Biofabrication of 3D breast cancer models for dissecting the cytotoxic response of human T cells expressing engineered MAIT cell receptors

Abstract

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune–cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune–cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune–cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

3D bioprinting and the revolution in experimental cancer model systems—A review of developing new models and experiences with in vitro 3D bioprinted breast cancer tissue-mimetic structures

“…In most of these studies, mainly the less aggressive MCF7 (luminal A) or the more aggressive triple-negative (MDA-MB-231) breast cancer cell lines were used. Only a few papers describe multi-cellular models, 3D models combined with vessels, adipocytes, fibroblasts or 3D bioprinted tissue mimetic in vitro structures in the field of breast cancer research ( 82 , 83 , 109 ). We also have experience printing triple-negative breast cancers using MDA-MB-23 and MDA-MD-468 cell lines.…”

“…Alginate-based bioinks stabilised and CaCl 2 stabilised preferably avoiding mutagenic UV for human cell printing (e.g., in the case of GELMA) ( 155 ). In the other part of these works, matrigels decellularized ECM with additional collagen or other gradients, GELMA, fibrinogen or hyaluronic acid (HA), and PEG- (polyethylene glycol) based materials are also used ( 82 , 109 , 124 ). The main problem with the published descriptions and protocols is that the used biomaterials are different and not fully characterised or specified in most of the papers.…”

Opportunities and challenges to engineer 3D models of tumor-adaptive immune interactions

“…To create a tumor-immune interaction model, Dey et al. printed a ring of T cells into a collagen bath and deposited a single MDA-MB-231/human dermal fibroblast spheroid in the center ( Figure 2D ) ( 73 ). In each of the print designs, cancer cells migrated from the central spheroid and invaded into the collagen matrix.…”

“…In one study, Dey et al. developed 3D tumor-T cell platforms that allowed for the study of complex immune-cancer interactions and the evaluation of genetically engineered CD8 + T cells expressing mucosal-associated invariant T (MAIT) cell receptors against breast cancer cells ( 73 ). Their study showed that the engineered T cells effectively eliminated tumors in 3D culture.…”

Bioprinted Multicomponent Hydrogel Co-culture Tumor-Immune Model for Assessing and Simulating Tumor-Infiltrated Lymphocyte Migration and Functional Activation

“…By providing such precise control, hydrogels enable the creation of in vitro models that more accurately recreate the histopathology of the infiltrated immune cells in the tumor, thus significantly improving the usefulness of these models in cancer research. Moreover, the use of bioprinting to exert spatial control over engineered cell-laden hydrogels has been demonstrated by several research groups to create 3D constructs, which can further enhance the realism and intricacy of in vitro cancer-TIL models. − …”