Pancreatic Tumor Cells Research Articles

In Vivo Flow Cytometric Evaluation of Circulating Metastatic Pancreatic Tumor Cells after High-Intensity Focused Ultrasound Therapy.

We examined our hypothesis that high‐intensity focused ultrasound (HIFU) treatment of pancreatic ductal adenocarcinoma (PDAC) in nude mice models may lead to an increased occurrence of hematogenous metastasis. The human PDAC cell line BxPC‐3 transfected with mCherry was implanted into nude mice to establish orthotopic and subcutaneous xenograft (OX and SX) tumor models. Mice were exposed to HIFU when tumor sizes reached approximately 200–300 mm3. The OX and SX tumor models were monitored continuously for tumor growth characteristics and hematogenous metastasis using in vivo flow cytometric (IVFC) detection of circulating tumor cells (CTCs) from the pancreas. We chose an appropriate mouse model to further examine whether or not HIFU increases the potential risk of hematogenous metastasis, using IVFC detection. Our results showed that the CTC number was greater in the OX model than in the SX model. The CTC number in the OX model increased gradually over time, whereas the CTC number in the SX model remained low. Therefore, the OX model was better for studying tumor metastasis by IVFC detection. We found significantly decreased CTC numbers and tumor volume after HIFU ablation. Our results showed the applicability of the PDAC OX tumor model for studying the occurrence of tumor metastasis due to the generation of CTCs. HIFU ablation substantially restricted PDAC hematogenous metastasis and provided effective tumor control locally. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals Inc., on behalf of International Society for Advancement of Cytometry.

A novel insight into the anticancer mechanism of metformin in pancreatic neuroendocrine tumor cells

The antidiabetic drug metformin displays anticancer properties in several neoplasms. In pituitary NETs, aryl hydrocarbon receptor-interacting protein (AIP) is up-regulated by the somatostatin analog octreotide.Metformin inhibited QGP-1 cell proliferation in a dose- and time-dependent manner, at concentrations similar to those achievable in treated patients (−31 ± 12%, p < 0.05 vs basal at 100 μM). Moreover, metformin decreased pancreatic neuroendocrine tumors (PAN-NETs) cell proliferation (−62 ± 15%, p < 0.0001 vs basal at 10 mM), without any additive effect when combined with octreotide. Both octreotide and metformin induced AIP up-regulation. AIP silencing abolished the reduction of mTOR phosphorylation induced by metformin and octreotide. Moreover, metformin decreased HSP70, increased Zac1 and AhR expression; these effects were abolished in AIP silenced QGP-1 cells.In conclusion, metformin acts as an anticancer agent in PAN-NET cells, its activity is mediated by AIP and its interacting proteins. These findings provide a novel insight into the antitumorigenic mechanism of metformin.

Inhibition of stearoyl‐CoA desaturase suppresses growth of pancreatic tumors in vitro and in vivo

Pancreatic ductal adenocarcinoma (PDAC) is a relatively rare cancer type, but is the fourth‐leading cause of cancer death in the US. Although chemotherapy and immunotherapy have been used for patients with PDAC, the overall five‐year survival rate is 8% and there is an urgent need for effective therapies. Stearoyl‐CoA desaturase (SCD) is an enzyme localized in the endoplasmic reticulum and generates monounsaturated fatty acid from saturated fatty acid by introducing a single double bond. In the present study, we examined the role of lipid metabolism regulated by SCD in PDAC growth and survival using Pdx1Cre; LSL‐KrasG12D mice and human PDAC cell lines. We isolated pancreatic tumors expressing epithelial cellular adhesion molecule by magnetic‐activated cell sorting from the Pdx1Cre; LSL‐KrasG12D mouse pancreas and formed tumor organoids in matrigel. We found that a specific SCD inhibitor (A939572) induces degeneration of the tumor organoids. Interestingly, addition of oleic acid (18:1), but not stearic acid (18:0), rescued the inhibitor‐induced degeneration of tumor organoids. Treatment with the SCD inhibitor increased mRNA expression of unfolded protein response (UPR) markers, such as Atf4, Atf6, Ddit3, spliced Xbp1, and Grp78 in organoids. The increased expression of these genes was reversed by addition of oleic acid, but not stearic acid. Similarly, the SCD inhibitor suppressed the proliferation of PANC‐1, a human PDAC cell line. The SCD inhibitor induced the expression of the UPR markers in PANC‐1 and their induction was suppressed by oleic acid, but not by stearic acid. After injection of the SCD inhibitor to Pdx1Cre; LSL‐KrasG12D mice bearing pancreatic tumors, we observed TUNEL+ cells in pancreatic tumors, but not in normal acinar cells. Our data suggest that the inhibition of SCD causes UPR and cell death in pancreatic tumors.Support or Funding InformationPilot project funding from NIH/NIAAA grant P50 AA011999, USC Dean’s pilot project program

