A novel therapeutic approach targeting PD-L1 in HNSCC and bone marrow-derived mesenchymal stem cells hampers pro-metastatic features in vitro: perspectives for blocking tumor-stroma communication and signaling.

Abstract

Background: Current conventional treatment regimens for head and neck squamous cell carcinoma (HNSCC), are poorly effective because of the emergence of resistance mechanisms. Many studies have reported how the tumor microenvironment influences tumor response to immune checkpoint inhibitors targeting PD-1/PD-L1. It has been reported that overexpression of PD-L1 correlates with and is involved in cancer progression by promoting epithelial-to-mesenchymal-transition (EMT) program, stemness and tumor cell invasiveness through AKT and MAPK pathways. In this study, we investigated how bone marrow mesenchymal stem cells (BM-MSCs) recruited and educated by HNSCC cells are able to promote tumor cell invasion and EMT program. In addition, we analyzed how the crosstalk between stromal cells and tumor cells can affect PD-L1 expression levels. In this context, we developed and characterized a novel anti-PD-L1 recombinant Fab (rFab') and tested its ability to potentiate the effect of cisplatin.

Methods: BM-MSCs and HNSCC cells co-cultures, cell migration and invasion were performed using Boyden chambers. The effect of treatments on cell viability and growth were analyzed by MTT and clonogenic assay, respectively. The anti-PD-L1 rFab' was prepared in E. Coli and tested for its binding on HNSCC cells and BM-MSCs by FACS analysis and fluorescence microscopy. PD-L1, p-AKT, p-ERK, N-cadherin and β-catenin expression levels were analyzed by western blotting.

Results: BM-MSCs were induced by tumor cells to migrate, invade and to trans-differentiate in cancer associated fibroblasts (CAFs) as demonstrated by increased expression levels of α-SMA and FAP-α. BM-MSCs contributed to HNSCC invasiveness by increasing p-AKT, p-ERK, N-cadherin and β-catenin expression levels. When BM-MSCs and HNSCC cells were co-cultured the level of PD-L1 expression was enhanced in both cells indicating a reciprocal support in favoring tumor aggressiveness. Tumor cell treatment with rFab' anti-PD-L1 reduced their viability, growth, migration and invasion and blunted the underlying signaling pathways. In addition, rFab' anti-PD-L1 was able to potentiate the antitumor effect of cisplatin on HNSCC cells.

Conclusions: BM-MSCs recruited and educated by HNSCC cells support tumor cell aggressiveness via PD-L1. A novel rFab' anti-PD-L1 reduces HNSCC proliferation, migration and invasion and potentiates the cisplatin effect suggesting its potential to be conjugated with drugs for immuno-cytotoxic therapy.