Background: Human pancreatic ductal adenocarcinoma (PDAC) responds poorly to immune checkpoint inhibitor (ICPi). While the mechanism is not completely clear, it has been recognized that tumor microenvironment (TME) plays key roles. We investigated if targeting CD47 with a monoclonal antibody could enhance the response of PDAC to ICPi by altering the TME. Methods: Using immunohistochemistry, we examined tumor-infiltrating CD68 + pan-macrophages (CD68 + M) and CD163 + M2 macrophages (CD163 + M2) and tumor expression of CD47 and PD-L1 proteins in 106 cases of PDAC. The efficacy of CD47 blockade was examined in xenograft models. CD45 + immune cells from syngeneic tumor models were subjected to single-cell RNA-sequencing (scRNA-seq) by using the 10x Genomics pipeline. Results: We found that CD47 expression correlated with the level of CD68 + M but not CD163 + M2. High levels of tumor-infiltrating CD68 + M, CD163 + M2, and CD47 expression were significantly associated with worse survival. CD47 high /CD68 + M high and CD47 high /CD163 + M2 high correlated significantly with shorter survival, whereas CD47 low / CD68 + M low and CD47 low /CD163 + M2 low correlated with longer survival. Intriguingly, CD47 blockade decreased the tumor burden in the Panc02 but not in the MPC-83 syngeneic mouse model. Using scRNA-seq, we showed that anti-CD47 treatment significantly remodeled the intratumoral lymphocyte and macrophage compartments in Panc02 tumor-bearing mice by increasing the pro-inflammatory macrophages that exhibit anti-tumor function, while reducing the anti-inflammatory macrophages. Moreover, CD47 blockade not only increased the number of intratumoral CD8 + T cells, but also remodeled the T cell cluster toward a more activated one. Further, combination therapy targeting both CD47 and PD-L1 resulted in synergistic inhibition of PDAC growth in the MPC-83 but not in Panc02 model. MPC-83 but not Panc02 mice treated with both anti-CD47 and anti-PD-L1 showed increased number of PD-1 + CD8 + T cells and enhanced expression of key immune activating genes. Conclusion: Our data indicate that CD47 targeting induces compartmental remodeling of tumor-infiltrating immune cells of the TME in PDAC. Different PDAC mouse models exhibited differential response to the anti-CD47 and anti-PD-L1 blockade due to the differential effect of this combination treatment on the infiltrating immune cells and key immune activating genes in the TME established by the different PDAC cell lines.

Human pancreatic ductal adenocarcinoma (PDAC) exhibits marginal responses to anti-PD-1/PD-L1 immunotherapy and its mechanism remains poorly understood. We have investigated the effect of anti-PD-L1 and c-Myc inhibition in PDAC. Using 87 patients with PDAC from our hospital database we found a significant correlation between the expression of PD-L1 and c-Myc. Moreover, the expression of both PD-L1 and c-Myc was associated with poor overall survival. In addition, we confirmed this finding with the PDAC patients in the TCGA database. Using several PDAC cell lines we demonstrated a significant correlation between the expression of PD-L1 and c-Myc. We also found that expression of PD-L1 correlated with high-grade histology. JQ1, an inhibitor of c-Myc inhibited PD-L1 expression and tumor growth. Using xenograft models, we demonstrated that the combination of JQ1 and anti-PD-L1 antibody exerted synergistic inhibition of PDAC growth. Our data demonstrated that the expression of PD-L1 and c-Myc may be helpful prognostic biomarkers, and their inhibition may potentially serve as an effective treatment for PDAC.

Objective V-domain Ig suppressor of T cell activation (VISTA) is a novel immune checkpoint protein that belongs to the B7 family. The aim of this study was to investigate the prognostic significance and therapeutic potential of VISTA in patients with pancreatic cancer. Methods Using immunohistochemistry (IHC), we examined the expression of VISTA and demonstrated the associations between the VISTA and overall survival in 223 PDAC patients from 2 different unrelated retrospective cohorts. The multiplex immunofluorescence was performed to illuminate the relationship between VISTA expression and tumor-infiltrating immune cell subclusters of PDAC. We also verified the findings in The Cancer Genome Atlas (TCGA) dataset. The anti-tumor effect of anti-VISTA therapy was studied by the mouse model with liver metastases of PDAC. Results The VISTA protein was highly expressed in 25.6% of tumor cells (TCs), 38.1% of immune cells, and 26.0% of endothelial cells in 223 PDAC tumor tissues. VISTA expression in TCs was significantly associated with prolonged overall survival. Multiplex immunofluorescence analysis revealed that VISTA level was positively correlated with CD68+ macrophages, CD3+ T cells, and CD19+ B cells in PDAC. However, a higher expression level of VISTA was detected in tumor-infiltrating CD68+ macrophages than in CD3+ T and CD19+ B cells. Furthermore, anti-VISTA antibody treatment significantly reduced the number of metastatic nodules in livers of mouse models of PDAC with liver metastases. Conclusion VISTA expressed in TCs is associated with a favorable prognosis in PDAC. Moreover, immunotherapy with anti-VISTA antibodies may potentially be an effective treatment strategy against PDAC.

Radiofrequency ablation (RFA) is an effective local therapy approach for treating solitary tumor of many types of malignancy. The impact of RFA on the tumor immune microenvironment on distant tumors after RFA treatment is still unclear. In this study, by using syngeneic tumor models and single-cell RNA and T-cell receptor sequencing, we have investigated the alterations of tumor-infiltrating immune cells in distant non-RFA tumors. Single-cell RNA sequencing identified six distinct lymphoid clusters, five distinct monocyte/macrophage clusters, three dendritic cells clusters, and one cluster of neutrophils. We found that RFA treatment reduced the proportions of immunosuppressive cells including regulatory T cells, tumorassociated macrophages and tumor-associated neutrophils, whereas increased the percentages of functional T cells in distant non-RFA tumors. Moreover, RFA treatment also altered gene expressions in single-cell level in each cell cluster. By using pseudo-time analysis, we have described the biological processes of tumor-infiltrating CD8 + T cells and monocytes/ macrophages based on the transcriptional profiles. In addition, the immune checkpoints including PD-1 and LAG3 were upregulated in the T cells in distant non-RFA tumors after RFA treatment. In conclusion, our data indicate that RFA treatment induced remodeling of tumor immune microenvironment in distant non-RFA tumors in pancreatic cancer mouse model and suggest that combining RFA with immune checkpoint inhibitors may be an effective treatment approach.

Background Most of acute pancreatitis (AP) are mild and self‐limiting, however, 15%–20% of patients develop severe acute pancreatitis (SAP) or moderately acute pancreatitis (MSAP) with local or systemic complications. Infection complications (ICs) result in 40%–70% morbidity and high mortality rates among SAP and MSAP patients. It is more important to early recognize of ICs of MSAP or SAP. Several studies have indicated that serum soluble programmed cell death protein (sPD‐1) or programmed cell death 1 ligand (sPD‐L1) levels were higher in patients with severe sepsis than in healthy volunteers and have a predictive capacity for mortality. However, the role of serum sPD‐1/sPD‐L1 in AP remains unclear. This study aimed to investigate whether the ICs of AP patients is associated with their sPD‐1 and sPD‐L1 levels, which were determined via enzyme‐linked immunosorbent assay of peripheral blood samples from 63 MSAP and SAP patients and 30 healthy volunteers. Results The serum sPD‐1 levels in AP patients on Days 1, 3, and 10 after onset were significantly increased in a time‐dependent manner compared with that in healthy volunteers. Moreover, the AP patients with ICs had significantly higher serum sPD‐1 levels than the AP without ICs. While serum sPD‐L1 levels in AP were similar to that in healthy volunteers. Besides, serum levels of sPD‐1/sPD‐L1 were negatively correlated with circulating lymphocytes. Univariate and multivariate regression analyses showed that the upregulated serum sPD‐1 level was an independent risk factor for ICs in AP. The area under the receiver operating characteristics curve indicated that combination with Acute Physiology and Chronic Health Evaluation II score and serum sPD‐1 level had a high accuracy in predicting ICs in AP. Conclusion Serum sPD‐1/sPD‐L1 may be involved in the immunosuppressive process in AP. Moreover, the serum sPD‐1 level may be an independent risk factor for predicting ICs in AP patients.

The serum soluble CD163 (sCD163) is elevated in patients with inflammatory disease and several types of cancer. However, the prognostic value of serum sCD163 in pancreatic ductal adenocarcinoma (PDAC) has not yet been investigated. In this study, serum level of sCD163 was measured by using the peripheral blood of 54 patients with PDAC, 20 patients with benign tumor of pancreas, and 30 healthy volunteers (healthy controls). The association between serum sCD163 level and overall survival was analyzed. Receiver operating characteristic (ROC) curves were generated, and areas under the curve (AUC) were compared to evaluate the diagnostic accuracy, including CA 19-9, CEA, CA 125, CA 153, and serum sCD163 level. Serum sCD163 level of patients with PDAC was significantly higher than patients with benign tumor (P = .002) and health controls (P < .001). Using ROC curves, we found that the AUC values of serum sCD163 were higher than those of CA 125 and CA 153, but lower than those of CA 19-9 and CEA. Serum sCD163 was negatively correlated with lymphocyte to monocyte ratio (LMR; r = −0.428, P = .001). In addition, the prognosis of PDAC patients with sCD163 ≥ median was worse than sCD163 < median by using univariate analysis (P = .027). Further, multivariate analysis showed that higher level of serum sCD163 was still associated with poorer overall survival (P = .020). In conclusion, the serum sCD163 has the potential as a new promising parameter to predict the prognosis in PDAC patients.