Abstract B61: High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types

Abstract Background: Checkpoint blockade therapies like PD-1 antibodies elicit durable long-lasting immunity in a subset of patients of certain tumor types. However, many patients across a range of tumor types are resistant to checkpoint blockade. Mechanisms that confer local response or resistance to checkpoint blockade are not well understood. Mounting evidence points to local suppression of T cell function as one of the most substantial barriers to effective antitumor immunity. Methods: The study involved multiparametric flow cytometry and genomic characterization of a subset of T cells in the tumors and lymphoid organs of syngeneic tumors, genetically engineered mouse model of cancers and human tumors. Results: A significant fraction (10-40%) of α/β TCR+ CD3+ T cells do not express CD4, CD8 or NKp46 in the tumors. The presence of double-negative T-cell subset (DN T cells) is restricted to tumor microenvironment and is relatively scarce in tumor draining lymph nodes and in spleen. To identify if DN T cells are antigen-specific, we performed AH-1 tetramer staining. While a substantial fraction of CD8 T cells are AH-1 tetramer positive, majority of DN T cells are AH-1 tetramer negative in CT26 tumors. Recently, it has been shown that higher frequency of CD39-negative bystander T cells correlated to lack of response to PD-1 therapy in non-small cell lung cancer patients. Mounting evidence also reveals presence of a specific subset of CD8 T cells with features of stem cell memory in tumors as defined by CXCR5 and TCF1 expression. Efforts are under way to understand if DN T cells exhibit bystander cell and/or memory stem-like phenotype. Majority of the DN T cells are proliferating (Ki67+) and express very low levels of PD-1 (in contrast to CD8 T cells) and Granzyme B. Treatment of mice bearing syngeneic tumors with PD-1 checkpoint blockade therapy does not impact the frequency of DN T-cell subset consistent with lower levels of PD-1 expression. Moreover, a substantial frequency (30-40%) of DN T cells in both syngeneic and KPC pancreatic GEMM tumors express CD103, a tissue resident marker. Furthermore, DN T cells are CD44+ CD127+ KLRG1- and may represent a memory precursor cell population. Upon ex vivo stimulation, these DN T cell subsets proliferate and express modest levels of PD-1. Preliminary analysis of single-cell RNA sequencing data from FACS sorted murine DN T cells reveals lack of CD4, CD8 expression and retention of CD2, CD44, CD69, CCR7 and CXCR5 expression. Flow cytometry analysis of human CD2+ TILs reveals presence of DN T cells in human tumors. Single-cell transcriptome analysis of CD2+ T cells isolated from human colorectal tumors revealed presence of DN T cells. Current efforts are focused on further characterization of this subset in both mouse and human tumors. Conclusions: In this study, we describe a tumor-resident CD4 and CD8 double-negative T-cell subset that may represent a bystander cell population with features of memory precursors and potential implications in modulation of response to checkpoint blockade therapy in cancer. Citation Format: Murali Gururajan, Ravi Kandasamy, Jessica Wong, Kalpit Shah, Shuoguo Wang, Adam Bata, Amy Truong, Sunil Kuppasani, Ching-Ping Ho, Robert Graziano, Michael Quigley, Anwar Murtaza, Jinqi Liu. High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B61.

