EX VIVO EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TILS) AND CANCER STEM CELLS FROM MALIGNANT GLIOMAS

Abstract

<h3>Background</h3> Gliomas are the most common type of primary malignant tumors of the Central Nervous System (CNS). Conventional therapies show low effectiveness and are frequently associated with important side effects. Glioblastoma (GBM) is the most lethal and frequent type of glioma, endowed with a subpopulation of cancer stem cells (CSC) responsible for resistance to conventional treatment and ensuing tumor relapse. Different forms of immunotherapy are increasingly recognized as promising therapeutic approach against aggressive tumors, including adoptive cell therapy with tumor infiltrating lymphocytes (TILs). However, studies accessing the potential of TILs in aggressive CNS tumors are limited. In this work, we describe the feasibility of isolating TILs and CSC from the same tumor specimen for ex vivo expansion and downstream immunotherapy application. <h3>Methods</h3> Fresh tumor samples were obtained from 10 patients (mean age: 60 years; 4 men and 6 women), diagnosed with malignant glioma and subjected to tumor resection in the Instituto do Câncer do Estado de São Paulo (ICESP), after informed consent approved by the Institutional Review Board. Brain tumor tissue was mechanically dissociated and enzymatically digested to generate a cell suspension. The CSC population was cultured in neurosphere formation media supplemented with EGF and FGF. TILs were expanded in culture medium supplemented with IL-2. <h3>Results</h3> Anatomopathological analyses of resected tumors confirmed diagnosis of 9 GBM and a grade I glioneural tumor. Tumorspheres could be generated in 8 out of 10 tumor samples, all of them GBM. No tumorspheres were formed in the respective cultures derived from the grade I glioneural tumor and from a GBM containing mutated ATRX. TILs could be expanded from all tumor samples tested, reaching a mean of 1,1 ± 0,7 × 10e6 total cells and mean cell viability of 60% ± 4%, after 5 days of in vitro expansion. <h3>Conclusion</h3> CSC could be expanded from all fresh glioma samples, except from a grade I glioma and a GBM with ATRX mutation, indicating a possible positive correlation with unfavorable clinical prognosis. Viable TILs could also be initially expanded from these glioma samples, indicating the possibility of using them to seed larger cultures subjected to typical rapid expansion protocols to attain clinical doses. Altogether, this procedure could help identify targetable CSC antigens, as well as screen for CSC-reactive TIL clones for possible adoptive cell therapy.