IAP inhibitor, Embelin increases VCAM-1 levels on the endothelium, producing lymphocytic infiltration and antitumor immunity.

There is an increasing unmet need for successful immunotherapeutic interventions. Lymphocyte extravasation via tumor tissue endothelial cells (TECs) is required for lymphocyte infiltration into tumor sites. This study aimed to investigate the clinical significance of dysfunctional TECs in pancreatic ductal adenocarcinoma (PDAC) and identify chemical compounds that boost tumor-infiltrating lymphocyte (TIL) numbers. We performed immunohistochemical detection and clinicopathological analysis of VCAM-1 on TECs, which is essential for lymphocyte trafficking. We characterized the gene expression profiles of TECs from fresh PDAC tissues. We isolated compounds that upregulated VCAM-1 and E-selectin expression in TECs and examined their biological activities. Compared to endothelial cells from chronic pancreatitis tissue, TECs showed significantly lower VCAM-1 and E-selectin expression and significant weaknesses in lymphocyte adhesion and transmigration, resulting in decreased T cell infiltration around vessels. Patients with a relatively high percentage of VCAM-1+ vessels among all vessels in PDAC tissue had an improved prognosis. A bioinformatics survey demonstrated that TNFR1 signaling was involved in abnormal VCAM-1 and E-selectin expression in TECs. We screened compounds affecting TNFR1 signaling, and the IAP inhibitor, Embelin, induced these molecules on TECs and enhanced T cell adhesion to TECs and transmigration. Furthermore, in vivo, Embelin enhanced tumor-infiltrating T cell numbers, leading to an antitumor immune response. Embelin accelerates TIL infiltration and the antitumor immune response by recovering VCAM-1 expression in TECs. Our strategy may be a therapeutic approach for accelerating the immunotherapeutic response in immune-quiescent tumors, leading to clinical trials' success.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

Tissue amino acid profiles are characteristic of tumor type, malignant phenotype, and tumor progression in pancreatic tumors.

Tissue amino acid profiles depend on the cell types and extracellular components that constitute the tissue, and their functions and activities. We aimed to characterize the tissue amino acid profiles in several types of pancreatic tumors and lesions. We examined tissue amino acid profiles in 311 patients with pancreatic tumors or lesions. We used newly developed LC-MS/MS methods to obtain the profiles, which were compared with clinicopathological data. Each tumor or lesion presented a characteristic tissue amino acid profile. Certain amino acids were markedly altered during the multistep pancreatic carcinogenesis and pancreatic ductal adenocarcinoma (PDAC) progression. A tissue amino acid index (TAAI) was developed based on the amino acids that were notably changed during both carcinogenesis and cancer progression. Univariate and multivariate survival analyses revealed that PDAC patients with a high TAAI exhibited a significantly shorter survival rate, and these findings were validated using a second cohort. We suggest that tissue amino acid profiles are characteristic for normal tissue type, tumor histological type, and pathological lesion, and are representative of the cancer grade or progression stage in multistep carcinogenesis and of malignant characteristics. The TAAI could serve as an independent prognosticator for patients with PDAC.

Reliable evaluation of tumor-infiltrating lymphocytes in pancreatic cancer tissue biopsies.

Tumor-infiltrating lymphocytes (TILs) represent cancer microenvironment. We previously reported TILs was prognosticators in pancreatic ductal adenocarcinoma (PDAC) patients by immunohistochemically measuring them in surgically-resected tissues. The aim of this study was to assess how best to evaluate TILs in PDAC tissue biopsies. First, we showed expression of CD3, CD4, or CD8 genes in PDAC tissue measured by quantitative RT-PCR (RT-qPCR) was prognostic using 241 surgically-resected specimens. We assessed whether the TILs in biopsied tissues can be effectively evaluated by comparing between immunohistochemistry and RT-qPCR. As a study model, we sampled twenty biopsies from surgically-resected PDAC specimen (n = 17). We investigated the variation levels of TILs in the different biopsies from the same specimen and evaluated using the intraclass correlation coefficient (ICC). The ICC value was 0.58 for CD3, 0.61 for CD4, and 0.46 for CD8, respectively; these ICC values meant correlations of "moderate" to "substantial" levels. To reach "near perfect", 3, 3, and 5 times biopsies were necessary for CD3, CD4, and CD8, respectively. When ICC values of immunolabeled TILs were of "low", ≥6 times biopsies were necessary to reach "moderate" levels. We found that TILs measured by RT-qPCR and repeated sampling increased reliability in TILs detected from biopsied PDAC tissues.

Tertiary Lymphoid Organs in Cancer Tissues.

Tertiary lymphoid organs (TLOs) are induced postnatally in non-lymphoid tissues such as those affected by chronic infections, autoimmune diseases, and chronic allograft rejection, and also in cancer tissues. TLOs are thought to provide important lymphocytic functional environments for both cellular and humoral immunity, similar to lymph nodes or Peyer's patches. TLOs have a structure similar to that of lymph nodes or Peyer's patches, including T cell zones, B cell follicles, and high endothelial venules (HEV) without encapsulation. Here, we review recent advances in our knowledge of TLOs in human solid cancers, including their location, structure, methods of evaluation, and clinicopathological impact. We also discuss the formation and/or maintenance of TLOs in cancer tissues in association with the tumor immune microenvironment, cancer invasion, and the tissue structure of the cancer stroma.

Arginase II expressed in cancer-associated fibroblasts indicates tissue hypoxia and predicts poor outcome in patients with pancreatic cancer.

An adequate level of arginine in the tissue microenvironment is essential for T cell activity and survival. Arginine levels are regulated by the arginine-catabolizing enzyme, arginase (ARG). It has been reported that arginase II (ARG2), one of two ARGs, is aberrantly expressed in prostate cancer cells, which convert arginine into ornithine, resulting in a lack of arginine that weakens tumor-infiltrating lymphocytes and renders them dysfunctional. However, immune suppression mediated by ARG2-expressing cancer cells in lung cancer has not been observed. Here we studied the expression of ARG2 in pancreatic ductal carcinoma (PDC) tissue clinicopathologically by examining over 200 cases of PDC. In contrast to prostate cancer, ARG2 expression was rarely demonstrated in PDC cells by immunohistochemistry, and instead ARG2 was characteristically expressed in α-smooth muscle actin-positive cancer-associated fibroblasts (CAFs), especially those located within and around necrotic areas in PDC. The presence of ARG2-expressing CAFs was closely correlated with shorter overall survival (OS; P  = 0.003) and disease-free survival (DFS; P  = 0.0006). Multivariate Cox regression analysis showed that the presence of ARG2-expressing CAFs in PDC tissue was an independent predictor of poorer OS (hazard ratio [HR]  = 1.582, P  = 0.007) and DFS (HR  = 1.715, P  = 0.001) in PDC patients. In addition to the characteristic distribution of ARG2-expressing CAFs, such CAFs co-expressed carbonic anhydrase IX, SLC2A1, or HIF-1α, markers of hypoxia, in PDC tissue. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 transcript after exposure to hypoxia, which had arginase activity. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not a general event and that the presence of ARG2-expressing CAFs is an indicator of poor prognosis, as well as hypoxia, in PDC tissue.

CXCL17 and ICAM2 are associated with a potential anti-tumor immune response in early intraepithelial stages of human pancreatic carcinogenesis.

BACKGROUND & AIMS: Anti-tumor immunity changes over the course of tumor progression; it is not clear how or when the developing tumor overcomes immune surveillance. Intraductal papillary mucinous neoplasm (IPMN) is an intraepithelial precursor lesion of pancreatic cancer that progresses from adenoma to carcinoma. We investigated when and how the human anti-tumor immune reaction changes during pancreatic tumor development. METHODS: Using immunohistochemical analysis of cells isolated from patients with IPMN, the numbers of tumor-infiltrating lymphocytes and dendritic cells and the maturation state of dendritic cells in the regional lymph nodes were investigated during tumor progression. Gene expression profiles were compared among epithelial neoplastic cells at each stage of tumor development. Biological functions of the selected gene products were analyzed using syngeneic mouse models. RESULTS: The anti-tumor immune reaction changed from an immune response to immune tolerance between the stages of intraductal papillary mucinous adenoma (IPMA) and intraductal papillary mucinous carcinoma (IPMC). Chemokine (C-X-C motif) ligand 17 (CXCL17) and intercellular adhesion molecule 2 (ICAM2) were involved in immune surveillance during tumor development-their expression levels were up-regulated exclusively in IPMA and disappeared from IPMC. CXCL17 and ICAM2 induced infiltration and accumulation of the tumor epithelial layer by immature myeloid dendritic cells. This was followed by a cellular immune reaction and ICAM2 simultaneously promoted the susceptibility of the tumor cells to cytotoxic T-cell-mediated cytolysis. These processes had a synergistic effect to increase the anti-tumor immune response. CONCLUSIONS: Immune surveillance occurs during the early intraepithelial stages of human pancreatic carcinogenesis and is mediated by expression of CXCL17 and ICAM2.

Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis.

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)-derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER alpha as well as downregulated gene expressions of ER beta. Under the physiological concentrations of estrogen (17beta-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing beta-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER alpha, rather than ER beta in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER alpha.