Combination therapy strategies with gemcitabine have often failed to improve the survival in patients with pancreatic cancer[15–18]. Notably, the addition of nab-paclitaxel has increased the survival of pancreatic cancer patients when treatment is combined with gemcitabine. However, the 2-year survival rate in patients that receive nab-paclitaxel plus gemcitabine is approximately 9% in comparison to 4% who received gemcitabine alone. Thus, there is a potent need to define therapeutic combinations that enhance the durability of clinical responses that ultimately extend the survival of pancreatic cancer patients. With the results described in this study, we believe that it is feasible to combine intralesional therapy with PV-10 with standard of care chemotherapy. We provide evidence that the potentiation of anti-tumor immune responses is necessary for profound tumor growth stabilization and regression. Specifically, treated Panc02 tumors were controlled upon i.l. therapy with PV-10, but untreated bystander tumors were unaffected (Fig. 3C-D). In contrast, in Panc02 tumors that express that immunogenic neoantigen, OVA, we observed that monotherapy with gemcitabine or PV-10 was equally as effective at controlling tumor growth in a single flank model (Fig. 4A). Moreover, PV-10 treatment alone or in combination with gemcitabine was capable of inducing the complete regression of treated tumors, while gemcitabine monotherapy failed to induce complete regressions. This was in contrast to the effectiveness of gemcitabine monotherapy in mice bearing a single Panc02OVA tumor. This suggests that the increased tumor burden in mice with lesions on both flanks diminished the efficacy of gemcitabine, which was overcome when combined with i.l. PV-10. In addition, the rate of tumor growth of untreated bystander tumors was slowed in mice that received PV-10 in combination with gemcitabine versus gemcitabine alone (Fig. 4B-C). This suggests that an immunogenic antigen potentiates the combination of PV-10 and gemcitabine which results in the regression of local and distally untreated tumors.
We next examined the efficacy of PV-10 combination therapy in Panc02 tumors that did not express OVA. We observed that PV-10 treatment alone had a modest effect in reducing tumor growth. Indeed, gemcitabine monotherapy effectively reduced tumor growth. However, the reduction of tumor growth was enhanced in mice that received combination therapy with PV-10 and gemcitabine (Fig. 5). Despite that Panc02 tumors are less immunogenic than Panc02OVA tumors, we were able to observe a significant improvement in tumor growth control and regression in mice that received combination therapy. Thus, the combination of gemcitabine with PV-10 can induce tumor regression even in less immunogenic tumors.
Treatment with gemcitabine is associated with the depletion of MDSCs and the promotion of tumoricidal activity by tumor-associated macrophages[24, 25]. Indeed, we observed that gemcitabine effectively reduced the frequency of bulk CD11b+ myeloid cells within spleens. However, there was a proportional shift characterized by the reduction of CD11b+Gr-1+ MDSCs and an increase in CD11b+Gr-1− myeloid cells. This reduction of MDSCs was ultimately associated with reduced tumor growth in mice that received gemcitabine alone or the combination with PV-10. We further investigated systemic changes that could impact the immune system in response to PV-10 treatment. Indeed, PV-10 treatment alone increased the abundance of HMGB1 within 24hrs after injection (Fig. 3B). We and others have shown that HMGB1 is an important mediator of DC activation and promotion of anti-tumor immunity[1, 6, 26]. Intriguingly, the increased abundance of HMGB1 and other DAMPs persisted in mice that received PV-10 in combination with gemcitabine (Fig. 7). Specifically, we observed that HMGB1, S100A8, and IL-1α were increased 9 days after treatment in mice that received PV-10 in combination with gemcitabine. Notably, mice that received the combination therapy exhibited the greatest reduction in tumor growth amongst all experimental groups, suggesting that the increased abundance of DAMPs in circulation is associated with better therapeutic responses. While HMGB1 can promote anti-tumor immune responses, it can simultaneously potentiate tumor cell survival mechanisms[27, 28]. Similarly, S100 proteins and IL-1α appear to have important roles in promoting pancreatic tumor progression. For instance, S100A8 and S100A9 enhances the production of IL-8 in pancreatic tumor cells which could promote the accumulation of immunosuppressive myeloid cells, including MDSCs[29–31]. Meanwhile, IL-1α can enhance the metastatic potential of pancreatic tumor cells by maintaining the constitutive activation of nuclear factor κ-B (NFκB) and promoting the secretion of hepatocyte growth factor (HGF)[32, 33]. Despite the roles of these DAMPs in promoting pancreatic tumor progression, we observed that the increase of these factors was associated with tumor regression in mouse models. Hence, the specific roles of DAMPs that are released by pancreatic tumors after PV-10 treatment remain unclear. Future studies will address the immunological consequences of these DAMPs on promoting anti-tumor immunity in the context of pancreatic cancer.
In conclusion, we demonstrate that intralesional therapy with PV-10 is a feasible strategy to augment therapeutic responses when combined with gemcitabine. Together, the results of this study provide support for future studies to investigate the induction of systemic anti-tumor immune responses after PV-10 treatment.