Abstract
Esophageal adenocarcinoma (EAC) is increasing exponentially year on year in Western civilisation, linked epidemiologically to GERD and obesity. It is characterized by a highly immunosuppressive tumour microenvironment (TME) and evasion of immune surveillance. A novel treatment option is upregulating immune mediated anti-tumour response via Immune Checkpoint Blockers (ICB).
Methods
The effects of immune checkpoint blockers were characterised in terms of proliferation, cytolysis and cancer cell viability. The basal expression of immune checkpoints (TIGIT, PD-1, PD-L1, PD-L2) and Damage Associated Molecular Patterns (Calreticulin, HMGB1) in EAC patients was profiled ex vivo using fresh tumor, blood and lymph-node tissue (n = 10) by flow cytometry. In an in-vitro study, T-lymphocytes were isolated and treated with Nivolumab, Pembrolizumab or Atezolizumab, activated and co-cultured for 48 hours with a panel of four esophageal cancer cell lines; OE33P, OE33R, FLO-1, FLO-1LM treated with 1.8Gy and 3.6Gy of radiation (Fig 1). Cytolysis was measured using a CCK8 assay.(n = 6).
Results
The expression of TIGIT, TIM-3, PD-1 and its ligands (PD-L1, PD-L2) were higher (p < 0.001) in EAC patients compared to age matched healthy controls. Similarly, when mimicking conditions of the TME including nutrient deprivation and hypoxia, this results in a significant (p < 0.001) increase in DAMP and ICB expression on CD3,4 and CD8 T cells in the TME when treated with radiation. T-lymphocytes induced by checkpoint blockers plus ionising radiation directly to the tumor resulted in the best repression of tumour growth with 3.6Gy inducing the highest rate of cytolysis (p < 0.001). ICB and radiation resulted in reduced cancer cell viability and proliferation.
Conclusion
Fractionated radiation can enhance immunologic function. In combination with ICB, this symbiotic relationship enhances the cytotoxic potential of T lymphocytes with conventional dosing, however, with hypofractionation, this signifies true immunogenicity, and as such this provides a basis for advocating for potential combination strategies with ICB in the multimodal treatment of EAC.
Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies
