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Purpose: Pancreatic ductal adenocarcinoma (PDAC) is considered a "cold" tumor because the tumor immune microenvironment (TIME) exhibits poor intratumoral T-cell infiltration. This study aimed to identify the marker genes associated with induction of cold TIME in PDAC cells., Methods: We orthotopically transplanted 10 primary cultures of PDAC derived from KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice into immunocompetent mice and evaluated TIME by immunohistochemistry (IHC) staining of CD8. We divided primary cultures into two groups: cold TIME group with low CD8 + T-cell infiltration and a hot TIME group with high infiltration. RNA sequencing was performed to identify specific genes in the cold TIME group, and single-cell RNA sequencing (scRNA-seq) data was used for validation. IHC was performed to evaluate expressions in human PDAC samples., Results: We identified six genes specific in PDAC cells to the cold TIME group by RNA sequencing; these were defined as "cold tumor induction-related genes". Human PDAC scRNA-seq data revealed that cold tumor induction-related genes were significantly and negatively correlated with the number of CD8 + T-cells (p = 0.0341). These genes included protocadherin 7 (PCDH7). High expression of PCDH7 significantly and negatively correlated with the number of CD8 + T-cells in scRNA-seq (p = 0.0474) and IHC (p = 0.0110) data using human PDAC samples. PCDH7 was an independent factor for poor prognosis in PDAC (overall survival: hazard ratio = 2.07, p = 0.0367)., Conclusion: PCDH7 is a prognostic marker associated with CD8 + T-cell infiltration for PDAC patients., Competing Interests: Declarations. Ethical approval: This study was approved by the Ethics Committee of Kyushu University (approval number: 22002-00). All animal experiments were conducted following the guidelines of the institutional animal committee of Kyushu University (approval number: A22-128 and A23-026). Consent to participate: Written informed consent was waived owing to the retrospective analysis of the study. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC) due to the suppressive tumor immune microenvironment. Autophagy, which has been shown to play a role in antitumor immunity, has been proposed as a therapeutic target for PDAC. In this study, single-cell RNA sequencing of autophagy-deficient murine PDAC tumors revealed that autophagy inhibition in cancer cells induced dendritic cell (DC) activation. Analysis of human PDAC tumors substantiated a negative correlation between autophagy and DC activation signatures. Mechanistically, autophagy inhibition increased the intracellular accumulation of tumor antigens, which could activate DCs. Administration of chloroquine, an autophagy inhibitor, in combination with Flt3 ligand-induced DC infiltration inhibited tumor growth and increased tumor-infiltrating T lymphocytes. However, autophagy inhibition in cancer cells also induced CD8+ T-cell exhaustion with high expression of immune checkpoint LAG3. A triple-therapy comprising chloroquine, Flt3 ligand, and an anti-LAG3 antibody markedly reduced tumor growth in orthotopic syngeneic PDAC mouse models. Thus, targeting autophagy in cancer cells and activating DCs sensitize PDAC tumors to immune checkpoint inhibitor therapy, warranting further development of this treatment approach to overcome immunosuppression in pancreatic cancer. Significance: Inhibiting autophagy in pancreatic cancer cells enhances intracellular accumulation of tumor antigens to induce dendritic cell activation and synergizes with immunotherapy to markedly inhibit the growth of pancreatic ductal adenocarcinoma., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: We aim to clarify the precise function of TGFβ1-activated kinase 1 (TAK1) in cancer-associated fibroblasts (CAF) within human pancreatic ductal adenocarcinoma (PDAC) by investigating its role in cytokine-mediated signaling pathways., Experimental Design: The expression of TAK1 in pancreatic cancer was confirmed by The Cancer Genome Atlas data and human pancreatic cancer specimens. CAFs from freshly resected PDAC specimens were cultured and used in a three-dimensional model for direct and indirect coculture with PDAC tumors to investigate TAK1 function. Additionally, organoids from [LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre (KPC)] mice were mixed with CAFs and injected subcutaneously into C57BL/6 mice to explore in vivo functional interactions of TAK1., Results: The Cancer Genome Atlas data revealed significant upregulation of TAK1 in PDAC, associating with a positive correlation with the T-cell exhaustion signature. Knockdown of TAK1 in CAFs decreased the inflammatory CAF signature and increased the myofibroblastic CAF signature both in vitro and in vivo. The absence of TAK1 hindered CAF proliferation, blocked several inflammatory factors via multiple pathways associated with immunosuppression, and hindered epithelial-mesenchymal transition and outgrowth in vitro in spheroid cocultures with PDAC cells. Additionally, TAK1 inhibitor restrained tumor growth, increased CD4+ and CD8+ T-cell abundance, and reduced immunosuppressive cells present in vivo., Conclusions: Blocking the TAK1+ CAF phenotype leads to the conversion of protumorigenic CAFs to antitumorigenic CAFs. This highlights TAK1 as a potential therapeutic target, particularly in CAFs, and represents a novel avenue for combined immunotherapy in PDAC., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Pancreatic ductal adenocarcinoma tumors exhibit resistance to chemotherapy, targeted therapies, and even immunotherapy. Dendritic cells use glucose to support their effector functions and play a key role in anti-tumor immunity by promoting cytotoxic CD8 + T cell activity. However, the effects of glucose and lactate levels on dendritic cells in pancreatic ductal adenocarcinoma are unclear. In this study, we aimed to clarify how glucose and lactate can impact the dendritic cell antigen-presenting function and elucidate the relevant mechanisms., Methods: Glycolytic activity and immune cell infiltration in pancreatic ductal adenocarcinoma were evaluated using patient-derived organoids and resected specimens. Cell lines with increased or decreased glycolysis were established from KPC mice. Flow cytometry and single-cell RNA sequencing were used to evaluate the impacts on the tumor microenvironment. The effects of glucose and lactate on the bone marrow-derived dendritic cell antigen-presenting function were detected by flow cytometry., Results: The pancreatic ductal adenocarcinoma tumor microenvironment exhibited low glucose and high lactate concentrations from varying levels of glycolytic activity in cancer cells. In mouse transplantation models, tumors with increased glycolysis showed enhanced myeloid-derived suppressor cell infiltration and reduced dendritic cell and CD8 + T cell infiltration, whereas tumors with decreased glycolysis displayed the opposite trends. In three-dimensional co-culture, increased glycolysis in cancer cells suppressed the antigen-presenting function of bone marrow-derived dendritic cells. In addition, low-glucose and high-lactate media inhibited the antigen-presenting and mitochondrial functions of bone marrow-derived dendritic cells., Conclusions: Our study demonstrates the impact of dynamic glycolytic reprogramming on the composition of immune cells in the tumor microenvironment of pancreatic ductal adenocarcinoma, especially on the antigen-presenting function of dendritic cells., (© 2024. The Author(s).)

Background: Tumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC., Methods: Microarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings., Results: Increased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8 + T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8 + T-cell exhaustion. Blocking Nectin2 improved CD8 + T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2 + TANs and OLR1 + TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs., Conclusions: TANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC., (© 2024. The Author(s).)

An association between periodontal disease and the development of pancreatic cancer has been pointed out since before. Advances in genome analysis technology have revealed that a pancreatic cancer-specific microbiome is formed in the intestines and tumors of pancreatic cancer patients and modifies the progression of pancreatic cancer. Disturbance of microbiome( dysbiosis)suppresses anti-tumor immunity against pancreatic cancer, promoting cancer progression. Therefore, attempts are being made to correct dysbiosis by administration of probiotics or transplantation of microbiome, which is especially activating immune checkpoint inhibitors against cancer. In addition, specific intratumor bacteria has been identified that create an immunosuppressive microenvironment through crosstalk with pancreatic cancer cells. In the future, analysis of the microbiome distribution in pancreatic cancers may determine the following treatment strategy as an individualized treatment. We hope that innovations in omics technology will reveal more detailed functions of microbiome and lead to the development of effective treatments for pancreatic cancer.

