MicroRNAs (miRNAs) have been reported to modulate macrophage colony-stimulating factor (M-CSF) and macrophages. The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages. Hepatocellular carcinoma (HCC) cell lines with decreased or increased expression of miR-26a were established in a previous study. M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay, and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro. Real-time PCR measured a panel of mRNAs expressed by macrophages. Xenograft models were used to observe tumor growth. Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC. Ectopic expression of miR-26a reduced expression of M-CSF. The conditioned medium (CM) from HepG2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate (PMA) increased expression of interleukin (IL)-12b or IL-23 mRNA and decreased expression of chemokine (C-C motif) ligand (CCL)22, CCL17, and IL-10 mRNA, in comparison to the medium from the parental HepG2 cells. These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002. Ectopic expression of miR-26a in HCC cells suppressed tumor growth, M-CSF expression, and infiltration of macrophages in tumors. Similar results were also found when using HCCLM3 cells. Furthermore, the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC. miR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

Immunosuppression is an important factor for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Follicular helper T cells (Tfh cells) play an anti-tumor role in various malignant solid tumors and predict better patient prognosis. In the present study, we aimed to determine the immunosuppressive mechanism associated with Tfh cells and explore a new strategy to improve the tumor microenvironment of PDAC. Flow cytometry was used to detect the infiltration and proportion of Tfh cells in tumor tissues and peripheral blood from patients with PDAC. The spatial correlations of Tfh cells with related immune cells were evaluated using immunofluorescence. The function of Tfh cells was examined using in vitro and in vivo model systems. The high infiltration of Tfh cells predicted better prognosis in patients with PDAC. Tfh cells recruited CD8⁺ T cells and B cells by secreting C-X-C motif chemokine ligand 13 (CXCL13), and promoted the maturation of B cells into antibody-producing plasma cells by secreting interleukin 21 (IL-21), thereby promoting the formation of an immunoactive tumor microenvironment. The function of Tfh cells was inhibited by the programmed cell death 1 ligand 1 (PD-L1)/programmed cell death 1 (PD-1) signaling pathway in PDAC, which could be reversed using neoadjuvant chemotherapy. Treatment with recombinant CXCL13, IL-21 and Tfh cells alleviated tumor growth and enhanced the infiltration of CD8⁺ T cells and B cells, as well as B cell maturation in a PDAC mouse model. Our results revealed the important role of Tfh cells in mediating anti-tumor cellular immunity and humoral immunity in PDAC via secreting CXCL13 and IL-21 and determined a novel mechanism of immunosuppression in PDAC.

Background: Tertiary lymphoid structures (TLS) are associated with favorable survival and play a critical role in most solid tumors. However, investigations of TLS are lacking in patients with grade 1 or grade 2 (G1/G2) non-functional pancreatic neuroendocrine tumors (NF-PanNETs). This study aimed to investigate the presence, cellular composition, association with tumor-infiltrating immune cells, and prognostic value of TLS in G1/G2 NF-PanNETs. Methods: Tumor tissues from a 182-patient Fudan cohort and a 125-patient external validation set were assessed by H&E staining, immunohistochemistry, and/or multispectral fluorescent immunohistochemistry. Results: TLS were identified in more than one-third of patients with G1/G2 NF-PanNETs and were located peritumorally, either just outside the tumor tissue or in the stromal area. TLS were mainly composed of B-cell follicles with germinal centers and T-cell zones with dendritic cells. Kaplan-Meier analyses showed that the presence of TLS correlated with both longer recurrence-free survival (RFS, p<0.001) and overall survival (OS, p=0.001), but the number of TLS had no prognostic significance. Multivariate Cox-regression analyses demonstrated that the presence of TLS, WHO classification, and 8th edition American Joint Committee on Cancer (AJCC^8th) tumor-node-metastasis (TNM) stage were independent prognostic factors for RFS (p=0.004, p=0.001, and p<0.001, respectively) and OS (p=0.009, p=0.008, and p=0.019, respectively). These results were confirmed using an external validation set. Finally, a nomogram incorporating the presence of TLS was constructed to predict the probability of 5-year RFS of resected G1/G2 NF-PanNETs, which improved on the current WHO classification and AJCC^8th TNM stage. Conclusions: The presence of TLS is an independent and favorable predictor of resected G1/G2 NF-PanNETs, which may play a role in cancer immunobiology.

Objective This study retrospectively characterized the immune infiltrating profile in non-functional pancreatic neuroendocrine tumors (NF-PanNETs). Methods Tumor tissues from the 109-patient Fudan cohort and 73-patient external validation set were evaluated by immunohistochemistry for nine immune cell types: tumor-infiltrating neutrophils (TINs), tumor-associated macrophages (TAMs), CD11c⁺ dendritic cells, anti-NCR1⁺ NK cells, CD4⁺ and CD8⁺ T cells, CD45RO⁺ memory T cells, FOXP3⁺ regulatory T cells (Tregs), and CD20⁺ B cells. Results TINs were primarily distributed in intratumoral area, dendritic cells and NK cells were scattered evenly in intratumoral and stromal areas, and Tregs were rarely detected. The remaining five cell types were primarily present in peritumoral stroma. Total TINs (P<0.001) and TAMs (P=0.002) increased as NF-PanNET-grade rose. Kaplan-Meier analyses showed that high intratumoral TINs, total TAMs, and stromal CD4⁺ T cell infiltration correlated with shorter recurrence-free survival (RFS, P=0.010, P=0.027, and P=0.035, respectively) and overall survival (OS, P=0.017, P=0.029, and P=0.045, respectively). Additionally, high intratumoral CD8⁺ T cell infiltration correlated with prolonged RFS (P=0.039). Multivariate Cox regression demonstrated that intratumoral TINs, WHO classification and AJCC^8th TNM were independent factors for RFS (P=0.043, P=0.023, and P=0.029, respectively), whereas intratumoral TINs and WHO classification were independent factors for OS (P=0.010 and P=0.007, respectively). Furthermore, combination of TINs, WHO classification and AJCC^8th TNM remarkably improved prognostic accuracy for RFS. Above results have been verified in the external validation set. Conclusion Intratumoral TINs are an independent and unfavorable predictor of postoperative NF-PanNETs. A combination of TINs, WHO classification, and AJCC^8th TNM could improve prognostic accuracy for RFS.