MiR-934 as a Prognostic Marker Facilitates Cell Proliferation and Migration of Pancreatic Tumor by Targeting PROX1.

BackgroundPancreatic cancer is an extremely lethal digestive cancer with late diagnosis and poor prognosis. miR-934 has been reported to serve as an oncogene in multiple cancers, such as ovarian cancer and bladder cancer. However, its role in pancreatic cancer remains undiscovered.Materials and MethodsThe expression data of miR-934 were obtained from the Gene Expression Omnibus database and from our own patient samples. The clinicopathological data and corresponding follow-up data were retrieved from The Cancer Genome Atlas database. CCK8 and colony formation assays were conducted to measure cell proliferation capacity in vitro. Wound healing and transwell assays were performed to detect the migration ability of pancreatic cancer cell.ResultsWe found that miR-934 was significantly upregulated in pancreatic tumor samples and cell lines. The expression of miR-934 was related to pathological stages. Upregulated miR-934 was associated with poor prognosis in patients with pancreatic cancer. Mir-934 inhibition reduced, while overexpression promoted, cell proliferation and migration. Mechanically, we found miR-934 could directly bind to 3ʹ-UTR of PROX1 leading to mRNA derogation. Furthermore, increased cell proliferation and migration caused by miR-934 overexpression could be reversed by forced PROX1 expression.ConclusionmiR-934 is an oncogene in pancreatic cancer and could serve as a prognosis indicator for patients with pancreatic cancer, suggesting that miR-934 is a promising therapeutic target for pancreatic cancer.

Abstract PR15: A novel NK cell-targeted therapeutic strategy against pancreatic cancer

Abstract Recent years have witnessed an increased incidence of pancreatic adenocarcinoma (PDAC). Pancreatic tumors are poorly immunogenic; current immunotherapeutic strategies largely focus on boosting adaptive immunity while ignoring immunosuppressed innate immune players such as natural killer cells (NKs). NK cells respond to tumor insults by generating IFN-γ for T-cell activation. Multiple studies suggest that tumor cells evade NK cell-mediated killing by 1) developing “escape variants” and 2) regulating inhibitory and activating receptors on NK cells, imparting anergic/exhaustive phenotype inside the pancreatic tumor microenvironment. We posit that a therapeutic combination that not only overturns tumor-mediated NKs dysfunction but also boosts cytotoxic CD8 T cells will provide long-lasting therapeutic benefits in PDAC. Given this, we explored the efficacy of a unique therapeutic combination, tumor antigen-targeted IgE antibody (humanized anti-MUC1.IgE) in combination of anti-PD-L1 (for relieving T-cell exhaustion) and PolyICLC (for dendritic cell maturation) in a preclinical model of pancreatic cancer using mice transgenic for human MUC1 and FcϵRI (hMUC1/hFcϵRI). This therapeutic combination induced MUC1 specific rejection of two different human MUC1-expressing pancreatic tumor cell lines (Panc02.MUC1, KPC.MUC1) and prolonged the overall survival of mice challenged with subcutaneous and orthotopic tumors as compared to control counterparts. Additionally, this combination generated CD8 T-cell memory response as evidenced by MUC1 specific rejection/delays of tumors in mice rechallenged with MUC1-expressing tumors. Cytokine/chemokine profiling of anti-MUC1.IgE+anti-PD-L1+PolyICLC treated tumors further demonstrates a reduction in proinflammatory cytokines as compared to control counterparts. Most importantly, NK and CD8 T cells were involved in cell-mediated antitumor responses, as in vivo depletion of these subtypes abrogated the tumor-protective benefits in mice bearing orthotopic tumors. Anti-MUC1.IgE+anti-PD-L1+PolyICLC combination appears to increase circulating NKs and reverse NK cell exhaustion inside pancreatic tumor microenvironment. Additional data suggest that this therapeutic combination boosts tumor cell killing by NK cells in antibody-dependent cell cytotoxicity assays (ADCC). In sum, this is the first study to show that specific stimulation of IgE/FcϵRI axis in combination with PolyICLC and anti-PD-L1 can activate both CD8 T and NK cell effector pathways and provide long-lasting tumor-protective benefits against pancreatic cancer. This abstract is also being presented as Poster A48. Citation Format: Kamiya Mehla, Thomas C. Caffrey, Kelly A. O'Connel, Raghupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. A novel NK cell-targeted therapeutic strategy against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR15.