Background: Although immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC., Methods: We assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment., Results: HRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8 + T cells, and cytotoxic CD8 + T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8 + T cell penetration and efficacy, overcoming resistance to ICB therapy., Conclusions: HRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy., (© 2024. The Author(s).)

Preoperative treatment is commonly carried out for borderline resectable pancreatic ductal adenocarcinoma (PDAC). However, the relationship between the combination of immune cells in the tumor microenvironment and their intratumoral heterogeneity along with their association with histological findings remains unclear, especially in patients receiving preoperative chemotherapy. We aimed to explore the therapeutic strategies for patients with PDAC with poor prognosis after receiving chemotherapy based on histological and immunological microenvironmental classifications. We investigated the correlation between the prognosis and histological immune microenvironmental factors of patients who initially underwent surgery (n = 100) and were receiving gemcitabine plus nab-paclitaxel (GEM + nabPTX) as preoperative chemotherapy (n = 103). Immune profiles were generated based on immune cell infiltration into the tumor, and their correlation with patient outcomes and histological features was analyzed. Tumor-infiltrating neutrophils (TINs) were identified as independent poor prognostic factors using multivariate analysis in both surgery-first and preoperative chemotherapy groups. The patients were further classified into four groups based on immune cell infiltration into the tumor. Patients with high CD15 infiltration into the tumor and immature stroma at the cancer margins showed the worst prognosis in the preoperative chemotherapy group. The analysis of mRNA expression and immunohistochemical features revealed that CXCR2, the receptor for CXCL8, was correlated with disease-free and overall survival. We inferred that patients with immature stroma at the margins and high infiltration of CD15 + neutrophils within the tumor showed the worst prognosis and they could particularly benefit from treatment with inhibitors targeting CXCR2 or CXCL8., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Although radical antegrade modular pancreatosplenectomy for pancreatic ductal adenocarcinoma (PDAC) has become the gold standard procedure in open distal pancreatectomy, there has been no gold standardized procedure for PDAC in minimally invasive distal pancreatectomy (MIDP). In this study, we analyzed our novel cranial-to-caudal approach (CC approach) for patients undergoing MIDP and provide a video clip illustrating the details of the CC approach., Methods: Ninety-four patients who underwent MIDP with splenectomy between 2016 and 2021 were included in this study. The CC approach was performed in 23 (24.5%) of the 94 patients. The concept of the CC approach is easy identification of Gerota's fascia from the cranial side of the pancreas and secure tumor removal (R0 resection) wrapped by Gerota's fascia. The short- and long-term outcomes were compared between the CC and non-CC approaches., Results: The median operation time and blood loss were similar between the two groups. The ratios of grade ≥ B postoperative pancreatic fistula and Clavien-Dindo grade ≥ III complications were also comparable. All patients in the CC approach group achieved R0 resection, and the R0 ratio was similar in the two groups (p = 0.345). The 2-year survival rate in CC and non-CC approach groups was 87.5% and 83.6%, respectively (p = 0.903)., Conclusions: The details of the CC approach for MIDP were demonstrated based on an anatomical point of view. This approach has the potential to become a standardized approach for left-sided PDAC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intratumor bacteria modify the tumor immune microenvironment and influence outcomes of various tumors. Periodontal pathogen Fusobacterium nucleatum has been detected in pancreatic cancer tissues and is associated with poor prognosis. However, it remains unclear how F. nucleatum affects pancreatic cancer. Here, we compared clinical features with F. nucleatum colonization in pancreatic cancer tissues. F. nucleatum was detected in 15.5% (13/84) of pancreatic cancer patients. The tumor size was significantly larger in the F. nucleatum-positive group than in the negative group. To clarify the biological effect of intratumor F. nucleatum on pancreatic cancer progression, we performed migration/invasion assays and cytokine array analysis of cancer cells cocultured with F. nucleatum. F. nucleatum promoted CXCL1 secretion from pancreatic cancer cells, leading to cancer progression through autocrine signaling. Intratumor F. nucleatum suppressed tumor-infiltrating CD8 + T cells by recruiting myeloid-derived suppressor cells (MDSCs) to the tumor in an F. nucleatum-injected subcutaneous pancreatic cancer mouse model, resulting in tumor progression. Furthermore, tumor growth accelerated by F. nucleatum was suppressed by MDSC depletion or cytokine inhibitors. Intratumor F. nucleatum promoted pancreatic cancer progression through autocrine and paracrine mechanisms of the CXCL1-CXCR2 axis. Blockade of the CXCL1-CXCR2 axis may be a novel therapeutic approach for patients with intratumor F. nucleatum-positive pancreatic cancer., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Purpose: This study aimed to demonstrate the involvement of angiogenesis in cancer-associated acinar-to-ductal metaplasia (CA-ADM) lesion of invasive front pancreatic ductal adenocarcinoma (PDAC) and investigate the possible mechanism., Methods: Tissue samples from 128 patients with PDAC and 36 LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre mice were analyzed. Immunohistochemical assay was performed using HE, anti-CK19 and anti-amylase to confirm the presence of CA-ADM lesions, using anti-CD34 and anti-CD31 to measure microvessel density (MVD), and using anti-CD68, anti-CD163, anti-iNOS, or anti-MMP9 to evaluate the immune microenvironment. We performed multiplex immunohistochemical assay to detect the co-expression of MMP9 and CD68 on macrophage. We examined clinical outcomes and other clinicopathological factors to determine the significance of high-level MVD of CA-ADM on survival and liver metastasis. We performed tube formation assay to evaluate the effect of macrophage on angiogenic capacity in vitro., Results: Angiogenesis was significantly abundant in CA-ADM lesions compared with that in PDAC lesions in human and mouse tissues. High-level MVD in CA-ADM lesions was an independent predictor of poor prognosis (P = 0.0047) and the recurrence of liver metastasis (P = 0.0027). More CD68-positive and CD163-positive macrophages were detected in CA-ADM lesions than in PDAC. The percentage of CD68-positive macrophages was positively correlated with MVD in CA-ADM lesions. Multiplex-immunostaining revealed that MMP9 was expressed in CD68-positive macrophages of CA-ADM lesions. In CA-ADM lesions, the percentage of macrophages was positively correlated with MMP9 expression, which positively correlated with microvessel density., Conclusion: CA-ADM related angiogenesis is a promising predictive marker for poor prognosis of PDAC and may provide an attractive therapeutic target for PDAC., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background/objectives: Pancreatic ductal adenocarcinoma (PDAC) is characterized by excessive desmoplasia and autophagy-dependent tumorigenic growth. Pancreatic stellate cells (PSCs) as a predominant stromal cell type play a critical role in PDAC biology. We have previously reported that autophagy facilitates PSC activation, however, the mechanism remains unknown. We investigated the mechanism of autophagy in PSC activation., Methods: We compared gene expression profiles between patient-derived PSCs from pancreatic cancer and chronic pancreatitis using a microarray. The stromal expression of target gene in specimen of PDAC patients (n = 63) was analyzed. The effect of target gene on autophagy and activation of PSCs was investigated by small interfering RNAs transfection, and the relationship between autophagy and ER stress was investigated. We analyzed the growth and fibrosis of xenografted tumor by orthotopic models., Results: In analysis of gene expression microarray, endoplasmic reticulum aminopeptidase 2 (ERAP2) upregulated in cancer-associated PSCs was identified as the target gene. High stromal ERAP2 expression is associated with a poor prognosis of PDAC patients. Knockdown of ERAP2 inhibited unfolded protein response mediated autophagy, and led to inactivation of PSCs, thereby attenuating tumor-stromal interactions by inhibiting production of IL-6 and fibronectin. In vivo, the promoting effect of PSCs on xenografted tumor growth and fibrosis was inhibited by ERAP2 knockdown., Conclusions: Our findings demonstrate a novel mechanism of PSCs activation regulated by autophagy. ERAP2 as a promising therapeutic target may provide a novel strategy for the treatment of PDAC., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2021 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy., Competing Interests: Declaration of interests K.K., G.M.W., T.D.M., B.J.P., N.L., N.S.G., X.Z.Z., and/or K.P.L. are inventors of a number of issued patents and/or pending patent applications on Pin1, Pin1 biomarkers, Pin1 inhibitors, and Pin1 inhibitor combination to treat human diseases; X.Z.Z. and K.P.L. are inventors of cis P-tau antibody technology, which was licensed by BIDMC to the startup Pinteon Therapeutics. X.Z.Z. and K.P.L. are the scientific founders and former scientific advisors of and own equity in Pinteon. Their interests were reviewed and are managed by BIDMC in accordance with its conflict of interest policy. G.M.W. reports research funding from Glaxo Smith Kline (institutional funding). N.S.G. is a founder, science advisory board member (SAB), and equity holder in Gatekeeper, Syros, C4, Allorion, Jengu, Inception, B2S, EoCys, Larkspur, and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, and Sanofi. All other authors do not have any competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Pancreatic stellate cells (PSCs) occupy the majority of the pancreatic cancer microenvironment, contributing to aggressive behavior of pancreatic cancer cells (PCCs). Recently, anti-fibrotic agents have proven to be an effective strategy against cancer, but clinical trials have shown little efficacy, and the driving mechanism remains unknown. N-acetyl-cysteine (NAC) is often used for pulmonary cystic fibrosis. Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, was habitually used for type II diabetes, but recently reported to inhibit metastasis of PCCs. However, few studies have focused on the effects of these two agents on cancer-stromal interactions., Method: We evaluated the expression of α-smooth muscle actin (α-SMA) and the number of lipid droplets in PSCs cultured with or without NAC. We also evaluated changes in invasiveness, viability, and oxidative level in PSCs and PCCs after NAC treatment. Using an indirect co-culture system, we investigated changes in viability, invasiveness, and migration of PSCs and PCCs. Combined treatment effects of NAC and Pioglitazone were evaluated in PSCs and PCCs. In vivo, we co-transplanted KPC-derived organoids and PSCs to evaluate the effects of NAC and Pioglitazone's combination therapy on subcutaneous tumor formation and splenic xenografted mouse models., Results: In vitro, NAC inhibited the viability, invasiveness, and migration of PSCs at a low concentration, but not those of PCCs. NAC treatment significantly reduced oxidative stress level and expression of α-SMA, collagen type I in PSCs, which apparently present a quiescent-like state with a high number of lipid droplets. Co-cultured PSCs and PCCs mutually promoted the viability, invasiveness, and migration of each other. However, these promotion effects were attenuated by NAC treatment. Pioglitazone maintained the NAC-induced quiescent-like state of PSCs, which were reactivated by PCC-supernatant, and enhanced chemosensitivity of PCCs. In vivo, NAC and Pioglitazone's combination suppressed tumor growth and liver metastasis with fewer stromal components and oxidative stress level., Conclusion: NAC suppressed activated PSCs and attenuated cancer-stromal interactions. NAC induces quiescent-like PSCs that were maintained in this state by pioglitazone treatment.

