Tumor Microenvironment
and Immunooncology

Solid tumors are surrounded and interact with the tumor microenvironment (TME), which is composed of extracellular matrix, fibroblasts, immune cells, lymphatic and vascular endothelial cells, and further stroma cells. Contrasting the classical tumor cell-oriented basic research, the TME has gained more and more attention in recent decades, opening new perspectives for therapeutic approaches (Balkwill et al., 2012).

Many immune cell types, such as T cells and myeloid cells (e.g., tumor-associated macrophages, TAMs), are part of the TME and have already been intensely investigated for their therapeutic suitability. Depending on the cell subtype, they can have both pro- or anti-tumor activity and be associated with a good or bad prognosis. Especially T cells harbor the potential to be used to destroy tumor cells in a patient-specific approach: T cells with engineered chimeric antigen receptors (CAR T cells) have been developed for adoptive T cell transfer therapy and showed clinical success for the treatment of B cell lymphomas.

In a healthy system, the immune checkpoints limit the immune response to prevent collateral tissue damage. However, in a tumorigenic environment, cancer cells can control these checkpoints to inhibit their own destruction by the immune system. The clinical approval of immunotherapy of specific cancer types, such as melanoma, uses an entirely new tumor treatment approach that blocks immune checkpoints (e.g., by anti-CTLA-4, anti-PD-1, or anti PD-L1) and has improved the outcome of thousands of patients. However, not all patients benefit from the immunotherapy, with some not responding at all or others gaining resistance. This implies that additional basic research is needed to evaluate a more patient-specific treatment strategy (Topalian et al., 2015; Waldman et al., 2020).

Various in vitro approaches exist for deciphering the communication and interaction mechanisms between tumor cells and their microenvironment: CAR-T cell killing assays, co-culture of cancer cells and immune cells (or other stromal cells), and rolling and adhesion assays, just to name a few, help researchers worldwide to gain knowledge of this important field of cancer research.

This image shows the three-dimensional growth of germ cell tumor cells (green, GFP) and immune cells (lymphocytes, red, mCherry) using the hanging drop technique. Cell nuclei appear in blue (DAPI). In 3D culture, a close interaction between the diverse cell types develops. The drops were transferred into an ibidi μ-Plate 96 Well. The image was acquired using a Zeiss LSM 710 confocal microscope with a 10x objective. Data by Gillian Ludwig, Daniel Nettersheim, Translational Urooncology, Department of Urology, University Hospital Düsseldorf, Germany.

Balkwill FR, Capasso M, Hagemann T. The tumor microenvironment at a glance. J Cell Sci. 2012;125(Pt 23):5591-5596. doi:10.1242/jcs.116392.
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Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015;27(4):450-461. doi:10.1016/j.ccell.2015.03.001.
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Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol. 2020;20(11):651-668. doi:10.1038/s41577-020-0306-5.
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Experimental Example: Automated Analysis of CAR-T Cell Killing Assays

CAR-T cells represent a promising new cancer therapy tool. Live cell imaging allows real-time T cell/cancer cell interaction analysis with single cell resolution. However, analysis of confluent cell layers is time-consuming and, therefore, impossible in high throughput screens. To facilitate high throughput label-free analysis of T cell potency in a live cell imaging setup, we generated arrays of homogenously distributed cancer cells using the ibidi Micropatterning technology. By combining optical analysis and advanced image processing, cytotoxic T cell activity over time on a single cell level can be evaluated without the use of any labeling.

Single Cells in a 2D Environment

Time lapse microscopy of a CAR-T cell killing assay with RCC-26 renal tumor cells and JB4 T cells on a single cell pattern (µ-Slide VI ^0.4, 20 µm squares, 120 µm distance, rectangular). Data were analyzed using FastTrack AI by MetaVi Labs.

Multi-Cell Spots in a 3D Collagen Matrix

RCC-26 renal cancer cells immobilized on multi-cell pads (µ-Slide VI ^0.4, 200 µm circles, 600 µm distance, hexagonal). Effector T cells applied in a collagen I matrix (Collagen Type I, Rat Tail) induce apoptotic body formation of cancer cells.

Selected References

3D chemotaxis assays of CD8⁺ peripheral blood T cells (PBTs) from patients with head and neck squamous cell carcinoma (HNSCC) using the µ-Slide Chemotaxis

Newton HS, Gawali VS, Chimote AA, et al. PD1 blockade enhances K⁺ channel activity, Ca2⁺ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer. J Immunother Cancer. 2020;8(2):e000844. doi:10.1136/jitc-2020-000844.
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Co-culture assay with tumor cells and macrophages in a breast cancer model using the µ-Dish ^{35 mm, high}

Sharma VP, Tang B, Wang Y, et al. Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination. Nat Commun. 2021;12(1):7300. Published 2021 Dec 15. doi:10.1038/s41467-021-27308-2.
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Co-culture assay with bone marrow-derived macrophages (BMDM) and tumor cells in the µ-Plate 96 Well Black

Luthria G, Li R, Wang S, et al. In vivo microscopy reveals macrophage polarization locally promotes coherent microtubule dynamics in migrating cancer cells. Nat Commun. 2020;11(1):3521. Published 2020 Jul 14. doi:10.1038/s41467-020-17147-y.
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