Impact of radiotherapy on the immune landscape in oesophageal adenocarcinoma

Abstract

Background: In the contemporary era of cancer immunotherapy, an abundance of clinical and translational studies have reported radiotherapy (RT) and immunotherapies as a viable option for immunomodulation of many cancer subtypes, with many related clinical trials ongoing. In locally advanced disease, chemotherapy or chemoradiotherapy followed by surgical excision of the tumour remain the principal treatment strategy in oesophageal adenocarcinoma (OAC), however, the use of the host immune system to improve anti-tumour immunity is rapidly garnering increased support in the curative setting.

Aim: To immunophenotype OAC patients' immune checkpoint (IC) expression with and without radiation and evaluate the effects of checkpoint blockade on cell viability.

Methods: In the contemporary era of cancer immunotherapy, an abundance of studies have demonstrated that combination RT and IC inhibitors (ICIs) are effective in the immunomodulation of many cancer subtypes, with many related clinical trials ongoing. Although surgical excision and elimination of tumour cells by chemotherapy or chemoradiotherapy remains the gold standard approach in OAC, the propagation of anti-tumour immune responses is rapidly garnering increased support in the curative setting. The aim of this body of work was to immunophenotype OAC patients' IC expression with and without radiation and to establish the impact of checkpoint blockade on cell viability. This study was a hybrid combination of in vitro and ex vivo models. Quantification of serum immune proteins was performed by enzyme-linked immunosorbent assay. Flow cytometry staining was performed to evaluate IC expression for in vitro OAC cell lines and ex vivo OAC biopsies. Cell viability in the presence of radiation with and without IC blockade was assessed by a cell counting kit-8 assay.

Results: We identified that conventional dosing and hypofractionated approaches resulted in increased IC expression (PD-1, PD-L1, TIM3, TIGIT) in vitro and ex vivo in OAC. There were two distinct subcohorts with one demonstrating significant upregulation of ICs and the contrary in the other cohort. Increasing IC expression post RT was associated with a more aggressive tumour phenotype and adverse features of tumour biology. The use of anti-PD-1 and anti-PD-L1 immunotherapies in combination with radiation resulted in a significant and synergistic reduction in viability of both radiosensitive and radioresistant OAC cells in vitro. Interleukin-21 (IL-21) and IL-31 significantly increased, with a concomitant reduction in IL-23 as a consequence of 4 Gray radiation. Similarly, radiation induced an anti-angiogenic tumour milieu with reduced expression of vascular endothelial growth factor-A, basic fibroblast growth factor, Flt-1 and placental growth factor.

Conclusion: The findings of the current study demonstrate synergistic potential for the use of ICIs and ionising radiation to potentiate established anti-tumour responses in the neoadjuvant setting and is of particular interest in those with advanced disease, adverse features of tumour biology and poor treatment responses to conventional therapies.

Keywords: Immunology; Immunotherapy; Oesophageal Cancer; Oncology; Radiotherapy; Surgery.