Purpose: The aim of this study was to investigate the genetic mutation profiles of Japanese pancreatic ductal adenocarcinoma (PDAC) patients., Methods: Next-generation sequencing was performed using FoundationOne® CDx on 17 PDAC patients who were treated by surgical resection at Kyushu University Hospital between February 2016 and January 2019. The tumor mutational burden and microsatellite instability status were also assessed., Results: There were 16 patients (94%) with KRAS mutations, 13 (76%) with TP53 mutations, three (18%) with SMAD4 mutations, and one (6%) with a CDKN2A mutation. All patients had at least one pathogenic variant or a likely pathogenic variant. No patient had targeted therapies that matched with any clinical benefit according to FoundationOne® CDx. An unresectable PDAC patient with BRCA2-mutant disease was successfully treated by conversion surgery using platinum-based neoadjuvant chemotherapy., Conclusions: Currently, FoundationOne® CDx might be difficult to use on PDAC patients, although further investigations with larger study populations are called for.

Introduction: Pancreatic adenocarcinoma (PAAD) is an aggressive malignancy, with a major mortality resulting from the rapid progression of metastasis. Unfortunately, no effective treatment strategy has been developed for PAAD metastasis to date. Thus, unraveling the mechanisms involved in PAAD metastatic phenotype may facilitate the treatment for PAAD patients., Objectives: PIK3CB is an oncogene implicated in cancer development and progression but less is known about whether PIK3CB participates in PAAD metastasis. Therefore, the objective of this study is to explore the mechanism(s) of PIK3CB in PAAD metastasis., Methods: In our study, we examined the PIK3CB expression pattern using bioinformatic analysis and clinical material derived from patients with PAAD. Subsequently, a series of biochemical experiments were conducted to investigate the role of PIK3CB as potential mechanism(s) underlying PAAD metastasis in vivo using nude mice and in vitro using cell lines., Results: We observed that PIK3CB was involved in PAAD progression. Notably, we identified that PIK3CB was involved in PAAD metastasis. Downregulation of PIK3CB significantly reduced PAAD metastatic potential in vivo. Furthermore, a series of bioinformatic analyses showed that PIK3CB was involved in cell adhesion in PAAD. Notably, PIK3CB depletion inhibited invasion potential specifically via suppressing cell adhesion to collagen I in PAAD cells., Conclusion: Collectively, our findings indicate that PIK3CB is involved in PAAD metastasis through cell-matrix adhesion. We proposed that PIK3CB is a potential therapeutic target for PAAD therapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Rationale: Pancreatic cancer is one of the most difficult cancers to manage and its poor prognosis stems from the lack of a reliable early disease biomarker coupled with its highly metastatic potential. Liver metastasis accounts for the high mortality rate in pancreatic cancer. Therefore, a better understanding of the mechanism(s) underlying the acquisition of the metastatic potential in pancreatic cancer is highly desirable. Methods: Microarray analysis in wild-type and highly liver metastatic human pancreatic cancer cell lines was performed to identify gene expression signatures that underlie the metastatic process. We validated our findings in patient samples, nude mice, cell lines and database analysis. Results: We identified a metastasis-related gene, laminin subunit alpha 4 ( LAMA4 ), that was upregulated in highly liver metastatic human pancreatic cancer cell lines. Downregulation of LAMA4 reduced the liver metastatic ability of pancreatic cancer cells in vivo . Furthermore, LAMA4 expression was positively correlated with tumor severity and in silico analyses revealed that LAMA4 was associated with altered tumor microenvironment. In particular, our in vitro and i n vivo results showed that LAMA4 expression was highly correlated with cancer-associated fibroblasts (CAFs) level which may contribute to pancreatic cancer metastasis. We further found that LAMA4 had a positive effect on the recruitment and activity of CAFs. Conclusions: These data provide evidence for LAMA4 as a possible biomarker of disease progression and poor prognosis in pancreatic cancer. Our findings indicate that LAMA4 may contribute to pancreatic cancer metastasis via recruitment or activation of CAFs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Progress in diagnostic modalities, surgical procedures, and multidisciplinary treatment for pancreatic diseases has increased the number of long-term survivors after pancreatic resection. Several reports have focused on high-risk lesions (HRLs), including high-grade pancreatic intraepithelial neoplasia (PanIN), pancreatic ductal adenocarcinoma, high-grade intraductal papillary mucinous neoplasm (IPMN), and IPMN with an associated invasive carcinoma, in the remnant pancreas after partial pancreatic resection for pancreatic cancer or IPMN. The etiology of HRLs in the remnant pancreas is thought to be either isolated local recurrence of the initial lesion in the remnant pancreas or a newly developed primary lesion. Although it is difficult to distinguish between local recurrence and a new primary lesion, comparison of genetic alterations between two lesions may help with this distinction. Early detection of HRLs in the remnant pancreas may improve the prognosis of patients, and several investigators have proposed predictive factors for HRLs in the remnant pancreas after partial pancreatic resection for pancreatic cancer or IPMN. The reported short- and long-term outcomes of surgical resection of HRLs in the remnant pancreas are relatively favorable. Life-long surveillance of the remnant pancreas is recommended after partial pancreatic resection for pancreatic cancer or IPMN.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to immunotherapy. As a factor of resistance, the dense fibrosis of this cancer acts as a barrier to inhibit immune cell infiltration into a tumor. We examined the influence of a Hedgehog signal inhibitor, Patched 1-interacting peptide, on fibrosis, infiltration of immune cells, and immunotherapeutic effects on PDAC. We found that this peptide inhibited proliferation and migration of cancer-associated fibroblasts and cancer cells. Furthermore, this peptide reduced the production of extracellular matrix and transforming growth factor β1 in cancer-associated fibroblasts and induced expression of HLA-ABC in PDAC cells and interferon-γ in lymphocytes. In vivo, the peptide suppressed fibrosis of PDAC and increased immune cell infiltration into tumors. The combination of this peptide and an anti-programmed death-1 antibody augmented the antitumor effect, and this combination showed the same effect in experiments using cancer cells and autologous lymphocytes. These results indicate that, in addition to the direct effect of tumor suppression, the Patched 1-interacting peptide increases the infiltration of immune cells by reducing fibrosis of PDAC and consequently enhances the effect of immunotherapy. Therefore, treatment with this peptide may be a novel therapy with 2 different mechanisms: direct tumor suppression and enhancing the immune response against PDAC.

