Anti-PD1 treatment to induce M1 polarization of tumor infiltrating macrophages in a 3D ex vivo system of lung cancer patients.

Abstract

e23090 Background: Nivolumab and pembrolizumab treatment targeting the PD1/PD-L1 axis has demonstrated increased survival benefit in subpopulations of patients with advanced non-small cell lung cancer (NSCLC). The impact of these therapeutics on tumor immune microenvironment is not fully understood. Classical M1 macrophages are critical components involved in the inflammatory response and antitumor immunity. In this study, we evaluated the effect of PD1/PD-L1 blockade on M1 polarization of tumor-associated macrophages (TAMs) and activation of cytotoxic T-cells in a 3D ex vivo system of NSCLC. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay. 3D tumor microspheres were treated with nivolumab or pembrolizumab at 10 mg/ml for 36 hours within an intact tumor microenvironment. Flow cytometry analysis was performed to evaluate treatment-mediated TAM polarization, activation of T-cells and changes in CD4 and CD8 subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by ex vivo treatment. Results: Both nivolumab and pembrolizumab treatment increased population of M1 macrophages (CD68+, CD80+, CD163-) and simultaneous release of MIP1b, IFN-ɣ, TNF-a, and GM-CSF cytokines as well as expression of genes related to the M1 phenotype that was accompanied by activation of CD8 cells assessed by Ki67 and CD107a expression and increased expression of genes involved in the IFNg pathway. Conclusions: Our studies showed that anti PD1/PD-L1 treatment leads to M1 macrophage polarization and T-cell activation in subgroups of NSCLC patients emphasizing the importance of comprehensive analysis of tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may help developing rationale combination treatments in NSCLC.