antigen Receptor
TIL: Tumor-Infiltrating T Lymphocyte
FDA: Food and Drug Administration
ALL: Acute Lymphoblastic Leukemia
LBCL: Large B-Cell Lymphoma
CLL: Chronic Lymphocytic Leukemia
MM: Multiple Myeloma
TRUCK: T-Cells Redirected for Universal Cytokine Killing
NSG: NOD Scid Gamma
PDX: Patient-Derived Xenograft
LDH: Lactate Dehydrogenase
ECM: Extracellular Matrix
CAF: Cancer-Associated Fibroblast
Treg: Regulatory T cell
PDAC: Primary Pancreatic Ductal Adenocarcinoma
EGF: Epidermal Growth Factor
bFGF: Basic Fibroblast Growth Factor
IGF: Insulin-Like Growth Factor
PEG: Polyethylene Glycol
PLGA: Poly-Lactic-Co-Glycolic Acid
VEGFR2: Vascular Endothelial Growth Factor Receptor 2
HER2: Human Epidermal Growth Factor Receptor 2
FRα: Folate Receptor Alpha
TAG-72: Tumor-Associated Glycoprotein 72
CD98hc: Cluster of Differenciation 98 Heavy Chain
B7-H3: B7 Homolog 3
HLA-A*02: Human Leukocyte Antigen A*02
CD19: Cluster of Differenciation 19
CD44: Cluster of Differenciation 44
EGFR: Epidermal Growth Factor Receptor
DCLK1: Doublecotrin-Like Kinase 1
CII: Collagenase II
MET: Mesenchymal-Epithelial Transition
NKG2D: Natural Killer Group 2 Member D
CD39: Cluster of Differenciation 39
CEA: Carcinoembryonic Antigen
CXCR3: C-X-C Chemokine Receptor Type 3
ROR1: Receptor Tyrosine Kinase-Like Orphan Receptor 1
TROP2: Trophoblast Cell Surface Antigen 2
EpCAM: Epithelial Cell Adhesion Molecule
IL13Rα2: Interleukin 13 Receptor Alpha 2
EphA3: Ephrin Type-A Receptor 3
P2X7: Purinergic Receptor P2X
CAIX: Carbonic Anhydrase IX
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Keywords: car-t, chimeric antigen receptor T cells, solid tumor, 3D culture, tumor microenvironment, spheroid, organoid, immunotherapy
Citation: Jassin M, Block A, Désiront L, Vrancken L, Grégoire C, Baron F, Ehx G, Nguyen TT and Caers J (2025) From spheroids to organoids: next-generation models for CAR-T cell therapy research in solid tumors. Front. Immunol. 16:1626369. doi: 10.3389/fimmu.2025.1626369
Received: 10 May 2025; Accepted: 23 June 2025;
Published: 11 July 2025.
Edited by:
Boris Gole, University of Maribor, SloveniaReviewed by:
Robert H Carnahan, Vanderbilt University Medical Center, United StatesLidija Gradisnik, University of Maribor, Slovenia
Copyright © 2025 Jassin, Block, Désiront, Vrancken, Grégoire, Baron, Ehx, Nguyen and Caers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jo Caers, Sm8uQ2FlcnNAY2h1bGllZ2UuYmU=