Abstract PR11: Tumor cell-intrinsic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer

Abstract Intertumoral heterogeneity—the biologic and functional differences among different individual tumors—poses a challenge for immunotherapy. To understand the tumor cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated the T cell-inflamed and non-T cell-inflamed tumor microenvironments in human patients, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T cell-inflamed phenotype was dominant over the T cell-inflamed phenotype in the local tumor microenvironment. Both quantitative and qualitative features, specifically expression of markers of prior TCR activation, of intratumoral CD8+ T cells predicted the response to immunotherapies. An integrated transcriptomic and epigenetic analysis revealed that tumor cell-intrinsic expression of the chemokine CXCL1 as a major determinant of the non-T cell-inflamed microenvironment, and ablation of tumor cell-intrinsic CXCL1, promoted T-cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. These results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor cell-intrinsic factors that can be manipulated to influence the outcome of immunotherapies. The observation that non-T cell-inflamed phenotype is dominant emphasized the importance of targeting mechanisms driving T-cell low phenotype for improving immunotherapy response. This experimental system will provide opportunities for understanding other aspects of tumor heterogeneity as well. This abstract is also being presented as Poster A45. Citation Format: Jinyang Li, Katelyn T. Byrne, Fangxue Yan, Taiji Yamazoe, Zeyu Chen, Timour Baslan, Lee P. Richman, Jeffrey Lin, Yu H. Sun, Shannon M. Liudahl, John W. Tobias, Scott Lowe, Lisa M. Coussens, John E Wherry, Robert H. Vonderheide, Ben Z. Stanger. Tumor cell-intrinsic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR11.

Combination of PD-1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer

Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PD-1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors. We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD-1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD-1 and OX40 mRNAs in PDACs were determined from The Cancer Genome Atlas and compared with patient survival times. In mice with orthotopic tumors, all those given control antibody or anti-PD-1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD-1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD-1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD-1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD-1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those rechallenged with mT4 cells survived an additional median 70 days and those rechallenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD-1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD-1 and anti-OX40 that survived after complete tumor rejection were rechallenged with KPC-Luc cells; those with depletion of CD4+ T cells before the rechallenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127 compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells, and positively associated with numbers of CD4+PD-1+ T cells, CD4+CD25+ T cells, and CD8+PD-1+ T cells. PDACs with high levels of OX40 and low levels of PD-1 were associated with longer survival times of patients. Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD-1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information can be used in development of immune-based combination therapies for PDAC.

A Comprehensive Molecular Characterization of the Pancreatic Neuroendocrine Tumor Cell Lines BON-1 and QGP-1.

