The introduction of immunotherapy and immune checkpoint inhibitors has revolutionized therapeutic approaches to treat cancer. These treatments harness the power and specificity of the immune system by potentiating the response of immune cells to tumor-specific antigens (also known as neoantigens). The discovery and approval of the first immune-checkpoint inhibitor treatments for cancer patients has paved the way to the development of additional novel immunotherapy strategies to improve patient prognosis.
For scientists on the forefront of I-O discovery, the first step in investigating the impact of immunomodulatory drugs is to develop a model system that faithfully mirrors interactions between immune cells and tumor cells.
With the advent of these new approaches to cancer therapy are conventional in vitro assays slowly becoming obsolete? Frankly, it is old news that 2D in vitro models, which are still the gold standard for oncology drug testing, do not adequately represent the tumor microenvironment (TME), which derives its hallmark characteristics from being both multicellular and 3 dimensional. The importance of testing even targeted small molecule therapeutics in the presence of a proper TME has been recognized by many labs. The TME becomes even more important for immuno-oncology (I-O) strategies, as these therapeutic agents tend to modulate interactions between multiple cell types. And with large protein-based therapeutics (> 25 Kd) tissue penetration and molecular stability, which ultimately impact drug availability, also become greater concerns.
For scientists on the forefront of I-O discovery, the first step in investigating the impact of immunomodulatory drugs to develop a physiologically relevant multicellular 3D model system that can faithfully mirror the interactions between immune cells and tumor cells. As molecular targets for new therapies expand to additional cell types, such as stromal cells and cancer stem cells, effective in vitro models will inevitably need to include these cellular components. Given that both innate and adaptive immunity play important roles in I-O therapeutic approaches, it remains to be seen whether specific T cell, B cell, dendritic and monocytic subtypes (required for both effector and suppressor functions) can be accurately modeled in vitro. Donor-matched cell types for multicellular in vitro models are considered essential by some investigators, but the actual impact of donor-mismatched cells has yet to be determined.
Despite these technical hurdles, there are strong arguments for continuing to include in vitro assays in the I-O testing tree. The ability to perform efficient in vitro I-O studies can cut down development time and costs, reduce risks and limitations associated with reliance on non-human models and tissues, as well as address some of the ethical concerns associated with in vivo animal studies.
The InSphero oncology team (and many other other cancer research groups) has been working hard to develop scalable, multicellular 3D in vitro models that contain important cell components, such as stromal fibroblasts and immune cells, suitable for I-O studies.
Informative endpoints for 3D immuno-oncology models. Histological characterization of immune-cell infiltration in 3D tumor microtissues produced from A549 tumor cells and fibroblast (human dermal fibroblasts hDF) treated with activated (a-CD3/a-CD28) and non-activated peripheral blood mononuclear cells (PBMCs). IHC markers are for proliferating cells (Ki67), apoptotic cells (Tunel); Lymphocytes (CD3); and CD45-associated molecules in T cell activation.
These 3D cell cultures can be generated in a scaffold and matrix-free system, thereby eliminating components that could limit diffusion/delivery of compounds. And unlike 2D cultures, the architecture of 3D models better recapitulates critical structural features of in vivo tumors that affect leukocyte infiltration and activation, such as ECM capsule deposition and limited exposure of target cell surface and tumor-associated antigens. 3D tumor models can also faithfully recapitulate cancer-associated metabolic changes known to crucially affect the maintenance and functionality of infiltrating immune cells.
By providing significantly improved in vivo-like conditions over standard 2D monolayer cell cultures, multicellular 3D models offer a highly improved platform to assess how immune cells elicit effective responses against tumors. The suitability of a 3D in vitro system for I-O studies can also be verified by immunohistochemistry, cytokine profiling and quantification of tumor-infiltrating immune cells.
Immunofluorescence images of immune cell infiltration in 3D tumor microtissues of HCT116 cells and fibroblast co-cultured with different grade of PBMCs stimulation-activation: naïve (1), with cytokines (2), with antibodies (3) and with Abs + cytokines (4). CD3 expressing lymphocytes are stained in red and in nuclear staining in Sytox green.
These assays can be customized to study the impact of immunomodulatory candidate drugs on specific immune cells. For example, the immune cells can be derived from HLA-matched donors, or they can consist of engineered immune cells such as CAR-T cells And with the inclusion of the proper immune cells these 3D models can be further adapted for use with immuno-activator antibodies (e.g., BiTEs).
CAR-Ts attack an A549 Tumor Microtissue in this brightfield microscopy image (well diameter = 1 mm)
With the adoption of multicellular 3D tumor models, such as 3D InSight™ Tumor Microtissues, the future of in vitro tumor for I-O therapeutics looks bright. Over the next months and years we expect to see a focus on applications that include specific populations of immune cells such an Tumor-Associated Macrophages (TAMs), specialized T cell subpopulations and even engineered immune cells that model adaptive immune responses. We also anticipate wider adoption of 3D tumor models generated from primary patient material, patient-derived xenografts and cancer stem cells.
It promises to be a very exciting time for oncology research!
Cover image: Live-cell imaging of immune cell infiltration in a 3D lung carcinoma model: A549-GFP (green) treated with immune cells (red), showing three treatment groups: no PBMCs (left), naïve PBMCs (center), and activated PBMCs (right). Visualized using a Yokogawa CQ1 High Content Analysis System. (Source: Wardwell-Swanson, J., Suzuki, M., et al. (2020) A Framework for Optimizing High Content Imaging of 3D Models for Drug Discovery. Manuscript submitted to SLAS Discovery for publication.)