Electroporation of 3D-cultured breast cancer cells elicits T lymphocyte-mediated killing

Cancer cells destruction induced by electro chemotherapy (ECT) can trigger a local and sys temic antitumor immune response as a result of releasing tumor antigens from electroporated cancer cells. 3D scaffolds composed of hyaluronic acid andionic-complementary self-assembling peptides can enable extracellular matrix organization, thus mimicking the complexity of the tumor microenvir onment. Theythereforerepresent avalid systemto study the anticancer effect of Electroporation (EP) protocols. We propose that the use of EP alone on cancer cells embedded in 3D scaffolds may trigger high antigens exposure, inducing T lymphocytes migra tion and activation that could lead to cancer cells elimination. We co-cultured HCC1954 breast carcinoma cells with Jurkat T cells in 3D scaffolds, applying two different EP conditions (600 V/cm and 1000 V/cm) commonly used in ECT protocols, to modulate T lymphocytes activation. Our results revealed that EP of co-cultures of HCC1954 cells with resting T cells significantly influenced the number and size of cancer cell-associated 3D structures (spheroids). T lymphocytes infiltration and cancer cells death were associated to the reduction in 3D cancer cell spheroids. The addition of PHA-M activated T cells significantly enhanced this overall effect. Follow ing these results, we co-cultured HCC1954 cells with human T lymphocytes to evoke a more clin ically relevant condition. Using flow cytometry, we confirmed the activation of T cells and their cyto toxic activity, mediated by the ability of EP to induce the release of IL-2, INF-gamma andTNF-alfaatthe mRNAlevel. Our study demonstrates that EP alone can exert anticancer effects by increasing tumor cell killing by activated T lymphocytes. We speculate that this is facilitated by EP-mediated increase in antigen ex posure on tumor cells.