ICB has become a promising immunotherapeutic modality for several refractory carcinomas, however its roles in PDAC is limited. The challenges majored in the identification of target patients, and finding effective combination therapeutic targets to amplify its clinical efficacy. In this study, we utilized published immune signature gene sets to depict the distinct immune features of PDAC and three immune subtypes are identified. The prognostic impact of immune gene sets, immune cell composition and immunomodulators are evaluated. The diversity of immune response reflects the inner cause of prognostic discrepancy and immunotherapy failure in PDAC. Meanwhile, our study firstly put forward that the high expression of TGM2 tracked with an immunosuppression-promoting phenotype and TGM2 is involved in the modulation of PD-L1 expression by regulating downstream transcription factor STAT3/NF-κB in pancreatic cancer cells. In addition, the enrichment disparity of IMs in each subtypes may suggest multiple potential combination immunotherapy strategies.
The prognostic impact of tumor immune feature is emerging to be concerned. Among all subtypes in our study, patients comprising C2 have the worst overall survival. The reasons for the prognostic discrepancy can be attributed to the following points. Firstly, the immune signatures in C2 are dominated by TGF-β and Wound healing modules which were associated with an immunosuppressive and pro-tumor phenotype [16, 17], and inversely correlated with patients prognosis based on our data. Meanwhile, the lowest enrichment degree in Lymphocyte module is another feature of C2. Consistent with that, the infiltration of T-, B-lymphocytes and NKT cells in C2 is less than the other subtypes, indicating a poor preexisting anti-tumor immunity . Moreover, the highest enrichment degree of IFN-γ module is found in C2. IFN-γ is a major mediator inducing the death of tumor cells. However, continuous exposure to IFN-γ also stimulates tumor cells to express multiple inhibitory modulators including PD-L1 which are majorly enriched in C2 and C3 in our study, thereby suppress the secretion of IFN-γ by effector T cells and result in T cell exhaustion [19, 20]. Together, a more prominent feature of immunosuppression may be promoted by these features of C2 and therefore the prognosis of C2 is worse. In addition to these molecular immune features, our results show that the overall burden of NKT cells is associated with a better prognosis in PDAC, while that is rarely reported before. Similar with CD8+ T cells, NKT cells is also regarded as one of the front-line anti-tumor forces . Apart from directly targeting on the tumor cells with CD1d positive, recent literature on mice model demonstrated that NKT cells could restrict the tumor evolution of PDAC indirectly by suppressing the pro-immunosuppression role of macrophages through prostaglandin E synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) . Consistent with that, subtype C2, with high fraction of macrophage and low fraction of NKT, shows more prominent immunosuppression phenotype than C1 and C3. These distinct molecular and cellular features across three subtypes show a diverse immune response of PDAC, which would be condition for prognostic evaluation and immunotherapy strategy designing.
To improve the clinical efficacy of anti-PD1/PDL1 therapy in PDAC, reasonable patient stratification and combination strategy designing should be concerned. For the former, prior studies indicated that the level of preexisting anti-tumor immunity and the expression level of PD-1/PD-L1 are two vital factors for the efficacy of anti-PD-1/PD-L1 treatment . In our study, tumor samples comprising C3 are rich in various anti-tumor cytokines, such as TNF and IFN-γ and have abundant infiltration of immune cells including NKT cells and T lymphocytes as well as B lymphocytes. Despite the anti-tumor effect of B cells remained unclear, a recent study in melanoma indicated that B cells may contribute to the response to ICB treatment by altering T cell activation and function . As well, C3 has the highest PD-1 and PD-L1 enrichment across the three subtypes. Thus, patients within C3 subtype seem to be more suitable for anti-PD treatment. Meanwhile, based on our data, C3 is also rich in CTLA4, TIGIT and HAVCR2 expression which may provide alternative options for combined targets. Compared with single-agent ICB therapy, combination treatment of anti-CTLA4 (ipilimumab) and anti-PDL1 (nivolumab or pembrolizumab) led to better tumor response and patient survival in melanoma, sarcoma and small cell lung cancer [25–29]. Besides, preclinical studies have demonstrated that anti-TIGIT or anti-HAVCR2 can effectively control tumor evolution, suggesting a promising combination target for anti-PD-1/PD-L1 treatment [30, 31].
In contrary with subtype C3, anti-PD-1/PD-L1 treatment may be not appropriate for the patients within subtype C1 due to the relatively low infiltration of T/B lymphocytes and the poorest enrichment in PD-1/PD-L1. Of note, C1 has the highest VTCN1 expression as well as favorable enrichment scores in NKT cells. As a newly discovered immune checkpoint expressed on APC cells and tumor cells, VTCN1 is expected to become a novel target for immunotherapy in the future despite its regulatory mechanism in cancer immunity remained to be further explored . Due to advantages of targeted on tumor cells and suppressive effect on graft versus host disease, NKT cells (majorly invariant NKT cells) is regarded as a viable vector for the CAR or rTCR treatments with multiple pre-clinical animal models supporting favorable anti-tumor effects in solid tumors [33–35].
Subtype C2 has favorable enrichment scores in PD-L1, CD276 and VTCN1 but is poor at lymphocytic infiltration. In addition to anti-PD-L1 treatment to restore the anti-tumor immunity, strategies to target oncogenic pathway is needed for patients in C2 to restrict tumor progression. TGM2 is a promising target for improving the response to chemotherapy in solid tumors including PDAC . While its role in the immune evade process of PDAC remains unclear. In this study, we find that TGM2 is positively related with the expression of multiple inhibitory immunomodulators, and the high-TGM2 group is mainly enriched in the immunosuppressive subtype C2, suggesting that TGM2 may be involved in the regulation of immunosuppression in PDAC. Through in vitro experiments, we verified that TGM2 has a positive impact on PD-L1 in PANC-1 and Mia PaCa-2 cells. The underlying mechanisms may refer to two aspects. Firstly, STAT3 has been reported to be involved in the regulation of PD-L1 expression in various cancer types [37–39]. However, whether TGM2 alters the expression of PD-L1 in PDAC via STAT3 signaling remains to be unknown. Our present study reveals that down-regulating TGM2 in PANC-1 and Mia PaCa-2 cells results in a decreased phosphorylation of STAT3, which indicates a potential pathway for the regulation of PD-L1 by TGM2. Secondly, previous studies revealed that TGM2 promotes the activation of AKT by suppressing PTEN, then result in the activation of downstream substances including transcription factor NF-κB in PDAC [10, 40]. Consistent with that, knocking down TGM2 in PANC-1 cells leads to a decrease in p-Akt and p-P65 expression. As a vital transcriptional factor, NF-κB can regulate the transcription of PD-L1 by directly binding to the promoter region of PD-L1 . Thus, TGM2 may take a positive impact on PD-L1 expression via Akt/ NF-κB pathway. These findings provide insights into the regulation network of PD-L1 expression in PDAC (Fig. 7f) and thereby TGM2 is expected to be a promising target for anti-PD-1/PD-L1 therapy.
Some limitations in this study need to be addressed. Firstly, as a retrospective study, the clinical value of these findings need to be further validated in a larger prospective cohort. Secondly, the potential selection bias of tumor specimens may exist. Thirdly, though transcriptional profiles in our study provided us underlying features of immune response in PDAC, with multi-omics data gathering, more solid evidence will promote our insight into tailored treatment for PDAC.
In summary, our study uncovers the specific immune features among PDAC patients. For the discrepant immune response mechanisms, more personalized ICB treatment should be considered. Moreover, TGM2 regulates the expression of PD-L1 in PDAC via STAT3 and Akt/NF-κB signaling pathway and predicts poorer survival of PDAC patients, indicating a potential role in immunotherapy.