Abstract
New successful anti-cancer strategies are based on the stimulation of immune reaction against tumors: however, preclinical testing of such treatments is still a challenge. To improve the screening of anti-cancer drugs, three-dimensional (3D) culture systems, including spheroids, have been validated as preclinical models. We propose the spheroid 3D system to test anti-tumor drug-induced immune responses. We show that colorectal carcinoma (CRC) spheroids, generated with the epithelial growth factor (EGF), can be co-cultured with Vδ2 T cells to evaluate the anti-tumor activity of these effector lymphocytes. By computerized image analysis, the precise and unbiased measure of perimeters and areas of tumor spheroids is achievable, beside the calculation of their volume. CRC spheroid size is related to ATP content and cell number, as parameters for cell metabolism and proliferation; in turn, crystal violet staining can check the viability of cells inside the spheroids to detect tumor killing by Vδ2 T cells. In this 3D cultures, we tested (a) zoledronate that is known to activate Vδ2 T cells and (b) the therapeutic anti-EGF receptor humanized antibody cetuximab that can elicit the antibody-dependent cytotoxicity of tumor cells by effector lymphocytes. Zoledronate triggers Vδ2 T cells to kill and degrade CRC spheroids; we detected the T-cell receptor dependency of zoledronate effect, conceivably due to the recognition of phosphoantigens produced as a drug effect on target cell metabolism. In addition, cetuximab triggered Vδ2 T lymphocytes to exert the antibody-dependent cellular cytotoxicity of CRC spheroids. Finally, the system reveals differences in the sensitivity of CRC cell lines to the action of Vδ2 T lymphocytes and in the efficiency of anti-tumor effectors from distinct donors. A limitation of this model is the absence of cells, including fibroblasts, that compose tumor microenvironment and influence drug response. Nevertheless, the system can be improved by setting mixed spheroids, made of stromal and cancer cells. We conclude that this type of spheroid 3D culture is a feasible and reliable system to evaluate and measure anti-tumor drug-induced immune responses beside direct anti-cancer drug effect.
Highlights
It is known that the immune system can control both survival and proliferation of tumors [1,2,3]
We propose the spheroid 3D culture system to evaluate anti-tumor drug-induced immune response; in particular, we analyzed the anti-tumor effects of Vδ2 T lymphocytes triggered by Zol and/or the anti-epidermal growth factor receptor (EGFR) humanized antibody cetuximab (Cet) on different CRC cell lines
It is of note that spheroids obtained with Caco2 cells appeared with a central portion darker than the periphery, suggesting that the culture conditions used can generate crypts-like structures (Figure 1d)
Summary
It is known that the immune system can control both survival and proliferation of tumors [1,2,3]. Pharmacological treatment with amino bisphosphonates (N-BPs), such as zoledronate (Zol), blocking the farnesyl pyrophosphate synthase of the mevalonate pathway, leads to IPP accumulation in tumor cells and, as a consequence, to the activation and expansion of Vγ9Vδ2 T lymphocytes [11,12,13]. N-BPs have been used in the treatment of many kind of bone diseases, including osteoporosis and bone tumors [14]. Due to their role as potent activators of γδ T lymphocytes, these compounds have been proposed for cancer immunotherapy [5, 14, 15]. Γδ T cells expressing the FcγRIIIA (CD16) can kill tumor cells by antibody-dependent cellular cytotoxicity (ADCC); this mechanism can be exploited using therapeutic antibodies [16]
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