Colorectal Cancer and Immunity: From the Wet Lab to Individuals

Abstract

Immunotherapy is a very promising field of research and application for treating cancers, in particular for those that are resistant to chemotherapeutics. Immunotherapy aims at enhancing immune cell activation to increase tumor cells recognition and killing. However, some specific cancer types, such as colorectal cancer (CRC), are less responsive than others to the current immunotherapies. Intrinsic resistance can be mediated by the development of an immuno-suppressive environment in CRC. The mutational status of cancer cells also plays a role in this process. CRC can indeed be distinguished in two main subtypes. Microsatellite instable (MSI) tumors show a hyper-mutable phenotype caused by the deficiency of the DNA mismatch repair machinery (MMR) while microsatellite stable (MSS) tumors show a comparatively more "stable" mutational phenotype. Several studies demonstrated that MSI CRC generally display good prognoses for patients and immunotherapy is considered as a therapeutic option for this type of tumors. On the contrary, MSS metastatic CRC usually presents a worse prognosis and is not responsive to immunotherapy. According to this, developing new and innovative models for studying CRC response towards immune targeted therapies has become essential in the last years. Herein, we review the in vitro and in vivo models used for research in the field of immunotherapy applied to colorectal cancer.

Keywords: colorectal cancer; immunotherapy; methods.

Figure 1

Schematic representation of the 2-dimensional (2D) co-culture methods that are suitable for in vitro study of immunotherapies. The in vitro 2D co-cultures using commercialized cell lines are a first approach to evaluate the activation, migration, or cytotoxic potential of immune cells following an immunomodulatory treatment because of its simplicity to set up, its low cost, and its reproducibility. Indirect co-culture consists of the transfer of conditioned medium from one cell to another. This allows the effects of soluble factors on immune cells biology to be studied. The indirect co-culture method using the Transwell assay allows the study of the migratory capacity of immune cells in the presence of tumor-derived conditioned media. Finally, the direct co-culture assay permits cell-to-cell interactions, thus allowing studies on immune cells activation and cytotoxic activity towards tumor cells.

Figure 2

Schematic representation of 3-dimensional (3D) cultures with cell lines (spheroids) or patient tumor tissues (organoids) helpful for studying immunotherapies. The growth of tumor cell lines in 3D allows the formation of spheroids characterized by a necrotic core and a proliferative and metabolic gradient mimicking the 3D structure of a tumor. The spheroid allows the easy assessment of immune cells infiltration and the evaluation of strategies with pro-immunogenic potential. The main limitation of this model is the lack of heterogeneity related to the use of cell lines. 3D models made from small pieces of tumor tissues, also called organoids, have shown their ability to mimic tumor heterogeneity in terms of cellular components, TME, or tumor histology. Co-cultures of 3D-cells isolated from tumor tissues with immune cells in the liquid–liquid interface (LLI) method allow immune cell infiltration, activation, and their associated anti-tumor effect to be studied in a context closely reproducing tumor complexity, heterogeneity, and histology. On the other hand, the air–liquid interface (ALI) culture method has been developed to preserve the micro-environmental cellular components to further improve studies on immunotherapies in a context as close as possible than those observed in clinical solid tumors.

Figure 3

Mouse models used for immunotherapy research. The use of in vivo animal models is crucial for studying anti-tumor molecules acting on the TME. For immunotherapies, the easiest and most efficient model is the so-called syngeneic mouse model. A murine cancer cell line is injected into an immunocompetent mouse and the anti-cancer activity of the molecule of interest assessed through tumor growth inhibition, immune cell infiltration, and activation. Humanized mice models allow the efficacy of immunotherapies used in clinic on mice expressing human immune cells to be studied. Mice are humanized by injecting PBMC (PBL model), hematopoietic stem cells (CD34 model) or hematopoietic stem cells with grafting of human fetal liver and thymus (BLT model). These humanized mice are then grafted with either human tumor cell lines or human tumors samples (patient-derived xenograft). As for syngeneic models, tumors can be harvested and analyzed ex vivo for measuring immune infiltrate, cytokine release, and performed mechanistic studies. PBMC: Peripheral Blood Mononuclear Cell, PBL: Peripheral Blood Leukocyte, BLT: Bone marrow, Liver, Thymus.