FLOT and CROSS chemotherapy regimens alter the frequency of CD27+ and CD69+ T cells in oesophagogastric adenocarcinomas: implications for combination with immunotherapy.

Combining immunostimulatory chemotherapies with immunotherapy is an attractive strategy to enhance treatment responses in oesophagogastric junctional adenocarcinoma (OGJ). This study investigates the immunostimulatory properties of FLOT, CROSS and MAGIC chemotherapy regimens in the context of OGJ using in vitro and ex vivo models of the treatment-naïve and post-chemotherapy treated tumour microenvironment. FLOT and CROSS chemotherapy regimens increased surrogate markers of immunogenic cell death (HMGB1 and HLA-DR), whereas the MAGIC treatment regimen decreased HMGB1 and HLA-DR on OGJ cells (markedly for epirubicin). Tumour-infiltrating and circulating T cells had significantly lower CD27 expression and significantly higher CD69 expression post-FLOT and post-CROSS treatment. Similarly, the supernatant from FLOT- and CROSS-treated OGJ cell lines and from FLOT- and CROSS-treated OGJ biopsies cultured ex vivo also decreased CD27 and increased CD69 expression on T cells. Following 48 h treatment with post-FLOT and post-CROSS tumour conditioned media the frequency of CD69+ T cells in culture negatively correlated with the levels of soluble immunosuppressive pro-angiogenic factors in the conditioned media from ex vivo explants. Supernatant from FLOT- and CROSS-treated OGJ cell lines also increased the cytotoxic potential of healthy donor T cells ex vivo and enhanced OGJ patient-derived lymphocyte mediated-killing of OE33 cells ex vivo. Collectively, this data demonstrate that FLOT and CROSS chemotherapy regimens possess immunostimulatory properties, identifying these chemotherapy regimens as rational synergistic partners to test in combination with immunotherapy and determine if this combinatorial approach could boost anti-tumour immunity in OGJ patients and improve clinical outcomes.

Chemotherapy regimens induce inhibitory immune checkpoint protein expression on stem-like and senescent-like oesophageal adenocarcinoma cells.

Use of immune checkpoint inhibitors (ICIs) with chemotherapy to enhance responses in oesophageal adenocarcinoma (OAC) is an attractive approach. We identified subpopulations of OAC cells expressing inhibitory immune checkpoint (IC) ligands (PD-L1, PD-L2 and CD160) and receptors (PD-1, TIGIT, TIM-3, LAG-3 and A2aR) in vitro and in ex vivo biopsies. Combination chemotherapy regimens FLOT and CROSS promote a more immune-resistant phenotype through upregulation of IC ligands and receptors on OAC cells in vitro. Importantly, this study investigated if OAC cells, enriched for ICs exhibited a more stem-like and senescent-like phentoype. FLOT preferentially upregulates PD-L1 on a stem-like OAC cell phenotype, defined by ALDH activity. Expression of senescence-associated ß-galactosidase is induced in a subpopulation of OAC cells following FLOT and CROSS chemotherapy treatment, along with enhanced expression of TIM-3 and A2aR ICs. Blockade of PD-1 signalling in OAC cells induced apoptosis and enhanced FLOT and CROSS chemotherapy toxicity in vitro. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance. Combination ICIs may be required to enhance the efficacy of chemotherapy and immunotherapy in OAC patients.