We have previously demonstrated that first-line chemotherapy combinations FLOT and CROSS upregulated ICs on the surface of OAC cells following 48h treatment in vitro21. Therefore, we sought to investigate how long this chemotherapy-induced upregulation of ICs on the surface of OAC cells is maintained by longitudinally profiling IC expression on the surface of OE33 cells following 48h treatment with vehicle control or FLOT chemotherapy regimen (Figure 1A).
48h treatment with FLOT significantly increased PD-L1 expression on the surface of OE33 cells compared with the vehicle control at 48h (50.85 ± 1.2 vs. 2.65 ± 0.3%, p<0.0001, Figure 1B). Interestingly, FLOT-induced PD-L1 upregulation on the surface of OE33 cells remained upregulated at 4 days (45.35 ± 1.5 vs. 1.63 ± 0.5%, p<0.0001) and 21 days (8.88 ± 0.3 vs. 0.97 ± 0.1%, p<0.0001, Figure 1B) post-treatment compared with the vehicle control (Figure 1B). Following subculture of recovered FLOT-treated OE33 cells PD-L1 was significantly upregulated compared with the vehicle control (3.15 ± 0.2 vs. 1.15 ± 0.1, p=0.007, Figure 1B).
Similarly, 48h treatment with FLOT significantly increased PD-L2 expression on the surface of OE33 cells compared with the vehicle control at 48h (38.45 ± 0.7 vs. 2.28 ± 0.2%, p<0.0001, Figure 1B). FLOT-induced PD-L2 upregulation on the surface of OE33 cells remained upregulated at 4 days (36.33 ± 6.8 vs. 2.5 ± 0.9, p=0.01) and 21 days (3.7 ± 0.1 vs. 2.58 ± 0.2, p=0.001, Figure 1B) post-treatment compared with the vehicle control (Figure 1B). In contrast to PD-L1, following subculture of recovered (day 24) FLOT-treated OE33 cells PD-L2 expression had returned to baseline with no significant difference when compared with the vehicle control (Figure 1B).
Although 48h treatment with FLOT did not significantly alter CD160 expression on the surface of OE33 cells compared with the vehicle control at 48h, CD160 was significantly upregulated 4 days post-FLOT treatment compared with the vehicle control (73.53 ± 5.6 vs. 3.75 ± 0.7, p=0.0008, Figure 1B). However, CD160 expression had returned to baseline 21 days post-FLOT treatment and following subculture of recovered FLOT-treated OE33 cells with no significant difference when compared with the vehicle control (Figure 1B).
Following 48h treatment with FLOT, PD-1 expression increased on the surface of OE33 cells on day 2 (46.18 ± 4.3 vs. 22.60 ± 4.4, p=0.06) and on day 4 compared with the vehicle control (28.95 ± 7.5 vs. 12.02 ± 5.5%, p=0.06, Figure 1C). Furthermore, following subculture of recovered FLOT-treated OE33 cells there was a reduction in PD-1 expression compared with the vehicle control (10.92 ± 0.8 vs. 17.63 ± 0.8, p=0.06, Figure 1C).
TIGIT expression increased on the surface of OE33 cells compared with the vehicle control at 48h (68.58 ± 3.0 vs. 57.0 ± 2.0%, p=0.06, Figure 1C) and remained upregulated at 4 days (67.28 ± 6.8 vs. 43.65 ± 3.1, p=0.01). However, 21 days post-FLOT treatment TIGIT expression returned to baseline and was comparable with the vehicle control (Figure 1C). Interestingly, following subculture of recovered FLOT-treated OE33 cells, TIGIT was significantly decreased compared with the vehicle control (9.17 ± 0.8 vs. 13.80 ± 0.9%, p=0.004, Figure 1C).
Similar to TIGIT, following 48h treatment with FLOT, TIM-3 expression increased on the surface of OE33 cells compared with the vehicle control at 48h (6.65 ± 0.9 vs. 1.8 ± 0.1, p=0.09, Figure 1C) and remained upregulated at 4 days (12.53 ± 1.3 vs. 1.51 ± 0.1%, p=0.003) (Figure 1C). Similarly, to TIGIT, following subculture of recovered FLOT-treated OE33 cells was a significant reduction in TIM-3 expression compared with the vehicle control (0.41 ± 0.1 vs. 1.95 ± 0.4%, p=0.05, Figure 1C).
