In recent years, three-dimensional (3D) cultures of tumor cells has emerged as an important tool in cancer research. The significance of 3D cultures, such as tumor spheroids, lies in their ability to mimic the in vivo tumor microenvironment more precisely, offering a nuanced understanding of immune responses within the context of tumor progression. In fact, the infiltration of cytotoxic lymphocytes is key to determining patients' prognosis in several types of cancer and response to immunotherapy. Therefore, harnessing the cytotoxic and infiltration potential of immune cells is a promising avenue for developing effective therapies. This protocol offers a straightforward approach for analyzing infiltrating lymphocytes in tumor spheroids by confocal microscopy imaging. Although it specifically involves utilizing tumor spheroids and culture with autologous tumor-infiltrating T lymphocytes (TILs), the protocol can be adapted for other immune cell types, such as NK cells.
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