Isolation, expansion and characterization of tumor infiltrating lymphocytes derived from metastatic colorectal carcinoma patients for adoptive immunotherapy

Colorectal carcinoma (CRC) is one of the most common type of cancer and the second cause of cancer death in the world. Conventional treatments appear to slow the tumor progression, but do not bring the patients to a disease-free state. Adoptive cellular immunotherapy is emerging as a new clinical strategy after the results obtained with patients with metastatic melanoma. In our study, we developed a new strategy for the extraction and expansion of both autologous TILs and tumor cells (TCs) from liver metastases of colorectal carcinoma. This was done by mechanical and enzymatic dissociation of tumor samples resected in the operating room. TCs were used for functional assays as well to determine TILs’ potency. Three protocols for TILs expansion were developed using different cocktails of cytokines (i.e. IL-2, IL-15, IL-21 and IL-7) and all showed promising results in terms of cytotoxic activity and expansion rate. We demonstrated how it is possible to obtain and expand TILs from metastatic colorectal carcinoma (mCRC) even when TILs’ recovered are low in number. TILs expanded with all three protocols were able to determine cytolysis on both an immortalized human CRC cell line (SW480) and against TCs and such activity remains sizeable even when a second round of antigen independent expansion is performed. We also observed how TILs which had prevalently expanded CD3+/CD4+ cells had a cytotoxic capacity compared to TILs which were prevalently CD3+/CD8+, showing the importance of CD3+/CD4+ in mediating CD3+/CD8+ cells anti-tumor activity and in endowing an anti-tumor response as well. Analysis of cell activation and exhaustion markers expressed by TIL in both CD3+/CD4+ and CD3+/CD8+ population documented a high heterogeneity of expression between TIL, with little difference between the different expansion protocols. Molecular assessment were performed on both TILs and TCs to further define the role of Ca2+ signalling pathways. In particular, our findings determined how Store Operated Ca2+ Entry in TILs is up-regulated and mainly depends on diacylglycerol kinase (DGK), which prevents the protein kinase C-dependent inhibition of Ca2+ entry in normal T cells. Notably, the pharmacological blockade of SOCE with the selective inhibitor, BTP-2, during target cell killing significantly increases cytotoxic activity at low TIL density, i.e., when TILs-mediated cancer cell death is rarer. We also determined how exogenous administration of H2S-releasing compounds has been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including CRC. We provided the evidence that NaHS – a H2S donor- induced extracellular Ca2+ entry in mCRC cells by activating the Ca2+-permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na+-dependent recruitment of the reverse-mode of the Na+/Ca2+ (NCX) exchanger. NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. Our findings support the notion that exogenous administration of H2S may prevent mCRC cell proliferation through an increase in [Ca2+]i.