Experimental models of neuroendocrine tumor disease are scarce, with only a few existing neuroendocrine tumor cell lines of pancreatic origin (panNET). Their molecular characterization has so far focused on the neuroendocrine phenotype and cancer-related mutations, while a transcription-based assessment of their developmental origin and malignant potential is lacking. In this study, we performed immunoblotting and qPCR analysis of neuroendocrine, epithelial, developmental endocrine-related genes as well as next-generation sequencing (NGS) analysis of microRNAs (miRs) on three panNET cell lines, BON-1, QGP-1, and NT-3. All three lines displayed a neuroendocrine and epithelial phenotype; however, while insulinoma-derived NT-3 cells preferentially expressed markers of mature functional pancreatic β-cells (i.e., INS, MAFA), both BON-1 and QGP-1 displayed high expression of genes associated with immature or non-functional β/δ-cells genes (i.e., NEUROG3), or pancreatic endocrine progenitors (i.e., FOXA2). NGS-based identification of miRs in BON-1 and QGP-1 cells revealed the presence of all six members of the miR-17–92 cluster, which have been implicated in β-cell function and differentiation, but also have roles in cancer being both oncogenic or tumor suppressive. Notably, both BON-1 and QGP-1 cells expressed several miRs known to be negatively associated with epithelial–mesenchymal transition, invasion or metastasis. Moreover, both cell lines failed to exhibit migratory activity in vitro. Taken together, NT-3 cells resemble mature functional β-cells, while both BON-1 and QGP-1 are more similar to immature/non-functional pancreatic β/δ-cells or pancreatic endocrine progenitors. Based on the recent identification of three transcriptional subtypes in panNETs, NT-3 cells resemble the “islet/insulinoma tumors” (IT) subtype, while BON-1 and QGP-1 cells were tentatively classified as “metastasis-like/primary” (MLP). Our results provide a comprehensive characterization of three panNET cell lines and demonstrate their relevance as neuroendocrine tumor models.

Tumor Cell-Intrinsic USP22 Suppresses Antitumor Immunity in Pancreatic Cancer.

Although immune checkpoint blockade (ICB) improves clinical outcome in several types of malignancies, pancreatic ductal adenocarcinoma (PDA) remains refractory to this therapy. Preclinical studies have demonstrated that the relative abundance of suppressive myeloid cells versus cytotoxic T cells determines the efficacy of combination immunotherapies, which include ICB. Here, we evaluated the role of the ubiquitin-specific protease 22 (USP22) as a regulator of the immune tumor microenvironment (TME) in PDA. We report that deletion of USP22 in pancreatic tumor cells reduced the infiltration of myeloid cells and promoted the infiltration of T cells and natural killer (NK) cells, leading to an improved response to combination immunotherapy. We also showed that ablation of tumor cell-intrinsic USP22 suppressed metastasis of pancreatic tumor cells in a T-cell-dependent manner. Finally, we provide evidence that USP22 exerted its effects on the immune TME by reshaping the cancer cell transcriptome through its association with the deubiquitylase module of the SAGA/STAGA transcriptional coactivator complex. These results indicated that USP22 regulates immune infiltration and immunotherapy sensitivity in preclinical models of pancreatic cancer.

Macropinocytosis Renders a Subset of Pancreatic Tumor Cells Resistant to mTOR Inhibition

SummaryPancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach.

A Virus-Infected, Reprogrammed Somatic Cell-Derived Tumor Cell (VIReST) Vaccination Regime Can Prevent Initiation and Progression of Pancreatic Cancer.

Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.

Orphan designation: Two allogeneic irradiated pancreatic tumour cell lines, Treatment of pancreatic cancer

Orphan designation: Two allogeneic irradiated pancreatic tumour cell lines, Treatment of pancreatic cancer

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling.

The irreversible ERBB1/2/4 inhibitor neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of neratinib.

The Microarchitecture of Pancreatic Cancer as Measured by Diffusion-Weighted Magnetic Resonance Imaging Is Altered by T Cells with a Tumor Promoting Th17 Phenotype.

Diffusion-weighted magnetic resonance imaging (DW-MRI) is a diagnostic tool that is increasingly used for the detection and characterization of focal masses in the abdomen, among these, pancreatic ductal adenocarcinoma (PDAC). DW-MRI reflects the microarchitecture of the tissue, and changes in diffusion, which are reflected by changes in the apparent diffusion coefficient (ADC), are mainly attributed to variations in cellular density, glandular formation, and fibrosis. When analyzing the T cell infiltrates, we found an association of a tumor-promoting subpopulation, characterized by the expression of interleukin (IL) 21 and IL26, with high ADC values. Moreover, the presence of IL21+ and IL26+ positive T cells was associated with poor prognosis. Pancreatic cancers—but not healthy pancreatic tissue—expressed receptors for IL21 and IL26, a finding that could be confirmed in pancreatic cell lines. The functionality of these receptors was demonstrated in pancreatic tumor cell lines, which showed phosphorylation of ERK1/2 and STAT3 pathways in response to the respective recombinant interleukins. Moreover, in vitro data showed an increased colony formation of tumor cells. In summary, our data showed an association of IL21+ and IL26+ immune cell infiltration, increased ADC, and aggressive tumor disease, most likely due to the activation of the key cancer signaling pathways ERK1/2 and STAT3 and formation of tumor colonies.

Enhanced signaling via ERBB3/PI3K plays a compensatory survival role in pancreatic tumor cells exposed to [neratinib + valproate

The ERBB1/2/4 inhibitor neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles; this effect is enhanced by HDAC inhibitors and the combination of [neratinib + sodium valproate] is now a phase I trial (NCT03919292). The present studies were performed to understand resistance mechanisms that evolve following [neratinib + valproate] exposure. Exposure of pancreatic tumor cells to [neratinib + sodium valproate] initially reduced the expression and phosphorylation of ERBB family receptors, c-MET and c-KIT. Following a 24 h drug exposure and a further 24 h culture in drug free conditions, the effects on c-MET, c-KIT and most ERBB family receptors had returned to near baseline levels. However, the expression and phosphorylation of ERBB3 were increased which was associated with elevated AKT T308 phosphorylation. Knock down of ERBB3 significantly enhanced [neratinib + valproate] lethality, which was associated with greater inactivation of AKT, mTOR, p70 S6K and ERK1/2. The PI3Kα/δ inhibitor copanlisib also significantly enhanced killing after [neratinib + valproate] exposure. Copanlisib enhanced [neratinib + valproate] lethality via autophagosome formation and autophagic flux. Our data argue for further in vivo exploration as to whether copanlisib can be safely combined with [neratinib + valproate].

Epithelial-mesenchymal transition: a hallmark in pancreatic cancer stem cell migration, metastasis formation, and drug resistance.

Metastasis, tumor progression, and chemoresistance are the major causes of death in patients with pancreatic ductal adenocarcinoma (PDAC). Tumor dissemination is associated with the activation of an epithelial-to-mesenchymal transition (EMT) process, a program by which epithelial cells lose their cell polarity and cell-to-cell adhesion, and acquire migratory and invasive abilities to become mesenchymal stem cells (MSC). These MSCs are multipotent stromal cells capable of differentiating into various cell types and trigger the phenotypic transition from an epithelial to a mesenchymal state. Therefore, EMT promotes migration and survival during cancer metastasis and confers stemness features to particular subsets of cells. Furthermore, a major problem limiting our ability to treat PDAC is the existence of rare populations of pancreatic cancer stem cells (PCSCs) or cancer-initiating cells in pancreatic tumors. PCSCs may represent sub-populations of tumor cells resistant to therapy which are most crucial for driving invasive tumor growth. These cells are capable of regenerating the cellular heterogeneity associated with the primary tumor when xenografted into mice. Therefore, the presence of PCSCs has prognostic relevance and influences the therapeutic response of tumors. PCSCs express markers of cancer stem cells (CSCs) including CD24, CD133, CD44, and epithelial specific antigen as well as the drug transporter ABCG2 grow as spheroids in a defined growth medium. A major difficulty in studying tumor cell dissemination and metastasis has been the identification of markers that distinguish metastatic cancer cells from cells that are normally circulating in the bloodstream or at sites where these cells metastasize. Evidence highlights a linkage between CSC and EMT. In this review, The current understanding of the PCSCs, signaling pathways regulating these cells, PDAC heterogeneity, EMT mechanism, and links between EMT and metastasis in PCSCs are summarised. This information may provide potential therapeutic strategies to prevent EMT and trigger CSC growth inhibition and cell death.

Detection of circulating tumor cells in blood of pancreatic ductal adenocarcinoma patients.

Aim: Previous studies suggest that circulating tumor cells (CTC) are present at very low frequencies in blood of pancreatic cancer (PC) patients. However, no technique has proven efficient for their detection, in part due to the lack of accurate tumor markers. Here, we evaluated the potential utility of two marker candidates - Mucin 16 (MUC16) and Tetraspanin 1 (TSPAN1) - identified through a detailed review of the literature.Methods: To evaluate the pattern of expression of both markers in pancreatic tumor cells vs. normal blood, we used cell lines derived from pancreatic cancer patients and blood from healthy adults.Results: Antibodies against both MUC16 and TSPAN1 showed expression in three pancreatic cancer (PC) cell lines while they were absent in blood cells. To evaluate the efficiency of isolating tumor cells from blood, PC cell lines were spiked at different frequencies in blood, sequentially stained with biotin-conjugated TSPAN1 and MUC16 antibodies and a streptavidin ferrofluids, followed by immunomagnetic enrichment. The recovery of spiked TSPAN1+ tumor cells was high with limited contamination by leukocytes. In contrast, no PC cells were isolated when the biotin MUC16 reagent was used because the biotin-conjugated clone did not recognize PC cells.Conclusion: The combination of MUC16, TSPAN1, and epithelial cell adhesion molecule (EpCAM) antibodies will likely increase the efficiency of capturing circulating tumor cell in blood of pancreatic ductal adenocarcinoma. To further develop a protocol for isolation of circulating tumor cell in blood of PC patients, high amounts of antibodies (5-10 mg) against EpCAM, MUC16, and TSPAN1 will be needed.

The Evaluation of Gene Oct4 Expression as a New Tumor Marker in Pancreatic Tumor and Non-Tumor Cell Lines

Introduction: Oct4 gene is one of the important genes involved in stem cell self-renewal control. Recently, this gene has been proposed as a novel molecular marker for cancer detection. Therefore, in this study we examined the expression of Oct4 gene in MIA Paca-2, PA-TU-8902 and AsPC-1 cell lines as well as pancreatic cancer.

Effects of Akkermansia muciniphila on the Proliferation, Apoptosis and Insulin Secretion of Rat Islet Cell Tumor Cells

To investigate the effects of Akkermansia muciniphila ( A. muciniphila) on the proliferation, apoptosis and insulin secretion of rat pancreatic islet cell tumor cells (INS-1). INS-1 cells were divided into three groups, normal, repair, and protect groups, and subsequently every group was subjected with A. muciniphila metabolites, live A. muciniphilaorpasteurized A. muciniphila for 48 h. A group that did not treat with anything was set as blank control. After intervention, the cell viability was determined by MTT method, the insulin secretion level stimulated by glucose was determined by ELISA, the expressions of the genes involved in insulin secretion and apoptosis were tested by qRT-PCR, and the expression of apoptosis related protein Bax was evaluated by Western blot. There was no significant change in INS-1 cell morphology after co-incubation with 3 types of A. Muciniphila interventions for 48 h. The proliferative activity of INS-1 cells was decreased in the repair group that treated with live A. muciniphila than that of control ( P<0.005). A. muciniphila intervention had no effect on insulin secretion in INS-1 cells in normal, repair or protection group ( P>0.05). A. muciniphila secretions promoted the expression of glucose transporter 2 ( Glut2) in 3 groups and the expression of glucokinase ( GCK) in repair group ( P<0.05). The expression of Baxof the INS-1 cell in the normal group was decreased after intervented with 3 kinds of A. muciniphila intervention materials ( P<0.001).The expression of Bax gene of the INS-1 cell in the repair group that treated with dead A. muciniphilawas decreased ( P<0.05). The expression of Bax protein of INS-1 cells that treated with A. muciniphila interventions was decreased. A. muciniphila can promote the expression of insulin secretion-related genes in INS-1 cells, inhibit the expression of apoptotic genes and apoptosis protein Bax.This research provides a new direction for applying A. muciniphila in improving type 2 diabetes.