Correlation of Vein-Rich Tumor Microenvironment of Intrahepatic Cholangiocarcinoma With Tertiary Lymphoid Structures and Patient Outcome.

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer composed of large-duct and small-duct types. Understanding the tumor immune microenvironment and its related vascular system is important for developing novel and efficient therapies. We focused on tertiary lymphoid structure (TLS) as a hallmark of antitumor immunity and investigated the clinicopathologic significance of TLSs and the influence of vascular microenvironment on TLS formation in iCCAs. We examined 261 iCCA cases clinicopathologically and analyzed the vascular system using immunohistochemistry. Single-cell (102,685 cells) and bulk RNA (33 iCCA cases) sequencing analyses were performed using data sets downloaded from public databases, and endothelial cell characteristics in iCCA tissues and functional networks related to the tumor microenvironment were bioinformatically examined. High densities of both intratumoral and peritumoral TLSs were significantly associated with prolonged survival only in large-duct-type iCCA. Multivariate analyses showed that peritumoral TLS was a prognostic factor for the large-duct type. TLS-rich iCCA had a significantly higher vein density and tumor-infiltrating T-cell count than TLS-poor iCCA. Both the presence of TLSs and high vein endothelial cells in iCCA tissues were significantly associated with molecular networks representing active immune responses in transcriptomic analysis. Vein density was a prognostic factor in patients with large-duct and small-duct types. This suggests that TLS formation is involved in a microenvironment with high vein density, which represents an antitumor-directed immune microenvironment.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Novel insights into immunohistochemical analysis for diagnosing serous neoplasm of the pancreas: aquaporin 1, stereocilin, and transmembrane protein 255B.

AIMS: Serous (cystic) neoplasm (SCN) of the pancreas is generally benign, and surgical treatment is recommended in only a limited number of cases. To avoid unnecessary surgery, an accurate diagnosis of SCN is essential. In the present study, we aimed to identify new immunohistochemical markers with which to distinguish SCN from other tumours. METHODS AND RESULTS: We compared the comprehensive gene expression profiles of SCN with those of normal pancreas and pancreatic ductal adenocarcinoma (PDAC). We selected the candidate molecules that were up-regulated in SCN, were minimally expressed or unexpressed in PDAC, and had specific and available antibodies suitable for immunohistochemistry, and then analysed their immunohistochemical expression in various tumours. We selected aquaporin 1 (AQP1), stereocilin (STRC), fibroblast growth factor receptor 3 (FGFR3), and transmembrane protein 255B (TMEM255B), which were diffusely expressed in SCN cells in 79%, 100%, 100% and 100% of SCN cases. AQP1 was not expressed in other tumours, except in 20% of mucinous cystic neoplasms (MCNs) and 19% of PDACs. STRC was rarely expressed in MCNs, neuroendocrine neoplasms (NENs), and PDACs. FGFR3 was expressed in 31% of intraductal papillary mucinous neoplasms (IPMNs), 50% of intraductal oncocytic papillary neoplasms, 40% of NENs, 30% of acinar cell carcinomas, 40% of solid pseudopapillary neoplasms, and 52% of PDACs. TMEM255B was not expressed in the other tumours, except in 50% of MCNs, 80% of gastric-subtype IPMNs, and 29% of PDACs. All antigens were usually expressed in a small proportion of cells when they were positive in tumours other than SCN. CONCLUSIONS: These findings indicate that AQP1 and STRC, and potentially TMEM255B, may act as SCN markers.

CD20+ tumor-infiltrating immune cells and CD204+ M2 macrophages are associated with prognosis in thymic carcinoma.

Thymic carcinoma is a rare malignant disease with no standard systemic chemotherapy. The purpose of the present study was to investigate tumor-infiltrating immune cells (TIIC) in the tumor microenvironment (TME), focusing on the impact of TIIC and program death-ligand 1 (PD-L1) expression on clinical outcomes in thymic cancer. Patients with thymic carcinoma resected between 1973 and 2017 were investigated. The tissue specimens were analyzed through immunohistochemical staining to elucidate the prognostic effects of TIIC, their ratios and PD-L1 in a preliminary cohort (n = 10). The density of TIIC as well as PD-L1 expression was evaluated in intraepithelial and tumor-stromal areas on the representative whole section of tumors. The immune factors showing significant association with disease-free survival (DFS) were evaluated in the total cohort (n = 42). TIIC in the preliminary population showed no significant difference between the two groups. However, CD8, CD20, CD204, FOXP3 and CD20/CD204 ratio demonstrated a tendency to act as predictive markers for recurrence. In the total cohort, significant differences were observed for CD8+ , CD20+ and CD204+ cells in tumor islets, and for CD8+ , CD20+ and FOXP3+ cells as well as the CD8/CD204 and CD20/CD204 ratios in the stroma, indicating their prognostic effect. The prognostic effect of the PD-L1 expression in tumor cells could not be established, possibly because of intratumoral heterogeneity. CD8, CD20 and CD204 positive TIIC in stroma were identified as possible better prognostic biomarkers, considering the heterogeneity of other biomarkers. The present study paves the way for exploring strategies of combination immunotherapy targeting B cell immunity in thymic carcinoma.

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.

Reduction of intrapancreatic neural density in cancer tissue predicts poorer outcome in pancreatic ductal carcinoma.

Neural invasion is one of the malignant features contributing to locally advanced and/or metastatic disease progression in patients with pancreatic ductal adenocarcinoma (PDAC). Few studies exist on the distribution and state of nerve fibers in PDAC tissue and their clinicopathological impacts. The aim of the present study was to investigate the clinicopathological characteristics and prognostic value of intrapancreatic neural alterations in patients with PDAC. We retrospectively analyzed 256 patients with PDAC who underwent macroscopic curative surgery. Nerve fibers, immunolabeled with a specific neural marker GAP-43, were digitally counted and compared among PDAC, chronic pancreatitis (CP) and normal pancreatic tissues. Interlobular nerve fibers were apparently hypertrophic in both CP and PDAC, although intrapancreatic neural density and nerve number decreased characteristically in PDAC. They tended to decrease toward the center of the tumor. Kaplan-Meier survival analyses revealed a statistically significant correlation between low neural density and shorter overall survival (OS) (P = 0.014), and between high neural invasion and shorter OS (P = 0.017). Neural density (P = 0.04; HR = 1.496; 95% CI 1.018-2.199) and neural invasion ratio (P = 0.064; HR = 1.439; 95% CI .980-2.114) were prognostic factors of shorter OS in the multivariate analysis. These findings suggest low intrapancreatic neural density in patients with PDAC as an independent prognosticator, which may represent aggressive tumor behavior. Furthermore, we propose a simple, practical and reproducible method (to measure neural density and the neural invasion ratio during conventional histopathological diagnosis of PDAC), which has been validated using another cohort (n = 81).

Tumor-associated CD204+ M2 macrophages are unfavorable prognostic indicators in uterine cervical adenocarcinoma.

Uterine cervical adenocarcinoma is rare, but its prevalence has increased. To improve outcomes and ensure the suitability of recent immunotherapies, the aim of this study was to evaluate the clinicopathological impact of the tumor immune microenvironment of uterine cervical adenocarcinoma. We investigated 148 adenocarcinoma cases, including 21 cases of adenocarcinoma in situ (AIS) and 127 cases of invasive adenocarcinoma, using immunohistochemistry to detect tumor-infiltrating immune cells and the expression of programmed cell death 1 ligand-1 (PD-L1) and p16 on tumor cells. We then carried out correlation and survival analyses. The density of immune cells and expression levels were compared between the tumor cell nest and stroma and between AIS and invasive adenocarcinoma using digital image analysis. A higher density of tumor-infiltrating CD204+ M2 macrophages was significantly associated with shorter disease-free survival, although no other tumor-infiltrating immune cells were prognostic, including CD4+ , CD8+ , FOXP3+ , and PD-1+ lymphocytes and CD68+ macrophages. The density of stroma-infiltrating lymphocytes and macrophages was significantly higher in invasive adenocarcinoma than in AIS. The density of tumor-infiltrating lymphocytes in p16-expressing human papillomavirus (HPV)-positive tumors was significantly higher than that in HPV-negative tumors. The HPV status was not associated with patient outcome. Expression of PD-L1 on tumor cells was found only in invasive adenocarcinoma cases (17.3%). A higher density of stroma-infiltrating lymphocytes and macrophages was found in PD-L1-positive tumors than in negative tumors. Patients with PD-L1-positive tumors tended to experience longer survival. It is suggested that tumor-infiltrating CD204+ M2 macrophages may predict poor prognoses in patients with cervical adenocarcinoma.

An Oncogenic ALK Fusion and an RRAS Mutation in KRAS Mutation-Negative Pancreatic Ductal Adenocarcinoma.

PURPOSE: Oncogenic mutations in the KRAS gene are a well-known driver event, occurring in >95% of pancreatic cancers. The objective of this study was to identify driver oncogene aberrations in pancreatic cancers without the KRAS mutation. METHODS: Whole-exome and transcriptome sequencing was performed on four cases of KRAS mutation-negative pancreatic ductal adenocarcinoma, which were identified in a cohort of 100 cases. RESULTS: One case harbored an oncogenic DCTN1-ALK fusion. The fusion gene enabled interleukin-3-independent growth of Ba/F3 cells and rendered them susceptible to the anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib and alectinib. The structure of the breakpoint junction indicated that the fusion was generated by nonhomologous end joining between a segment of DCTN1 exon DNA and a segment of ALK intron DNA, resulting in the generation of a cryptic splicing site. Another case harbored an oncogenic RRAS mutation that activated the GTPase of the RRAS protein. CONCLUSION: Rare oncogenic aberrations, such as the ALK fusion and RRAS mutation, may drive pancreatic carcinogenesis independent of the KRAS mutation. The Oncologist 2017;22:158-164Implications for Practice: The oncogenic DCTN1-ALK fusion and the RRAS mutation were associated with the development of pancreatic ductal adenocarcinoma (PDAC) in the absence of the KRAS mutation. Constitutional activation of DCTN1-ALK fusion protein was suppressed by the anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib and alectinib. Thus, a small subset of PDAC patients might benefit from therapy using these inhibitors.

Clinical significance of tumor-infiltrating immune cells focusing on BTLA and Cbl-b in patients with gallbladder cancer.

The host immune system plays a significant role in tumor control, although most cancers escape immune surveillance through a variety of mechanisms. The aim of the present study was to evaluate the clinicopathological significance of a novel co-inhibitory receptor, B and T lymphocyte attenuator (BTLA), the anergy cell marker Casitas-B-lineage lymphoma protein-b (Cbl-b), and clinical implications of tumor-infiltrating immune cells in gallbladder cancer (GBC) tissues. We investigated 211 cases of GBC, 21 cases of chronic cholecystitis (CC), and 11 cases of xanthogranulomatous cholecystitis (XGC) using immunohistochemistry to detect tissue-infiltrating immune cells and their expression of BTLA and Cbl-b, and carried out correlation and survival analyses. The density of infiltrating T cells was significantly higher in CC and XGC than in GBC. The density ratio of BTLA(+) cells to CD8(+) T cells (BTLA/CD8) and that of Cbl-b(+) cells to CD8(+) T cells (Cbl-b/CD8) were significantly higher in GBC than in CC and XGC. The FOXP3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly correlated with each other, and also with malignant phenotypes. Survival analyses revealed that a lower density of tumor-infiltrating CD8(+) cells, and higher Foxp3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly associated with shorter overall survival and disease-free survival in GBC patients. Multivariate analyses showed that M factor, perineural invasion, BTLA/CD8, and Cbl-b/CD8 were closely associated with shorter overall survival. These findings suggest that higher ratios of BTLA/CD8 and Cbl-b/CD8 are independent indicators of unfavorable outcome in GBC patients, and that upregulation of BTLA in cancer tissues is involved in inhibition of antitumor immunity.

Novel Insights Into Immunohistochemical Analysis For Acinar Cell Neoplasm of The Pancreas: Carboxypeptidase A2, Carboxypeptidase A1, and Glycoprotein 2.

Acinar cell carcinoma (ACC) is a rare and highly malignant pancreatic tumor. Owing to histologic similarity, ACC is often difficult to distinguish from other solid medullary pancreatic tumors, particularly neuroendocrine neoplasm (NEN) and intraductal tubulopapillary neoplasm (ITPN). We aimed to identify new immunohistochemical markers commonly expressed in tumor cells with acinar cell differentiation and useful for both surgical and small biopsy specimens. Candidate molecules exclusively expressed in neoplastic or non-neoplastic acinar cells in pancreatic tissues with specific and available antibodies suitable for immunohistochemistry were selected. We selected carboxypeptidase A1 (CPA1), carboxypeptidase A2 (CPA2), and glycoprotein 2 (GP2), which were expressed in 100%, 100%, and 96% of cases, respectively, in ACC (n=27) or neoplasia with acinar cell differentiation, including mixed acinar-neuroendocrine carcinoma (n=9), mixed acinar-ductal carcinoma (n=3), pancreatoblastoma (n=4), and acinar cystic transformation (n=2), in the cytoplasm of tumor cells with a granular pattern. Both CPA2 and CPA1 were not expressed in any other tumors without acinar cell differentiation, including NEN (n=44), pancreatic ductal adenocarcinoma (n=44), and ITPN (n=4). GP2 was not expressed in these tumors except in rare cases, including 14% of NEN, 15% of intraductal papillary-mucinous neoplasm, 25% of intraductal oncocytic papillary neoplasm, 25% of ITPN, and 7% of pancreatic ductal adenocarcinoma, wherein a small proportion of tumor cells expressed GP2 in their apical cell membrane. NEN cases also showed cytoplasmic GP2 expression. Therefore, CPA2, CPA1, and potentially GP2 may act as ACC markers.

Osteoblast-secreted factors enhance the expression of dysadherin and CCL2-dependent migration of renal carcinoma cells.

Renal cell carcinoma (RCC) frequently metastasizes to the bone marrow. These metastases are characterized by extensive osteolytic lesions. The mechanism, however, by which RCC cells metastasize to bone marrow remains poorly understood. To unravel the role of bone marrow cells in this context, we performed cell adhesion and migration assays using human RCC cell lines to analyze the influence of resident bone marrow cells on renal tumor cells. The strongest adhesion of RCC cells was observed to osteoblasts. Moreover, conditioned medium of osteoblasts (OB-CM) significantly increased RCC cell migration. By gene expression analysis dysadherin was identified as a transcript whose expression could be elevated more than twofold in RCC cells when exposed to OB-CM. Suppression of dysadherin expression in RCC cells by siRNA reduced their ability to migrate in the presence of OB-CM. Furthermore, the RCC cells secreted high amounts of the chemokine CCL2 when tumor cells migrated under the influence of osteoblast-secreted factors. CCL2 neutralization strongly reduced the migratory ability of the RCC cells. Silencing the expression of dysadherin in RCC cells resulted in a twofold reduction of CCL2 protein expression indicating a dysadherin-dependent expression of the chemokine. Taken together, our data show that osteoblasts are the major cell type of the bone marrow that affect RCC cells by secreting factors that increase the expression of dysadherin and CCL2 in the tumor cells leading to enhanced cell migration. These data suggest an osteoblast-induced autocrine mechanism for a facilitated homing of RCC cells to the bone marrow.

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.

Plasma biomarker discovery and validation for colorectal cancer by quantitative shotgun mass spectrometry and protein microarray.

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting-edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94,803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex-matched healthy controls using 2D image-converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex-matched healthy controls) using high-density reverse-phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94×10(-4) and 1.43×10(-12) (Student's t-test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage-I and -II diseases (P=3.01×10(-3) and P=1.13×10(-5) , respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer.

Association between the expression of core 3 synthase and survival outcomes of patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and metastatic type of malignant carcinoma that is associated with high mortality rates and is difficult to detect at early stages. Core 3 structure is a mucin-type O-glycans synthesized by ß1,3-N-acetylglucosaminyltransferase 6 (core 3 synthase), which plays an important role in the digestive system, in particular gastrointestinal goblet cells. It has been reported that core 3 synthase-expressing cells show lower migratory and invasive rates, and lower metastatic activity. A immunohistochemical study also showed that this enzyme was expressed in normal epithelial cells of the colon, but completely disappeared in colorectal cancer cells. The present study aimed to identify biomarkers that could be used to predict the prognosis of patients with CCA. Pathological specimens of 185 CCA tissues were immunohistochemically stained with two antibodies, G8-144 and MECA-79, which recognize core 3 synthase and 6-sulfated N-acetyllactosamine on the extended core-1 O-glycans, respectively. The association between G8-144 or MECA-79 positivity and patient prognosis was statistically analyzed. Positive expression of G8-144 was associated with improved prognosis in patients with distal CCA (dCCA). Patients with dCCA positive for G8-144 showed lower mortality rates than those with negative expression. However, the positive expression of MECA-79 was associated with CCA progression and metastasis, indicating that it is a poor prognostic marker for CCA. In conclusion, as both antibodies resulted in mirror-image staining, the involvement of G8-144 and MECA-79 in O-glycan synthesis could be considered as potential favorable and unfavorable biomarkers, respectively, for CCA prognosis.

BP180 Is a Prognostic Factor in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Prognosis plays a vital role in head and neck squamous cell carcinoma (HNSCC) patient management and decision-making. This study aimed to identify the role of BP180 as a prognostic factor in HNSCC. PATIENTS AND METHODS: Protein expression of bullous pemphigoid antigen II (BP180) was verified by immunohistochemistry (IHC) in a tissue microarray study of 202 cases. RESULTS: IHC analysis revealed that protein expression of BP180 among HNSCC patients differed significantly in the presence and absence of neural invasion, and according to T status in laryngeal and pharyngeal cancer subgroups. Overall survival and multivariate analysis showed that positive BP180-IHC and advanced clinical stage were significant independent positive predictors of mortality in HNSCC patients. In addition, in the oral cancer subgroup, independent positive predictors were positive BP180-IHC, advanced N status and neural invasion. In laryngeal and pharyngeal cancer subgroups, predictors were positive BP180-IHC and advanced clinical stage. CONCLUSION: BP180 is a prognostic factor in head and neck squamous cell carcinoma.

On-tissue polysulfide visualization by surface-enhanced Raman spectroscopy benefits patients with ovarian cancer to predict post-operative chemosensitivity.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm-1; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm-1, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance.

Clinicopathological significance of core 3 O-glycan synthetic enzyme, ß1,3-N-acetylglucosaminyltransferase 6 in pancreatic ductal adenocarcinoma.

Mucin-type O-glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of ß1,3-N-acetylglucosaminyltransferase 6 (ß3Gn-T6), an essential enzyme for the synthesis of core 3 O-glycan and several other O-glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of ß3Gn-T6 and several O-glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O-glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N-acetyllactosamine on extended core 1 O-glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. ß3Gn-T6 was expressed in ~20% of PDAC cases and 30-40% of PanINs but not in NPDEs. Higher expression of ß3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of ß3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that ß3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that ß3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of ß3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis.

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.

A Novel Role of Interleukin 13 Receptor alpha2 in Perineural Invasion and its Association with Poor Prognosis of Patients with Pancreatic Ductal Adenocarcinoma.

Perineural invasion (PNI) is one of the major pathological characteristics of pancreatic ductal adeno-carcinoma (PDAC), which is mediated by invading cancer cells into nerve cells. Herein, we identify the overexpression of Interleukin-13 Receptor alpha2 (IL-13Rα2) in the PNI from 236 PDAC samples by studying its expression at the protein levels by immunohistochemistry (IHC) and the RNA level by in situ hybridization (ISH). We observe that ≥75% samples overexpressed IL-13Rα2 by IHC and ISH in grade 2 and 3 tumors, while ≥64% stage II and III tumors overexpressed IL-13Rα2 (≥2+). Interestingly, ≥36 % peripancreatic neural plexus (PL) and ≥70% nerve endings (Ne) among PNI in PDAC samples showed higher levels of IL-13Rα2 (≥2+). IL-13Rα2 +ve PL and Ne subjects survived significantly less than IL-13Rα2 -ve subjects, suggesting that IL-13Rα2 may have a unique role as a biomarker of PNI-aggressiveness. Importantly, IL-13Rα2 may be a therapeutic target for intervention, which might not only prolong patient survival but also help alleviate pain attributed to perineural invasion. Our study uncovers a novel role of IL-13Rα2 in PNI as a key factor of the disease severity, thus revealing a therapeutically targetable option for PDAC and to facilitate PNI-associated pain management.

Tyrosine-phosphorylation of the 12th armadillo-repeat of beta-catenin is associated with cadherin dysfunction in human cancer.

Tyrosine phosphorylation of beta-catenin, an intracytoplasmic cadherin-binding protein, causes disruption of the cadherin-mediated cell adhesion system in cancer cells. We identified that the c-erbB-2 protein activated by TGFalpha was capable of binding to the 12th armadillo repeat (arm12) of beta-catenin with increased phosphorylation-state level. We produced a monoclonal antibody, 4G7, which recognizes a phosphorylated-tyrosine residue (Tyr-654) located at arm12 of beta-catenin. Tyrosine phosphorylation of the arm12 was detected after stimulation with TGFalpha in TMK-1. Immunoprecipitation analyses using 4G7, anti-beta-catenin, alpha-catenin, the c-erbB-2 gene product and phosphotyrosine antibodies indicated that tyrosine phosphorylation of the arm12 was closely correlated with dissociation between E-cadherin/beta-catenin and alpha-catenin or the c-erbB-2 gene product, but not with dissociation between beta-catenin and E-cadherin. Immunocytochemical staining of TMK-1 cells using the 4G7 antibody revealed that tyrosine phosphorylation of the arm12 was localized at cell-cell contact sites of cancer cells transiently and only after TGFalpha treatment. Immunohistochemical localization of the 4G7 antibody was observed in cancer cells at the invasive front where they detached from cancer nests. These findings indicated that the tyrosine phosphorylation of arm12 is a key marker of cadherin dysfunction and the 4G7 antibody may be useful in screening for a beta-catenin phosphorylation ligand or tyrosine kinases.