Cancer‑associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor‑stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

Background: Necroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer., Methods: We used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells., Results: RIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis., Conclusion: These findings indicate that necroptosis at the pancreatic cancer invasion front can promote cancer cell migration and invasion via the CXCL5-CXCR2 axis., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Histopathological imaging is widely used for the analysis and diagnosis of multiple diseases. Several methods have been proposed for the 3D reconstruction of pathological images, captured from thin sections of a given specimen, which get nonlinearly deformed due to the preparation process. The majority of the available methods for registering such images use the degree of matching of adjacent images as the criteria for registration, which can result in unnatural deformations of the anatomical structures. Moreover, most methods assume that the same staining is used for all images, when in fact multiple staining is usually applied in order to enhance different structures in the images., Methods: This paper proposes a non-rigid 3D reconstruction method based on the assumption that internal structures on the original tissue must be smooth and continuous. Landmarks are detected along anatomical structures using template matching based on normalized cross-correlation (NCC), forming jagged shape trajectories that traverse several slices. The registration process smooths out these trajectories and deforms the images accordingly. Artifacts are automatically handled by using the confidence of the NCC in order to reject unreliable landmarks., Results: The proposed method was applied to a large series of histological sections from the pancreas of a KPC mouse. Some portions were dyed primarily with HE stain, while others were dyed alternately with HE, CK19, MT and Ki67 stains. A new evaluation method is proposed to quantitatively evaluate the smoothness and isotropy of the obtained reconstructions, both for single and multiple staining., Conclusions: The experimental results show that the proposed method produces smooth and nearly isotropic 3D reconstructions of pathological images with either single or multiple stains. From these reconstructions, microanatomical structures enhanced by different stains can be simultaneously observed.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is characterized by cystic dilation of the pancreatic duct, caused by mucin hypersecretion, with slow progression via the adenoma-carcinoma sequence mechanism. Mutation of GNAS at codon 201 is found exclusively in IPMNs, occurring at a rate of 41-75%. Recent advances in molecular biological techniques have demonstrated that GNAS mutation might play a role in the transformation of IPMNs after the appearance of neoplastic cells, rather than in the tumorigenesis of IPMNs. GNAS mutation is observed frequently in the intestinal subtype of IPMNs with MUC2 expression, and less frequently in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC). Research has focused on assessing GNAS mutation status in clinical practice using various samples. In this review, we discuss the clinical application of GNAS mutation assessment to differentiate invasive IPMNs from concomitant PDAC, examine the clonality of recurrent IPMNs in the remnant pancreas using resected specimens, and differentiate pancreatic cystic lesions using cystic fluid collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), duodenal fluid, and serum liquid biopsy samples.

Lymph node metastasis is an independent prognostic factor in pancreatic cancer. However, the mechanisms of lymph node colonization are unknown. As a mechanism of lymphatic metastasis, it has been reported for other types of cancer that spheroids from tumor cells cause circular chemorepellent‑induced defects (CCIDs) in lymphatic endothelial monolayers. In pancreatic cancer, such mechanisms of metastasis have not been elucidated. The present study evaluated the involvement of this new mechanism of metastasis in pancreatic cancer and investigated the associated factors. In human pancreatic cancer tissue, it was observed that clusters of cancer cells penetrated the wall of lymphatic ducts around the primary tumor. An in vitro co‑culture system was then used to analyze the mechanisms of tumor cell‑mediated disruption of lymphatic vessels. Time‑lapse microscopic imaging revealed that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. CCID formation ability differed depending on the cell line. Neither aggregation of spheroids nor adhesion to lymphatic endothelial cells (LECs) exhibited a significant correlation with this phenomenon. The addition of supernatant from cultured cancer cells enhanced CCID formation. Microarray analysis revealed that the expression of S100 calcium binding protein P (S100P) was significantly increased when LECs were treated with supernatant from cultured cancer cells. Addition of a S100P antagonist significantly suppressed the migration of LECs and CCID formation. The present findings demonstrated that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. These CCIDs in pancreatic cancer were partly regulated by S100P, suggesting that S100P may be a promising target to inhibit lymph node metastasis.

Background: Extracellular signal-regulated kinases (ERKs) have been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer and colorectal cancer. ERK1/2 inhibitor can suppress growth of KRAS-mutant pancreatic tumors by targeting cancer cell. However, no studies have shown the expression of ERK1/2 on pancreatic stromal and its effect on pancreatic cancer-stromal interaction., Methods: Immunohistochemistry and western blotting were performed to detect the expression of p-ERK1/2 in pancreatic tissues and cells. Cell viability assay was used to study IC50 of ERK inhibitor on pancreatic cancer cells (PCCs) and primary cancer-associated pancreatic stellate cells (PSCs). Transwell migration, invasion, cell viability assay, senescence β-galactosidase staining were performed to determine the effect of ERK inhibitor on PCCs and PSCs in vitro and in vivo. The expression of key factors involved in autophagy and epithelial-to-mesenchymal transition (EMT) process were evaluated by western blotting. The expression of key factors related to cell invasiveness and malignancy were confirmed by qRT-PCR. Co-transplantation of PCC Organoid and PSC using a splenic xenograft mouse model was used to evaluated combined treatment of ERK inhibitor and autophagy inhibitor., Results: Immunohistochemical staining in pancreatic tumor samples and transgenetic mice detected p-ERK1/2 expression in both cancer cells and stromal cells. In pancreatic tissues, p-ERK1/2 was strongly expressed in cancer-associated PSCs compared with cancer cells and normal PSCs. PSCs were also significantly more sensitive to ERK1/2 inhibitor treatment. Inhibition of ERK1/2 suppressed EMT transition in HMPCCs, upregulated cellular senescence markers, activated autophagy in cancer-associated PSCs; and suppressed cancer-stromal interaction, which enhanced invasiveness and viability of cancer cells. We also found that chloroquine, an autophagy inhibitor, suppressed ERK inhibition-induced autophagy and promoted PSC cellular senescence, leading to significantly decreased cell proliferation. The combination of an ERK inhibitor and autophagy inhibitor suppressed liver metastasis in a splenic pancreatic cancer organoid xenograft mouse model., Conclusions: These data indicate that inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer-stromal interaction and metastasis.

Purpose: This study aimed at investigating the function and significance of CD110 expression in pancreatic cancer., Methods: We performed immunohistochemical staining for CD110 expression in tumor samples from 86 patients with pancreatic cancer. We evaluated clinical outcomes and other clinicopathological factors to determine the significance of CD110 on survival and liver metastasis. We examine thrombopoietin-CD110 signaling in cancer cell extravasation in vitro and in vivo. We investigated the effects of CD110 knockdown on liver metastasis in a splenic xenograft mouse model., Results: CD110 expression in cancer cells was associated with low-histological-grade invasive ductal carcinoma, and patients with high CD110 expression had poorer prognosis (P = 0.0003). High CD110 expression was an independent predictor of liver metastasis (P = 0.0422). Knockdown of CD110 expression significantly attenuated cell migration and invasion. Treatment with thrombopoietin promoted pancreatic cancer cell extravasation. In the presence of thrombopoietin, CD110 increased cell viability through the activation of the ERK-MYC signaling pathway. Knockdown of CD110 expression inhibited liver metastases in the mouse model., Conclusions: CD110 promotes pancreatic cancer progression and it may serve as a predictive factor for liver metastasis.

Background: It is difficult to determine whether a second high-risk lesion, including pancreatic ductal adenocarcinoma or high-grade pancreatic intraepithelial neoplasm, is a metachronous multifocal lesion or represents local recurrence after resection of the first high-risk lesion. This study attempts to clarify the characteristics of second high-risk lesions in the remnant pancreas using genetic analyses., Methods: Clinicopathologic data were collected from 12 patients who underwent pancreatectomy for a second high-risk lesion in the remnant pancreas. We performed mutational and immunohistochemical analyses of 4 major genes-KRAS, TP53, CDKN2A, and SMAD4-associated with pancreatic ductal adenocarcinoma progression, as well as targeted next-generation sequencing., Results: Mutations in the four genes in the second high-risk lesion were consistent with the first lesion in four patients but were inconsistent in the remaining eight patients, and thus we considered that the latter eight patients likely had metachronous multifocal high-risk lesions and the other four patients had local recurrence. The estimated cumulative recurrence rate after resection of the second high-risk lesion was greater in the local recurrence group compared with the metachronous multifocal group, and the estimated cumulative disease-specific survival rate was greater in the metachronous multifocal group. Targeted next-generation sequencing demonstrated that the second lesions in the metachronous multifocal high-risk lesion group showed differences in founder mutations compared with the first lesion. In the local recurrence group, the founder mutations in the second lesion were common with those in the first lesion., Conclusion: Genetic assessment might help discriminate metachronous multifocal high-risk lesions from local recurrence., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although recent studies revealed that adipose tissue accelerates pancreatic tumor progression with excessive extracellular matrix, key players for desmoplasia in the adipose microenvironment remains unknown. Here, we investigated the roles of adipose tissue-derived stromal cells (ASCs) in desmoplastic lesions and tumor progression by in vitro and in vivo experiments. In a three-dimensional (3-D) organotypic fat invasion model using visceral fat from CAG-EGFP mice, GFP-positive fibroblastic cells infiltrated toward cancer cells. When tumor cells were inoculated into transplanted visceral fat pads in vivo, tumor weights and stromal components were enhanced compared to subcutaneous and orthotopic tumor cells inoculated without fat pads. Expression of αSMA in established human ASCs was lower compared to cancer associated fibroblasts, and the 3-D collagen matrices produced by ASCs cultured in cancer cell-conditioned medium changed from loose to dense structures that affected the motility of cancer cells. Microarray analyses revealed upregulation of S100A4 in ASCs, while S100A4-positive stromal cells were observed at extrapancreatic invasion sites of human pancreatic cancer. The present findings indicate that ASCs are recruited to extrapancreatic invasion sites and produce dense collagen matrices that lead to enhanced tumor progression. Both inhibition of ASCs recruitment and activation could lead to a novel antistromal therapy., (© 2018 UICC.)

The pancreas is an organ prone to inflammation, fibrosis, and atrophy because of an abundance of acinar cells that produce digestive enzymes. A characteristic of pancreatic cancer is the presence of desmoplasia, inflammatory cell infiltration, and cancer-associated acinar atrophy (CAA) within the invasive front. CAA is characterized by a high frequency of small ducts and resembles acinar-to-ductal metaplasia (ADM). However, the clinical significance of changes in acinar morphology, such as ADM with acinar atrophy, within the tumor microenvironment remains unclear. Here, we find that ADM within the invasive front of tumors is associated with cell invasion and desmoplasia in an orthotopic mouse model of pancreatic cancer. An analysis of resected human tumors revealed that regions of cancer-associated ADM were positive for TGFα, and that this TGFα expression was associated with primary tumor size and shorter survival times. Gene expression analysis identified distinct phenotypic profiles for cancer-associated ADM, sporadic ADM and chronic pancreatitis ADM. These findings suggest that the mechanisms driving ADM differ according to the specific tissue microenvironment and that cancer-associated ADM and acinar atrophy contribute to tumor cell invasion of the local pancreatic parenchyma., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Three-dimensional culture systems reflect biological environments better than conventional two-dimensional culture. Additionally, three-dimensional culture is a strong experimental tool to analyze direct interactions between cancer cells and stromal cells in vitro. Herein, we describe protocols for an organotypic fat invasion model that is a novel culturing system mimicking the extrapancreatic invasion of pancreatic adenocarcinoma (PDAC). This novel model is based on the collagen I gel embedding method and enables us to analyze the functional and histological interactions between cancer cells and adipose tissue.

Background: Preoperative nutritional and immunological patient factors have been found to be associated with prognostic outcomes of malignant tumors; however, the clinical significance of these factors in pancreatic ductal adenocarcinoma (PDAC) remains controversial., Objective: The aim of this study was to evaluate the prognostic value of nutritional and immunological factors in predicting survival of patients with PDAC., Methods: Retrospective studies of 329 patients who underwent surgical resection for PDAC and 95 patients who underwent palliative surgery were separately conducted to investigate the prognostic impact of tumor-related factors and patient-related factors, including Glasgow Prognostic Score (GPS), modified GPS, Prognostic Nutritional Index (PNI), neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio, and lymphocyte/monocyte ratio., Results: In multivariate analysis for patients with surgical resection for PDAC, PNI was an independent factor for overall survival (OS) and disease-free survival. The median OS of patients with PNI ≤ 45 was significantly shorter than that of patients with PNI > 45 (17.5 and 36.2 months, respectively; p < 0.001). In multivariate analysis for patients undergoing palliative surgery for PDAC, only NLR was an independent prognosis factor. The median OS of patients with NLR > 5 was significantly shorter than that of patients with NLR ≤ 5 (2.7 and 8.9 months, respectively; p < 0.001)., Conclusions: PNI in patients with surgical resection and NLR in patients with palliative surgery for PDAC may be useful prognostic factors.

Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruction and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Specific cell populations leading the local invasion of cancer are called "leading cells". However, the underlying mechanisms are unclear. Here, we identified leading cells in pancreatic cancer and determined how these cells lead and promote cancer cell invasion in the extracellular matrix (ECM). Using three-dimensional matrix remodeling assay, we found that pancreatic stellate cells (PSCs) frequently invaded the collagen matrix with pancreatic cancer cells (PCCs), which invaded behind the invading PSCs. In addition, invading PSCs changed the alignment of collagen fibers, resulting in ECM remodeling and an increase in the parallel fibers along the direction of invading PSCs. Endo180 expression was higher in PSCs than in PCCs, Endo180 knockdown in PSCs attenuated the invasive abilities of PSCs and co-cultured PCCs, and decreased the expression level of phosphorylated myosin light chain 2 (MLC2). In mouse models, Endo180-knockdown PSCs suppressed tumor growth and changes in collagen fiber orientation in co-transplantation with PCCs. Our findings suggest that PSCs lead the local invasion of PCCs by physically remodeling the ECM, possibly via the function of Endo180, which reconstructs the actin cell skeleton by phosphorylation of MLC2., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Contrast agents with greater specificity and sensitivity are required for the diagnosis of pancreatic cancers by magnetic resonance imaging (MRI). In this study, small heat shock protein 16.5 (Hsp16.5)-based nanocages conjugated to gadolinium(III)-chelated contrast agents and iRGD peptides (which target neuropilin-1 expressed on pancreatic cancer cells) were developed. To investigate whether template size influences relaxivity, nanocages with one to four hydrophobic domains were designed. MRI data showed that larger nanocages had higher T 1 relaxivity than smaller nanocages, which resulted from a reduction in molecular tumbling rates caused by an increase in nanocage size, and a robust cage structure resulting from the introduction of hydrophobic domains. For in vivo MRI studies, the engineered nanocages were evaluated using the Kras G12D ; Trp53 R172H ; Pdx-1Cre (KPC) transgenic mouse models, which develop clinically relevant pancreatic tumor under normal processes of angiogenesis, immune function and inflammation. Molecular MRI with protein nanocages was enabled to detect neuropilin-1-positive cells and to produce strong signal enhancement of spontaneous pancreatic tumors in KPC genetically engineered mouse models. Novel iRGD-modified nanocages displayed potential as a specific and sensitive MRI contrast agent for the diagnosis of pancreatic tumors for clinical translation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: The development of an effective screening method for pancreatic ductal adenocarcinoma (PDAC) is of paramount importance. This study assessed the diagnostic utility in pancreatic diseases of duodenal markers during upper gastrointestinal endoscopy (GIE) or endoscopic ultrasonography., Methods: This study prospectively enrolled 299 consecutive participants, including 94 patients with PDACs, 144 patients with other pancreatic diseases, and 61 normal individuals as control subjects. All subjects underwent upper GIE or endoscopic ultrasonography either at Kyushu University Hospital (Fukuoka, Japan) or the Mayo Clinic (Jacksonville, Fla) from October 2011 to July 2014. Duodenal fluid (DF) was collected without secretin stimulation and of carcinoembryonic antigen and S100 calcium-binding protein P (S100P) concentrations were measured., Results: Concentrations of S100P in DF were significantly higher in patients with PDAC and chronic pancreatitis than in control subjects (P < 0.01). A logistic regression model that included age found that the sensitivity and specificity of S100P concentration in diagnosing stages 0/IA/IB/IIA PDAC were 85% and 77%, respectively, with an area under the receiver operating characteristic curve of 0.82. Carcinoembryonic antigen concentrations in DF of patients with pancreatic disease did not differ significantly from control subjects., Conclusions: Analysis of S100P concentration in DF, in combination with routine screening upper GIE, may facilitate the detection of PDAC.

Background: The pattern of recurrence affects the clinical outcome in tumor patients. However, the clinical significance of lung metastasis as the primary recurrence site after resection in patients with pancreatic cancer remains unclear. This study aimed to clarify the clinical significance of the primary recurrence site in patients with pancreatic cancer, in terms of prognosis and clinicopathological features., Methods: This retrospective cohort study included 220 patients with postoperative recurrence after pancreatectomy for pancreatic cancer and classified by primary site of recurrence. We focused on patients with lung metastasis as the primary recurrence and investigated its correlation with prognosis and clinicopathological factors., Results: Twenty-four (11%) patients had lung metastasis as the primary recurrence. This recurrence pattern had the best prognosis among all recurrence patterns, including liver metastasis and local recurrence. Patients with lung metastasis as the primary recurrence had favorable overall survival and survival from the date of recurrence compared with patients with other primary recurrence sites in both univariate (P = 0.0008 and P = 0.0005) and multivariate analyses (P = 0.0051 and P = 0.0068). In terms of clinicopathological features of resected pancreatic tumors, lung metastasis as the primary recurrence was associated with lower tumor stage and histologic grade, and less vascular invasion and residual tumor volume than liver metastasis., Conclusions: Pancreatic cancer patients with lung metastasis as the primary recurrence after pancreatectomy have a better prognosis than those with other recurrence patterns.

Background: Salinomycin has cytotoxic effects on various types of malignancy and induces autophagy. However, it has not been clarified whether autophagy induced by salinomycin treatment has a protective or cytotoxic role. We investigated whether salinomycin affects autophagy in pancreatic cancer cells and whether autophagy induced by salinomycin treatment has a protective or cytotoxic role in these cells., Methods: We investigated the effect of salinomycin using three pancreatic cancer cell lines. We investigated effect on proliferation and the CD133 positive fraction using flow cytometry. In addition, we monitored the change in autophagic activity after salinomycin treatment using fluorescent immunostaining, western blotting, and flow cytometry. Finally, knockdown of ATG5 or ATG7 by siRNA was used to investigate the impact of autophagy inhibition on sensitivity to salinomycin., Results: Salinomycin suppressed the proliferation of pancreatic cancer cells in a concentration dependent manner, and reduced the CD133 positive fraction. Salinomycin enhanced autophagy activity in these cells in a concentration dependent manner. Autophagy inhibition made pancreatic cancer cells more sensitive to salinomycin., Conclusions: Our data provide the first evidence indicating that autophagy induced by salinomycin have a protective role in pancreatic cancer cells. A new therapeutic strategy of combining salinomycin, autophagy inhibitors, and anticancer drugs could hold promise for pancreatic cancer treatment., (Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Pancreatic stellate cells (PSCs) change from a quiescent to activated state in the tumor environment and secrete extracellular matrix (ECM) molecules and cytokines to increase the aggressiveness of tumors. However, it is not clear how PSCs are activated to produce these factors, or whether this process can be inhibited. PSCs have morphologic and functional similarities to hepatic stellate cells, which undergo autophagy to promote fibrosis and tumor growth. We investigated whether autophagy activates PSCs, which promotes development of the tumor stroma and growth of pancreatic tumors in mice., Methods: We used immunofluorescence microscopy and immunohistochemistry to analyze pancreatic tumor specimens from 133 patients who underwent pancreatectomy in Japan from 2000 to 2009. PSCs were cultured from pancreatic tumor tissues or tissues of patients with chronic pancreatitis; these were analyzed by immunofluorescence microscopy, immunoblots, quantitative reverse transcription polymerase chain reaction, and in assays for invasiveness, proliferation, and lipid droplets. Autophagy was inhibited in PSCs by administration of chloroquine or transfection with small interfering RNAs. Proteins were knocked down in immortalized PSCs by expression of small hairpin RNAs. Cells were transplanted into pancreatic tails of nude mice, and tumor growth and metastasis were quantified., Results: Based on immunohistochemical analyses, autophagy was significantly associated with tumor T category (P = .018), histologic grade (P = .001), lymph node metastases (P < .001), stage (P = .009), perilymphatic invasion (P = .001), and perivascular invasion (P = .003). Autophagy of PSCs was associated with shorter survival times of patients with pancreatic cancer. PSC expression of microtubule-associated protein 1 light chain 3, a marker of autophagosomes, was associated with poor outcomes (shorter survival time, disease recurrence) for patients with pancreatic cancer (relative risk of shorter survival time, 1.56). Immunoblots showed that PSCs from pancreatic tumor samples expressed higher levels of markers of autophagy than PSCs from chronic pancreatitis samples. Inhibitors of autophagy increased the number of lipid droplets of PSCs, indicating a quiescent state of PSCs, and reduced their production of ECM molecules and interleukin 6, as well as their proliferation and invasiveness in culture. PSCs exposed to autophagy inhibitors formed smaller tumors in nude mice (P = .001) and fewer liver metastases (P = .018) with less peritoneal dissemination (P = .018) compared to PSCs not exposed to autophagy inhibitors., Conclusions: Autophagic PSCs produce ECM molecules and interleukin 6 and are associated with shorter survival times and disease recurrence in patients with pancreatic cancer. Inhibitors of PSC autophagy might reduce pancreatic tumor invasiveness by altering the tumor stroma., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: The clinical importance of intraoperative peritoneal cytology in patients with pancreatic cancer remains incompletely understood., Methods: Peritoneal washing samples were collected from 411 consecutive patients with pancreatic ductal adenocarcinoma from 1996 to 2014. Of the 411 patients, 335 underwent macroscopically curative resection and 76 with noncurative factors did not undergo resection. We compared long-term outcomes between patients with positive cytology (cytology+) and those with negative cytology (cytology-) and investigated the importance of clinicopathologic factors., Results: Of 335 patients with curative resection, 300 (89.6%) were cytology- and 35 (10.4%) were cytology+. The median overall survival of cytology+ patients was less than that of cytology- patients (16 vs 31 months, respectively; P < .0001). The median overall survival of cytology+ patients with noncurative factors was significantly worse than that of cytology+ patients with curative resection (6.9 vs 16.0 months, respectively; P = .0023). The median disease-free survival of cytology+ patients was less than that of cytology- patients (6.5 vs 16 months, respectively; P < .0001). In the multivariate analysis, cytology+ was an independent prognostic factor for overall survival and disease-free survival., Conclusion: Cytology+ without noncurative factors was a predictive factor for a poor prognosis. Therefore, it is important to regard patients with pancreatic cancer characterized by cytology+ as a special group that may warrant more aggressive adjuvant therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pancreatic cancer progression involves components of the tumor microenvironment, including stellate cells, immune cells, endothelial cells, and the extracellular matrix. Although peripancreatic fat is the main stromal component involved in extra-pancreatic invasion, its roles in local invasion and metastasis of pancreatic cancer remain unclear. This study investigated the role of adipose tissue in pancreatic cancer progression using genetically engineered mice (Pdx1-Cre; LSL-KrasG12D; Trp53R172H/+) and an in vitro model of organotypic fat invasion. Mice fed a high fat diet had significantly larger primary pancreatic tumors and a significantly higher rate of distant organ metastasis than mice fed a standard diet. In the organotypic fat invasion model, pancreatic cancer cell clusters were smaller and more elongated in shape and showed increased fibrosis. Adipose tissue-derived conditioned medium enhanced pancreatic cancer cell invasiveness and gemcitabine resistance, as well as inducing morphologic changes in cancer cells and increasing the numbers of lipid droplets in their cytoplasm. The concentrations of oleic, palmitoleic, and linoleic acids were higher in adipose tissue-derived conditioned medium than in normal medium, with these fatty acids significantly enhancing the migration of cancer cells. Mature adipocytes were smaller and the concentration of fatty acids in the medium higher when these cells were co-cultured with cancer cells. These findings indicate that lipolytic and fibrotic changes in peripancreatic adipose tissue enhance local invasiveness and metastasis via adipocyte-released fatty acids. Inhibition of fatty acid uptake by cancer cells may be a novel therapy targeting interactions between cancer and stromal cells.

The interaction between the cancer cells and the peritoneal mesothelial cells (PMCs) plays an important role in the peritoneal dissemination in several types of cancer. However, the role of PMCs in the peritoneal dissemination of pancreatic cancer remains unclear. In the present study, we investigated the interaction between the pancreatic cancer cells (PCCs) and the PMCs in the formation of peritoneal dissemination in vitro and in vivo. The tumor-stromal interaction of PCCs and PMCs significantly enhanced their mobility and invasiveness and enhanced the proliferation and anoikis resistance of PCCs. In a 3D organotypic culture model of peritoneal dissemination, co-culture of PCCs and PMCs significantly increased the cells invading into the collagen gel layer compared with mono-culture of PCCs. PMCs pre-invaded into the collagen gel, remodeled collagen fibers, and increased parallel fiber orientation along the direction of cell invasion. In the tissues of peritoneal dissemination of the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+;Pdx-1-Cre) transgenic mouse, the monolayer of PMCs was preserved in tumor-free areas, whereas PMCs around the invasive front of peritoneal dissemination proliferated and invaded into the muscle layer. In vivo, intraperitoneal injection of PCCs with PMCs significantly promoted peritoneal dissemination compared with PCCs alone. The present data suggest that the cancer-associated PMCs have important promoting roles in the peritoneal dissemination of PCCs. Therapy targeting cancer-associated PMCs may improve the prognosis of patients with pancreatic cancer.

Cancer-associated fibroblasts (CAFs) are heterogeneous cell populations that influence tumor initiation and progression. CD146 is a cell membrane protein whose expression has been implicated in multiple human cancers. CD146 expression is also detected in pancreatic cancer stroma; however, the role it plays in this context remains unclear. This study aimed to clarify the function and significance of CD146 expression in pancreatic cancer. We performed immunohistochemical staining to investigate the prevalence of CD146 expression in stromal fibroblasts in pancreatic cancer. We also examined the influence of CD146 on CAF-mediated tumor invasion and migration and CAF activation using CD146 small interfering RNA or overexpression plasmids in primary cultures of CAFs derived from pancreatic cancer tissues. CD146 expression in CAFs was associated with high-grade pancreatic intraepithelial neoplasia and low histological grade invasive ductal carcinoma of the pancreas, while patients with low CD146 expression had a poorer prognosis. Blocking CD146 expression in CAFs significantly enhanced tumor cell migration and invasion in a co-culture system. CD146 knockdown also promoted CAF activation, possibly by inducing the production of pro-tumorigenic factors through modulation of NF-κB activity. Consistently, overexpression of CD146 in CAFs inhibited migration and invasion of co-cultured cancer cells. Finally, CD146 expression in CAFs was reduced by interaction with cancer cells. Our findings suggest that decreased CD146 expression in CAFs promotes pancreatic cancer progression. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia-targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT-PCR to evaluate the expression of calpain-1 and calpain-2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT-PCR indicated that PCCs and PSCs expressed calpain-2 mRNA. Calpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer-stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer-stromal interaction., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Radical antegrade modular pancreatosplenectomy (RAMPS) is a modification of standard retrograde pancreatosplenectomy (SRPS) used to achieve the dissection of N1 lymph nodes, early vascular control, and negative posterior margins. However, there have been few comparative studies regarding the clinical outcomes of the RAMPS and SRPS procedures., Methods: Ninety-three patients underwent distal pancreatectomy for the treatment of pancreas body and tail adenocarcinoma between 2000 and 2014. Clinicopathologic data were retrospectively analyzed in this study. We compared short- and long-term outcomes between RAMPS and SRPS. In addition, we investigated the significance of clinicopathologic factors in left-sided pancreatic cancers., Results: Fifty-three patients underwent RAMPS and 40 patients underwent SRPS. RAMPS revealed a larger number of retrieved lymph nodes [28.4 ± 11.6 vs 20.7 ± 10.1; P = 0.0016], more frequent R0 resection [90.5 vs 67.5 %; P = 0.0053], less intraoperative bleeding than SRPS [485.4 ± 63.3 vs 682.3 ± 72.8 ml; P = 0.0444], and shorter operating time (267.3 ± 11.5 vs 339.4 ± 13.2 min; P < 0.0001) as compared with SRPS. In comparing RAMPS and SRPS, RAMPS showed a tendency for improvement of the median survival times than SRPS (47 vs 34 months) (P = 0.1920). In the multivariate analysis, R1 resection, histologic grade, and vascular invasion for overall survival (OS) were found to be independent factors., Conclusions: There were a decrease of intraoperative bleeding and an increase in the number of retrieved lymph nodes and the R0 resection rate using RAMPS as compared with SRPS.

Objectives: To clarify the usefulness of molecular biomarkers for distinguishing invasive carcinoma derived from intraductal papillary mucinous neoplasms (IPMNs [Inv-IPMN]) from concomitant pancreatic ductal adenocarcinoma (PDAC)., Methods: Data from 19 patients with resected concomitant PDAC were retrospectively reviewed. KRAS/GNAS mutations and immunohistochemical (IHC) expression of p53 and p16/CDKN2A were assessed in both IPMN and distinct PDAC. As controls, KRAS/GNAS mutations and IHC labeling were assessed between invasive and noninvasive components in 1 lesion of 22 independent patients., Results: KRAS/GNAS mutation status of invasive and noninvasive components in Inv-IPMN was consistent in 18 (86%) of 21 patients. Conversely, mutational patterns in IPMN and distinct PDAC in the same pancreas differed from each other in 17 (89%) of 19. There were 10 (53%) and 8 (42%) of 19 patients who showed the same p53 and p16/CDKN2A staining between concomitant PDAC and distinct IPMN. In the Inv-IPMN cohort, 19 (86%) of 22 patients showed the same IHC expression pattern between the noninvasive and invasive components., Conclusions: It may be possible to distinguish Inv-IPMN from concomitant PDAC by assessing these molecular biomarkers. More precise distinction of Inv-IPMN and concomitant PDAC will lead to adequate recognition of the natural history of IPMNs and hence optimal management.

Metastasis is the main cause of cancer-associated death, and metastasis of pancreatic cancer remains difficult to treat because of its aggressiveness. MicroRNAs (miRNAs) play crucial roles in the regulation of various human transcripts, and many miRNAs have been reported to correlate with cancer metastasis. We identified an anti-metastatic miRNA, miR-5100, by investigating differences in miRNA profiling between highly metastatic pancreatic cancer cells and their parental cells. Overexpression of miR-5100 inhibited colony formation (P<0.05), cell migration (P<0.0001) and invasion (P<0.0001) of pancreatic cancer cells. In addition, we identified a possible target of miR-5100, podocalyxin-like 1 (PODXL), and demonstrated miR-5100 directly binds to the 3' untranslated region of PODXL and post-transcriptionally regulates its expression in pancreatic cancer cells. Silencing PODXL resulted in diminished cell migration (P<0.0001) and invasion (P<0.05). We also clarified the close relationship between expression of PODXL in human pancreatic cancer specimens and liver metastasis (P=0.0003), and determined that post-operative survival was longer in the low-PODXL expression group than in the high-PODXL expression group (P<0.05). These results indicate that miR-5100 and PODXL have considerable therapeutic potential for anti-metastatic therapy and could be potential indicators for cancer metastases in patients with pancreatic cancer.

Pancreatic stellate cells (PSCs) enhance the malignant behavior of pancreatic cancer by interacting with cancer cells and producing extracellular matrix (ECM). To date, several stroma-targeted therapies for pancreatic cancer have been attempted, but these therapies are still not in practical use. Integrins expressed in stromal cells are involved in fibrosis of several organs, as well as promoting tumor malignancy. We investigated whether CD51, also known as integrin αV, expressed in PSCs was associated with stromal formation of pancreatic cancer and enhancement of tumor malignancy. We also assessed the effects of suppression of CD51 in PSCs on pancreatic cancer. Immunohistochemistry for CD51 in resected pancreatic cancer tissues showed that high expression of CD51 in the tumor stroma was associated with lymph node metastasis (P=0.025), positive pathologic margin (P=0.025), and shorter patient survival times (P=0.043). Lentivirus-mediated short hairpin RNA knockdown of CD51 decreased the proliferation and migration of PSCs. Quantitative real-time polymerase chain reaction showed that expression levels of genes related with ECM and tumor-stromal interactions were decreased by CD51 knockdown in PSCs. In a co-implantation model of pancreatic cancer cells and PSCs, tumor growth in vivo was inhibited by CD51 knockdown in PSCs (P<0.05). We also found reduced tumor stroma and decreased proliferation of cancer cells in implanted cancer tissues with CD51-silenced PSCs (P<0.05). Our results showed that CD51 expression in pancreatic cancer stroma is associated with enhanced tumor malignancy and that CD51 may be a potential therapeutic target for pancreatic cancer.

Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The cell surface protein Transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors, and its expression on tumor cells is implicated in cancer cell metastasis and patient prognosis. The role of TM4SF1 in malignant tumors remains poorly understood, particularly in pancreatic cancer. We performed immunohistochemical staining to analyze the expression of TM4SF1 in resected pancreatic tissues and investigated the correlation between TM4SF1 expression and prognosis. The function of TM4SF1 in the invasion and migration of pancreatic cancer cells was analyzed in vitro using an RNA interference technique. In pancreatic cancer tissues, TM4SF1 expression was detected in cancer cells, and patients with high tumor levels of TM4SF1 showed longer survival times than those with low TM4SF1 levels (P=0.0332). In vitro, reduced TM4SF1 expression enhanced the migration (P<0.05) and invasion (P<0.05) of pancreatic cancer cells partially via decreased E-cadherin expression. TM4SF1 protein levels were also reduced after TGF-β1-induced epithelial-mesenchymal transition (EMT).TM4SF1 expression is associated with better prognosis in pancreatic cancer. Loss of TM4SF1 contributes to the invasion and migration of pancreatic cancer cells.

We describe the development of neuropilin 1-binding peptide (iRGD)-nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD-nanocages for their target cells. An engineered L30-iRGD-nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD-nanocage by heating the mixture. The OSU03012-loaded L30-iRGD-nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD-nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery.