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.

Silica nanoparticles are increasingly used in the biomedical fields due to their excellent solubility, high stability and favorable biocompatibility. However, despite being considered of low genotoxicity, their bio-related adverse effects have attracted particular concern from both the scientific field and the public. In this study, human cervical adenocarcinoma cells (HeLa line) were exposed to 0.01 or 1.0 mg/mL of hydrophilic silica nanoparticles. The ¹H NMR spectroscopy coupled with multivariate statistical analysis were used to characterize the metabolic variations of intracellular metabolites and the compositional changes of the corresponding culture media. At the early stage of silica nanoparticles-exposure, no obvious dose–effect of HeLa cell metabolome was observed, which implied that cellular stress-response regulated the metabolic variations of HeLa cell. Silica nanoparticles induced the increases of lipids including triglyceride, LDL, VLDL and lactate/alanine ratio and the decreases of alanine, ATP, choline, creatine, glycine, glycerol, isoleucine, leucine, phenylalanine, tyrosine, and valine, which involved in membrane modification, catabolism of carbohydrate and protein, and stress-response. Subsequently, a complicated synergistic effect of stress-response and toxicological-effect dominated the biochemical process and metabolic response, which was demonstrated in the reverse changes of some metabolites including acetate, ADP, ATP, choline, creatine, glutamine, glycine, lysine, methionine, phenylalanine and valine between 6 and 48 h post-treatment of silica nanoparticles. The toxicological-effects induced by high-dosage silica nanoparticles could be derived from the elevated levels of ATP and ADP, the utilization of glucose and amino acids and the production of metabolic end-products such as glutamate, glycine, lysine, methionine, phenylalanine, and valine. The results indicated that it is important and necessary to pursue further the physiological responses of silica nanoparticles in animal models and human before their practical use. NMR-based metabolomic analysis helps to understand the biological mechanisms of silica nanoparticles and their metabolic fate, and further, it offers an ideal platform for establishing the bio-safety of existing and new nanomaterials.

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.

Impairment of the blood-brain barrier (BBB) in severe acute pancreatitis (SAP) could result in life-threatening pancreatic encephalopathy. Interleukin-10 (IL-10) is a classical cytokine that is well-known for its strong immunoregulatory and anti-inflammatory abilities. However, whether and how IL-10 protects the BBB in SAP are still unclear. This study includes in vivo experiments using a SAP rat model and in vitro experiments using an in vitro BBB model consisting of a monolayer of brain microvascular endothelial cells (BMECs). The study groups are divided into the control, SAP (in vivo)/TNF-α (in vitro), IL-10 treatment, IL-10 + signal transducer and activator of transcription 3 (STAT3) inhibitor S3I-201 treatment groups. Pancreatic pathological scores, serum amylase, serum TNF-α levels and BBB permeability by Evan’s blue assay in SAP rat models were evaluated. BMEC apoptosis in SAP rats or induced by TNF-αin vitro was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and flow cytometry, separately. Expression levels of claudin-5 and proteins involved in the STAT3 signaling pathway were measured by Western blotting. Location and changes of junctional structure of claudin-5 on BMECs were assessed by immunohistochemistry and immunofluorescence. In vivo, IL-10 alleviated the severity of inflammation, attenuated the increased BBB permeability in SAP rat models by reducing BMEC apoptosis via the STAT3 pathway and ameliorated the down-regulation of claudin-5 expression in BMECs; in vitro, IL-10 improved BBB integrity against TNF-α by attenuating BMEC apoptosis via the STAT3 pathway, the impairment of tight junction structure and the down-regulation of claudin-5 expression in BMECs. IL-10 improves BBB properties in SAP by attenuating the down-regulation of claudin-5 expression and the impairment of tight junctions and by STAT3 pathway-mediated anti-apoptotic effects on BMECs.