As an emerging technique for local tissue ablation, IRE has shown excellent efficacy in animal experiments and during clinical studies of pancreatic cancer [10,23–25]. However, its clinical use is hampered by the high recurrence rate[15,16]. This study demonstrated the efficacy of IRE on pancreatic cancer. Then, integrated omics analyses involving transcriptomics, proteomics, and scRNA-seq revealed that IRE significantly altered the cell distribution and functional characteristics of CD4+ T cells. The present study offers an in-depth analysis of changes in pancreatic cancer at the molecular level after IRE procedures and provide a reference for identifying potential strategies for IRE combination treatments.
The ability of IRE to destroy tumors and achieve local tumor control has been confirmed[23,25]. However, Narayanan et al. revealed that only 20% of pancreatic cancer mouse models showed no tumor recurrence after IRE[26,27]. Our study revealed similar results: the subcutaneous tumors showed a significant reduction in tumor volume after IRE; however, all of them regrew again. Moreover, most patients who received IRE for pancreatic cancer underwent tumor recurrence and metastatic disease[14,16]. While new techniques of IRE are applied in the clinic, such as real-time current monitoring and high-frequency IRE, this phenomenon will persists[28]. Therefore, IRE alone is evidently insufficient to eradicate pancreatic cancer.
The immunomodulatory effects of IRE on tumors have attracted the attention of numerous scholars, and most current studies focused on CD8+ T cells due to their important role in anti-tumor immunity [23,29]. However, CD4+ T cells are also critical in pancreatic cancer development[30,31]. Some CD4+ T cell subsets, such as CD4+ effector T cells, can enhance the antibody synthesis and secretion by B cells and proliferation of natural killer cell cells, while some lead to tumor immune escape through immune resistance, such as CD4+ Tregs[32,33]. Some studies assessed changes in CD4+ T cells for pancreatic cancer before and after IRE, but the results are not conclusive. He et al. found a transient decrease followed by a steady increase in CD4+ T cells in peripheral blood samples of patients with pancreatic cancer [34]. A clinical trial involving 10 patients with pancreatic cancer showed that the number of CD4+ T cells in their blood remained unchanged after IRE treatment [35]. Furthermore, an experimental study of pancreatic cancer in mice reported no significant difference in the total number of infiltrating CD4+ T cells in tumor tissues before and after IRE ablation, but CD4+ memory T cells increased significantly after IRE ablation [36]. The different conclusions drawn by these studies could be attributed to the varied parameters used for IRE and the conditions of the patients with pancreatic cancer or the animal models. In our study, the parameters used for IRE ablation were as follows: voltage = 1000 V, pulse duration = 100 ms, pulse frequency = 1 Hz, and pulse number = 90, which could largely cover the tumors of mice. After IRE, the transcriptomics and proteomics of orthotopic tumors indicated that Th1, Th2, and Th17 cell differentiation were the most enriched signaling pathways in DEGs and DEPs, indicating that CD4+ T cells might be the central immunoregulatory mediator in pancreatic cancer after IRE.
We further assessed the distribution and function of CD4+ T cells in tumors before and after IRE treatments using scRNA-seq. The results showed that the proportion of CD4+ effector T cells in CD4+ T cells significantly decreased, whereas the proportion of naive and Tregs in CD4+ T cells significantly increased in pancreatic cancer post IRE. Furthermore, the GSEA revealed a significant downregulation of TNF and IL-17 pathways in CD4+ effector T cells and CD4+ naive T cells post IRE. As TNF can induce tumor cell death and IL-17 can enhance innate and adaptive immunity as an early initiator, the results indicated the downregulation of the release of cytokines and killing capacity [37,38]. Therefore, IRE did not augment the anti-tumor effects of CD4+ T cells in pancreatic cancer in our study. Although the results and the specific mechanism need to be further verified, it is a potential treatment method to enhance the proliferation and differentiation of CD4+ effector T cells after IRE, which can serve as an integrative approach to enhance the anti-tumor immunity and improve the efficacy of IRE.
Tregs, an inhibitory subset of CD4+ T cells, contribute to tumor immune escape by inhibiting anti-tumor immunity [32,39]. Their presence often leads to poor clinical prognosis. Various studies have examined changes in Tregs in pancreatic cancer post IRE. During animal experiments, Guo et al. reported that the proportion of Tregs in the blood of mice significantly decreased after IRE treatments, which might be related to a reduced tumor burden [40]. However, they also observed an increase in the proportion of Tregs in pancreatic cancer tumor tissues and spleen by 2 days after IRE. During clinical trials, Pandit et al. analyzed Tregs in the peripheral blood of 11 patients with pancreatic cancer who received IRE treatment. The results showed that the number of Tregs decreased 1 day after the treatment but significantly increased in peripheral blood 3–5 days thereafter [41]. In contrast, a clinical trial involving 34 patients with pancreatic cancer revealed that the number of Tregs in the blood of pancreatic cancer patients with pancreatic cancer showed a transient increase by 3 days after IRE treatments, followed by a decrease on day 7 [34]. Similar to the findings of this study, we found that Tregs increased remarkably in pancreatic cancer after IRE. The expression of the co-repressive genes Ctla-4 and Tigit in Tregs also increased significantly. These results indicated that IRE ablation might induce the proliferation of Tregs in pancreatic cancer, which might augment the immune resistance. Moreover, the post-IRE stress induced by trauma must be considered, as it could cause a serious inflammatory response. The increase of Tregs after IRE might serve as an important process to maintain organismal homeostasis.
Although the exact reason remains unclear, an increase in Tregs is generally considered unfavorable for the prognosis of the pancreatic cancer patients. Immunotherapies targeting Tregs after IRE may be a valuable strategy. Currently, CTLA-4 antibodies are the most successful treatment targeting Tregs [42,43]. The combination of IRE and CTLA-4 antibodies is yet to be applied in research studies on treatment for pancreatic cancer. Burbach et al. attempted using IRE combined with CTLA-4 antibodies to treat prostate cancer in mice, indicating that the combined treatment could promote the production of CD8+ memory T cells in various tissues and tumor regression [44]. The data of this study indicated that CTLA-4 antibodies were highly expressed in the Tregs of pancreatic cancer model mice after IRE, which provided a good basis for treatments based on CTLA-4 antibodies.
In addition, our study found that IRE treatment-related DEGs and DEPs were concentrated in the signaling pathways for ECM and ECM-receptor interactions. The deposition of large amounts of ECM is a well-known histological indicator of pancreatic cancer, which acts as a natural physical barrier that severely hampers immune cell infiltration and drug delivery [45,46]. Hence, various studies have attempted to reshape the tumor microenvironment by targeting the ECM of pancreatic cancer tissues [47]. However, the more widely used ECM-targeting drugs—matrix metalloproteins and hyaluronic acid inhibitors—have not produced good therapeutic effects in patients with pancreatic cancer[48–50]. As a local ablation therapy, IRE can physically destroy and remodel the ECM of pancreatic cancer tissues, yielding therapeutic effects that may be more significant than those of drug therapy. Currently, studies on the application of IRE to the ECM of pancreatic cancer are relatively scarce. Fujimori et al. applied IRE and MWA to healthy porcine lungs and found that the pulmonary blood vessels and ECM proteins treated with MWA were destroyed 2 days later [51]. By comparison, approximately 50% of the blood vessels and ECM scaffolds in the porcine lungs were preserved after IRE treatment. In addition, the number of macrophages and T cells in the lungs of the porcine IRE group was significantly higher than that in the MWA group. Compared with the destruction of thermal ablation, IRE preserved the blood vessels and ECM scaffolds in porcine lungs, thereby effectively increasing immune cell infiltration. Therefore, we hypothesize that an IRE ablation might also promote an anti-tumor immune response by remodeling the ECM of pancreatic cancer tissues. However, further experimental confirmation is required.