Following 48h treatment with FLOT, LAG-3 was significantly increased on the surface of OE33 cells compared with the vehicle control at 48h (4.19 ± 0.1 vs. 1.90 ± 0.4%, p=0.03, Figure 1C) and remained upregulated at 21 days (19.50 ± 0.1 vs. 4.43 ± 0.3%, p=0.009, Figure 1C). Similar, to findings observed for TIGIT and TIM-3, following subculture of recovered FLOT-treated OE33 cells, LAG-3 expression decreased compared with the vehicle control (0.76 ± 0.2 vs. 1.71 ± 0.4%, p=0.06, Figure 1C).
Although 48h treatment with FLOT did not significantly alter A2aR expression on the surface of OE33 cells compared with the vehicle control at 48h, A2aR was significantly upregulated 4 days post-FLOT treatment (5.41 ± 0.6 vs. 1.41 ± 0.2%, p=0.004) and 21 days (1.78 ± 0.1 vs. 1.12 ± 0.2%, p=0.02, Figure 1C). Similarly, following subculture of recovered FLOT-treated OE33 cells, A2aR expression was decreased compared with the vehicle control (0.90 ± 0.1 vs. 2.16 ± 0.4%, p=0.06, Figure 1C).
Overall, several ICs were significantly upregulated on the surface of OE33 cells longitudinally including PD-L1, PD-L2, CD160, TIGIT, TIM-3, LAG-3 and A2aR. Interestingly, PD-L1 remained increased compared with the vehicle control following subculture of FLOT-recovered OE33 cells at 24 days, whilst PD-L2 and CD160 returned to baseline expression levels. Interestingly, PD-1, TIGIT, TIM-3, LAG-3 and A2aR expression were decreased compared with the vehicle control following subculture of FLOT-recovered OE33 cells at 24 days.
Pro-survival MEK signalling upregulates ICs on the surface of OAC cells following chemotherapy treatment
Chemotherapy-induced upregulation of ICs on the surface of OAC cells suggests these tumour cells may be employing ICs as an adaptive survival mechanism to overcome genotoxic stress. However, the signalling pathways mediating FLOT-induced IC upregulation remain unknown. Therefore, we sought to investigate if the pro-survival signalling pathway MEK may be regulating the chemotherapy-induced upregulation of ICs.
Inhibition of MEK signalling significantly reduced the basal expression of PD-L1 on the surface of SK-GT-4 cells compared with the vehicle control (0.82 ± 0.3 vs. 2.13 ± 0.1%, p=0.02) (Figure 2A). Moreover, inhibition of MEK signalling significantly decreased FLOT-induced PD-L1 upregulation on the surface of OE33 cells (28.24 ± 6.8 vs. 31.63 ± 7.1%, p=0.03) and SKGT-4 cells (8.75 ± 1.6 vs. 31.38 ± 6.5%, p=0.008) compared with FLOT treatment alone (Figure 2A).
Inhibition of MEK signalling significantly increased the expression of PD-L2 on the surface of OE33 cells (3.51 ± 0.7 vs. 1.72 ± 0.1%, p=0.02) and SK-GT-4 cells (5.79 ± 0.9 vs. 2.93 ± 0.4%, p=0.001) compared with the vehicle control (Figure 2A). However, inhibition of MEK signalling did not alter the expression of FLOT-induced PD-L2 expression compared with FLOT treatment alone in either cell line.
Similarly, inhibition of MEK signalling significantly increased the expression of CD160 on the surface of OE33 cells (2.69 ± 0.1 vs. 1.4 ± 0.1%, p=0.002) and SK-GT-4 cells (1.32 ± 0.1 vs. 0.60 ± 0.1%, p=0.04) compared with the vehicle control (Figure 2A). Moreover, inhibition of MEK signalling in combination with FLOT treatment significantly increased the expression of CD160 on the surface of OE33 cells compared with FLOT treatment alone (3.90 ± 0.8 vs. 2.84 ± 0.6%, p=0.03) compared with the vehicle control (Figure 2A).
Inhibition of MEK signalling significantly increased the expression of PD-1 on the surface of OE33 cells (11.15 ± 2.5 vs. 6.97 ± 1.3%, p=0.01) compared with the vehicle control (Figure 1B). In contrast, inhibition of MEK signalling significantly decreased the expression of PD-1 on the surface of SK-GT-4 cells (15.39 ± 4.3 vs. 20.20 ± 2.8%, p=0.001) compared with the vehicle control (Figure 2B). Inhibition of MEK signalling did not significantly alter the expression of TIGIT on the surface of OE33 or SK-GT-4 cells basally or in combination with FLOT treatment (Figure 2B).
Inhibition of MEK signalling significantly reduced the expression of basal levels of TIM-3 on the surface of SK-GT-4 cells compared with the vehicle control (1.36 ± 0.3 vs. 2.26 ± 0.2%, p=0.05) (Figure 2A). Inhibition of MEK signalling significantly decreased FLOT-induced TIM-3 upregulation on the surface of OE33 cells (3.92 ± 0.2 vs. 24.53 ± 3.8%, p=0.03) and SKGT-4 cells (3.35 ± 0.3 vs. 9.09 ± 0.5%, p=0.001) compared with FLOT treatment alone (Figure 2A).
MEK inhibition significantly increased the expression of basal levels of LAG-3 on the surface of OE33 cells compared with the vehicle control (1.77 ± 0.1 vs. 0.96 ± 0.1%, p=0.02) (Figure 2A). In contrast, inhibition of MEK signalling significantly decreased FLOT-induced LAG-3 upregulation on the surface of SKGT-4 cells (5.80 ± 0.8 vs. 9.03 ± 0.8%, p=0.002) compared with FLOT treatment alone (Figure 2A).
Additionally, inhibition of MEK signalling significantly decreased the expression of basal levels of A2aR on the surface of SK-GT-4 cells compared with the vehicle control (2.08 ± 0.3 vs. 2.60 ± 0.3, p=0.04) (Figure 2A). Similarly, inhibition of MEK signalling significantly decreased FLOT-induced A2aR upregulation on the surface of SKGT-4 cells (4.47 ± 0.9 vs. 8.80 ± 1.6, p=0.002) compared with FLOT treatment alone (Figure 2A). In addition, there were a reduction in FLOT-induced A2aR upregulation on the surface of OE33 cells compared with FLOT treatment alone (8.74 ± 0.9 vs. 16.83 ± 2.7, p=0.06, Figure 2A).
Overall, MEK signalling regulated FLOT-induced upregulation of PD-L1, TIM-3, LAG-3 and A2aR on the surface of OAC cells. Of note, inhibition of the STAT3 signalling pathway did not significantly affect the expression of ICs on the surface of OAC cells (data not shown).
Blockade of PD-L1, PD-1 and A2aR intrinsic signalling in OAC cells enhances the toxicity of the FLOT regimen
Given the observation that the pro-survival MEK signalling pathway regulated FLOT-induced PD-L1 and A2aR upregulation on the surface of OAC cells, we investigated if blockade of PD-1 (nivolumab), PD-L1 (atezolizumab) or A2aR signalling axes in OAC cells might enhance the toxicity of FLOT chemotherapy regimen (Figure 3).
Single agent nivolumab and single agent A2aR antagonist significantly decreased the viability of OE33 cells compared with untreated cells (84.12 ± 5.2 vs. 102.1 ± 1.4%, p=0.02 and 84.56 ± 4.1 vs. 102.4 ± 1.8%, p=0.003, respectively Figure 3A). Similar results were observed in the SK-GT-4 cell line; single agent nivolumab, single agent atezolizumab and single agent A2aR antagonist decreased the viability of SK-GT-4 cells compared with untreated cells (91.13 ± 2.3 vs. 100.0 ± 0.9%, p=0.004, 94.15 ± 2.8 vs. 100.0 ± 0.9%, p=0.07 and 91.70 ± 2.7 vs. 100.0 ± 0.9%, p=0.008, respectively Figure 3A).
Combining A2aR antagonist with FLOT significantly decreased the viability of OE33 cells compared with FLOT treatment alone (71.69 ± 3.1 vs. 72.60 ± 7.0%, p=0.03, Figure 3A). Combining nivolumab with FLOT significantly decreased the viability of SK-GT-4 cells compared with FLOT treatment alone (36.01 ± 4.4 vs. 38.72 ± 2.7%, p=0.01, Figure 3A).
Combining FLOT with nivolumab significantly decreased the viability of OE33 cells (51.57 ± 14.50 vs. 84.12 ± 5.2%, p=0.02) and SK-GT-4 cells (36.01 ± 4.4 vs. 91.13 ± 2.3%, p<0.0001), compared with nivolumab treatment alone (Figure 3A). Combining FLOT with atezolizumab significantly decreased the viability of SK-GT-4 cells (37.61 ± 3.8 vs. 94.15 ± 2.8%, p=0.0001), compared with atezolizumab treatment alone (Figure 3A). Combining FLOT with A2aR antagonism significantly decreased the viability of OE33 cells (71.69 ± 3.1 vs. 84.56 ± 4.1%, p=0.0005), and SK-GT-4 cells (37.02 ± 2.2 vs. 91.70 ± 2.7%, p<0.0001), compared with A2aR antagonism alone (Figure 3A).
Overall, single agent nivolumab and A2aR antagonism significantly decreased the viability of OAC cells alone. Interestingly, combining nivolumab or A2aR antagonist with the FLOT regimen significantly enhanced the reduction in viability of OAC cells compared with FLOT treatment alone. In addition, combining FLOT chemotherapy with single agent nivolumab, atezolizumab or A2aR antagonism significantly enhanced the reduction in viability of OAC cells compared with ICB treatment alone.
Given these findings we sought to investigate how blockade of the PD-1, PD-L1 or A2aR signalling axes alone and in combination with FLOT might affect the proliferation of OE33 cells longitudinally (Figure 3B). Single agent nivolumab significantly decreased Ki67 expression in OE33 cells at days 4 days (72.68 ± 0.2 vs. 100.0 ± 0.4%, p<0.0001) and 21 days (71.07 ± 0.2 vs. 100.0 ± 3.0%, p=0.01) compared with vehicle treated cells (Figure 3C). Similarly, single agent atezolizumab significantly decreased Ki67 expression in OE33 cells 4 days (61.34 ± 0.4 vs. 100.0 ± 3.0%, p<0.0001) and 21 days (61.34 ± 0.4 vs. 100.0 ± 3.0%, p=0.005) compared with vehicle treated cells (Figure 3C). Single agent A2aR antagonist significantly increased Ki67 expression in OE33 cells at 4 days (139.9 ± 0.3 vs. 100.0 ± 3.0%, p<0.0001) and decreased Ki67 expression at 21 days (63.68 ± 0.2 vs. 100.0 ± 3.0%, p=0.007) compared with vehicle treated cells (Figure 3C).
Interestingly, 48h FLOT treatment significantly increased Ki67 expression in OE33 cells compared with the vehicle control 2 days (224.4 ± 12.4 vs. 100.0 ± 4.0%, p=0.0008), 4 days (183.3 ± 1.8 vs. 100.0 ± 0.4%, p<0.001) and 21 days (145.95 ± 5.2 vs. 100.0 ± 3.0%, p=0.002) post-treatment (Figure 3C). However, Ki67 expression was significantly decreased in the FLOT treated cells 21 days post-treatment compared with FLOT treated cells 4 days post-treatment (145.95 ± 5.2 vs. 183.3 ± 1.8 vs. 100.0 ± 0.4%, p=0.01) and compared with FLOT-treated cells 2 days post-treatment (145.95 ± 5.2 vs. 224.4 ± 12.4%, p=0.03) (Figure 3C).
Interestingly, single agent nivolumab in combination with FLOT treatment significantly decreased Ki67 expression in OE33 cells compared with FLOT treatment alone 2 days post-treatment (159.8 ± 6.1 vs. 224.4 ± 12.36%, p=0.005) and 4 days post-treatment (170.3 ± 0.4 vs. 183.3 ± 1.8%, p=0.003) (Figure 3C). Similarly, single agent atezolizumab in combination with FLOT treatment significantly decreased Ki67 expression in OE33 cells compared with FLOT treatment alone 2 days post-treatment (151.9 ± 3.7 vs. 224.4 ± 12.36%, p=0.02) and 21 days post-treatment (112.2 ± 4.6 vs. 145.9 ± 5.2%, p=0.02) (Figure 3C).
Additionally, single agent A2aR antagonist in combination with FLOT treatment significantly decreased Ki67 expression in OE33 cells compared with FLOT treatment alone 2 days post-treatment (155.8 ± 5.8 vs. 224.4 ± 12.36%, p=0.02), 4 days post-treatment (152.7 ± 2.5 vs. 183.3 ± 1.8%, p=0.0001) and 21 days post-treatment (80.49 ± 4.6 vs. 145.9 ± 5.2%, p=0.0007) (Figure 3C).
Overall, single agent nivolumab, atezolizumab and A2aR antagonism significantly decreased the proliferation of OAC cells alone. Interestingly, combining single agent nivolumab, atezolizumab and A2aR antagonism with the FLOT regimen significantly decreased the proliferation of OAC cells compared with FLOT treatment alone. Taken together these findings suggest that inhibition of the PD-1 axis or A2aR axis decreases the survival of OAC cells and when combined with the FLOT regimen synergistically enhance the toxicity of FLOT against OAC cells in vitro.
Given these findings we next aimed to investigate how blockade of PD-1, PD-L1 or A2aR signalling axes alone and in combination with FLOT might affect OAC cell apoptosis and cell death (Figure 4).
Single agent nivolumab significantly induced cell death in OE33 cells compared with the vehicle control (9.71 ± 0.1 vs. 9.19 ± 0.04%, p=0.02), demonstrated by a significant increase in late stage apoptotic cells (Figure 4). Similarly, single agent A2aR antagonist significantly induced cell death in OE33 cells (19.25 ± 0.2 vs. 9.19 ± 0.04%, p<0.0001) and in SK-GT-4 cells (6.88 ± 0.1 vs. 5.21 ± 0.1%, p=0.001) compared with the vehicle control, demonstrated by a significant increase in late stage apoptotic cells (Figure 4). In addition, single agent nivolumab (9.13 ± 0.3 vs. 5.8 ± 0.4%, p=0.005) and A2aR antagonist (12.10 ± 0.6 vs. 5.8 ± 0.4%, p=0.0004) significantly increased the percentage of early stage apoptotic SK-GT-4 cells compared with untreated cells (Figure 4).
Combining single agent nivolumab (23.78 ± 2.4 vs. 18.33 ± 2.2%, p=0.04), atezolizumab (27.23 ± 4.1 vs. 18.33 ± 2.2%, p=0.04) or A2aR antagonist (28.45 ± 0.8 vs. 18.33 ± 2.2%, p=0.04) with FLOT significantly induced cell death in OE33 cells compared with FLOT treated cells, demonstrated by a significant increase in late stage apoptotic cells (Figure 4). Similarly, combining single agent atezolizumab (10.28 ± 0.4 vs. 8.44 ± 0.3%, p=0.04) or A2aR antagonist (12.00 ± 0.7 vs. 8.44 ± 0.3%, p=0.04) with FLOT significantly induced cell death in SK-GT-4 cells compared with FLOT treated cells, demonstrated by a significant increase in late stage apoptotic cells (Figure 4).
Although, combining single agent nivolumab with FLOT did not significantly enhance SK-GT-4 cell death, a significant increase in early stage apoptotic SK-GT-4 cells was observed using combination nivolumab with FLOT compared with FLOT alone (27.15 ± 1.7 vs. 13.73 ± 1.6%, p=0.0002) (Figure 4). Similarly, combining single agent atezolizumab (27.15 ± 1.7 vs. 13.73 ± 1.6%, p=0.001) or A2aR antagonist (24.90 ± 1.9 vs. 13.73 ± 1.6%, p=0.0003) with FLOT significantly induced increased the percentage of early stage apoptotic SK-GT-4 cells compared with FLOT treated cells (Figure 4).
Overall, these findings highlight that single agent PD-1, PD-L1 and A2aR IC blockade induced apoptosis and OAC cell death. Furthermore, combining ICB with the FLOT chemotherapy regimen synergistically enhanced induction of apoptosis in OAC cells and OAC cell death.
Blockade of IC signalling in OAC cells decreases the formation of γH2AX and expression of DNA repair genes
We have shown that PD-1, PD-L1 and A2aR signalling confers OAC cells with a survival advantage as their blockade alone reduces OAC cell viability and can enhance FLOT chemotherapy toxicity. Interestingly, studies have implicated a role for PD-L1 intrinsic signalling in mediating DNA repair in colon cancer22. Therefore, to achieve a greater understanding of the mechanisms of action behind enhanced FLOT cytotoxicity in combination with ICB we assessed if blockade of these IC pathways might alter the formation of γH2AX alone and in combination with FLOT chemotherapy (Figure 5). Tumour cells rapidly proliferate and typically acquire DNA damage during replication generating genotoxic stress in the cells, which ultimately leads to tumour cell death if left unrepaired. Formation of γH2AX is an important step in the initiation of DNA repair.
Single agent nivolumab, atezolizumab and A2aR antagonist significantly decreased the levels of γH2AX expression in OE33 cells following 24h treatment compared with the vehicle control (nivolumab: 1352 ± 15.09 vs. 1507 ± 8.51%, p=0.005, atezolizumab: 1383 ± 6.8 vs. 1507 ± 8.51%, p=0.002 and A2aR antagonist: 1416 ± 13.6 vs. 1507 ± 8.51%, p=0.02) (Figure 5A). Similar findings were observed in the SK-GT-4 cell line where single agent nivolumab, atezolizumab and A2aR antagonist significantly decreased the levels of γH2AX expression in OE33 cells following 24h compared with the vehicle control (nivolumab: 4167 ± 50.85 vs. 4491 ± 32.9%, p=0.001, atezolizumab: 4129 ± 33.1 vs. 4491 ± 32.9%, p=0.002 and A2aR antagonist: 2791 ± 38.6 vs. 4491 ± 32.9%, p<0.0001) (Figure 5A).
Following 24h treatment with FLOT the levels of γH2AX expression in OE33 cells was significantly increased compared with the vehicle control (2756 ± 29.05 vs. 1507 ± 8.5%, p<0.0001). Interestingly, combining single agent nivolumab, atezolizumab and A2aR antagonist with the FLOT regimen significantly decreased the levels of γH2AX expression in OE33 cells following 24h compared with FLOT treated cells (nivolumab: 1937 ± 9.9 vs. 2756 ± 29.0%, p=0.0001, atezolizumab: 1836 ± 12.1 vs. 2756 ± 29.0%, p=0.0001 and A2aR antagonist: 2232 ± 11.8 vs. 2756 ± 29.0%, p=0.0002) (Figure 5A).
Similar findings were observed in the SK-GT-4 cell line (Figure 5A). Following 24h treatment with FLOT the levels of γH2AX expression in SK-GT-4 cells was significantly increased compared with the vehicle control (5694 ± 49.6 vs. 4491 ± 32.9%, p=0.0001). Similarly, combining single agent nivolumab with the FLOT regimen significantly decreased the levels of γH2AX expression in SK-GT-4 cells following 24h compared with FLOT treated cells (4414 ± 13.1 vs. 5694 ± 49.6%, p<0.0001) (Figure 5A). On the contrary, combining single agent nivolumab with the FLOT regimen significantly increased the levels of γH2AX expression in SK-GT-4 cells following 24h compared with FLOT treated cells (nivolumab: 4.44 ± 4.1 vs. 4.44 ± 4.1%, p<0.0001) (Figure 5A). Overall, similar trends were also observed at 48h and at 72h in which single agent nivolumab, atezolizumab and A2aR antagonism decreased γH2AX expression in OE33 and SK-GT-4 cells compared with the vehicle control (Figure 5A). Similarly, at 48h and 72h timepoints, combining single agent nivolumab, atezolizumab and A2aR antagonist with the FLOT regimen significantly decreased the levels of γH2AX expression in OAC cells compared with FLOT treated cells (Figure 5A).
Furthermore, Tu et al.,23 demonstrated that intracellular PD-L1 acts as an RNA binding protein enhancing the mRNA stability of NBS1 and BRCA1, thus upregulating the expression of DNA repair proteins NBS1 and BRCA1. Therefore, we assessed if ICB might alter the expression of well described DNA repair genes PARP1, SMUG1, MLH1 and MMS19 alone and in combination with FLOT chemotherapy (Figure 5). Single agent atezolizumab significantly reduced the mRNA expression levels of PARP1 and SMUG1 compared with the vehicle control (PARP1: 0.41 ± 0.1 vs. 0.78 ± 0.2%, p=0.005 and SMUG1: 0.45 ± 0.1 vs. 0.83 ± 0.1%, p=0.008) (Figure 5B). Interestingly, combining A2aR antagonist with the FLOT regimen significantly increased the mRNA expression levels of MMS19 compared with FLOT treated cells (1.12 ± 0.1 vs. 0.13 ± 0.06%, p=0.03) (Figure 5B).
Overall, single agent nivolumab, atezolizumab and A2aR antagonist decreased γH2AX expression in OAC cells and decreased the gene expression of DNA repair genes. Similarly, combining single agent nivolumab, atezolizumab with FLOT chemotherapy decreased γH2AX expression in OAC cells and expression of DNA repair genes. Interestingly, although single agent A2aR antagonist decreased γH2AX expression in OAC cells, an increase in expression of DNA repair genes was observed.