scholarly journals The Identification of the Anthracycline Aclarubicin as an Effective Cytotoxic Agent for Pancreatic Cancer

2020 ◽  
Author(s):  
Thomas Brouwer ◽  
Sabina Y van der Zanden ◽  
Jaap DH van Eendenburg ◽  
Bert A Bonsing ◽  
Noel FCC de Miranda ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Lines ◽  
Dose Range ◽  
Standard Of Care ◽  
New Combination ◽  
Patient Treatment ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Short Term Exposure ◽  
Low Passage

Abstract BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low passage PDAC cell lines.MethodsProliferation and colony formation assays were performed to determine the anti-cancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low passage PDAC cell lines. In addition, the effect of standard of care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. The assays involved short-term exposure to the drugs in order to mimic pharmacokinetics in a patient.ResultsAclarubicin showed superior anti-tumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low passage PDAC cell line. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. Subsequent testing in commercial cell lines showed similar cytotoxic effects of aclarubicin in two out of three cell lines. Gemcitabine and doxorubicin had variable responses between the cell lines, but their effect never exceeded that of aclarubicin.ConclusionsAclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re-)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.

scholarly journals Identification of the Neurokinin-1 Receptor as Targetable Stratification Factor for Drug Repurposing in Pancreatic Cancer

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2703
Author(s):  
Iris Beirith ◽  
Bernhard W. Renz ◽  
Shristee Mudusetti ◽  
Natalja Sergejewna Ring ◽  
Julian Kolorz ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Lines ◽  
Splice Variants ◽  
Drug Repurposing ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Neurokinin 1 Receptor ◽  
Pdac Tissue ◽  
Dependent Growth ◽  
Neurokinin 1

The SP/NK1R-complex plays an important role in tumor proliferation. Targeting of the neurokinin-1 receptor in previous studies with its antagonist aprepitant (AP) resulted in anti-tumoral effects in colorectal cancer and hepatoblastoma. However, there is still a lack of knowledge regarding its effects on pancreatic cancer. Therefore, we treated human pancreatic ductal adenocarcinoma (PDAC) cell lines (Capan-1, DanG, HuP-T3, Panc-1, and MIA PaCa-2) and their cancer stem cell-like cells (CSCs) with AP and analyzed functional effects by MTT-, colony, and sphere formation assays, respectively; moreover, we monitored downstream mechanisms by flow cytometry. NK1R inhibition resulted in dose-dependent growth reduction in both CSCs and non-CSCs without induction of apoptosis in most PDAC cell lines. More importantly, we identified striking AP dependent cell cycle arrest in all parental cells. Furthermore, gene expression and the importance of key genes in PDAC tumorigenesis were analyzed combining RT-qPCR in eight PDAC cell lines with publicly available datasets (TCGA, GEO, CCLE). Surprisingly, we found a better overall survival in patients with high NK1R levels, while at the same time, NK1R was significantly decreased in PDAC tissue compared to normal tissue. Interestingly, there is currently no differentiation between the isoforms of NK1R (truncated and full; NK1R-tr and -fl) in any of the indicated public transcriptomic records, although many publications already emphasize on important regulatory differences between the two isoforms of NK1R in many cancer entities. In conclusion, analysis of splice variants might potentially lead to a stratification of PDAC patients for NK1R-directed therapies. Furthermore, we presume PDAC patients with high expressions of NK1R-tr might benefit from treatment with AP to improve chemoresistance. Therefore, analysis of splice variants might potentially lead to a stratification of PDAC patients for NK1R-directed therapies.

Biologically optimized schedule of gemcitabine and nab-paclitaxel regimen in metastatic pancreatic adenocarcinoma.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. TPS4168-TPS4168
Author(s):  
Laith I. Abushahin ◽  
Anne M. Noonan ◽  
John L. Hays ◽  
Pannaga G. Malalur ◽  
Ashish Manne ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Cells ◽  
Pancreatic Adenocarcinoma ◽  
Cell Lines ◽  
Dose Intensity ◽  
Standard Of Care ◽  
Significance Level ◽  
Pancreatic Cancer Cells ◽  
Metastatic Pancreatic Adenocarcinoma

TPS4168 Background: Metastatic pancreatic adenocarcinoma has a poor prognosis, and improvements in therapy have been challenging. Alongside efforts in developing novel agents, there is a need to optimize and maximize the benefit of currently approved drugs. Gemcitabine + nab-paclitaxel is a frequently used regimen for pancreatic adenocarcinoma. Nab-paclitaxel is albumin–bound chemotherapy; hence the role of albumin uptake is critical for its effect. Caveolae are small membrane invaginations essential for transendothelial albumin uptake. Cav-1 is the principal structural component of caveolae. Williams and colleagues have published a series of preclinical studies demonstrating that tumor cell-specific Cav-1 expression directly correlates with albumin and albumin-bound chemotherapy uptake and subsequent apoptotic response in tumor cells. In vitro studies showed that exposure of pancreatic cancer cells to Gemcitabine resulted in up-regulation of Cav-1 peaking 48 hours after gemcitabine exposure. This Cav-1 up-regulation correlated with increased temporal albumin cellular uptake. In addition, Williams and colleagues noted that exposure of pancreatic cancer cell lines to Gemcitabine resulted in a time–specific re-entry of cells into the G2/M phase (nab-paclitaxel cytotoxicity phase) between 48-60 hours after gemcitabine treatment. Collectively this data suggest that infusing nab-paclitaxel after 48 hours of gemcitabine infusion would be optimal for both increased uptake as well as increased susceptible tumor cells. We had previously shown this effect on multiple cell lines as well as mouse models. Methods: This is a phase II trial; patients will receive a standard of care chemotherapy regimen consisting of FDA-approved Gemcitabine + nab-paclitaxel with modification of the schedule to deliver nab-paclitaxel 48 hours (2 days) after gemcitabine infusions. The primary endpoint is ORR, with a null hypothesis of 20% vs. a target of 35%. Employing a 2-stage design (minimax) and assuming 80% power and a 0.05 significance level, a total of 53 patients will be required. In the first stage, if at least 7/31 patients respond to therapy, an additional 22 patients will be added for a total of 53 patients. The study will be terminated early if ≤ six patients respond in the first stage. Observation of response in at least 16/53 patients would be required to warrant further investigation of this infusion schedule of combination therapy. The secondary endpoints include the safety of the regimen schedule, Relative dose intensity, disease control rate, PFS, and OS. The trial opened to enrollment in June 2020 and is accepting patients. Clinical trial information: NCT04115163.

scholarly journals Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Sébastien A. Felt ◽  
Gaith N. Droby ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Oncolytic Virus ◽  
Ductal Adenocarcinoma ◽  
Mechanisms Of Resistance ◽  
Pdac Cell ◽  
Ldl Uptake ◽  
Vesicular Stomatitis ◽  
Pdac Cell Line

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.

scholarly journals Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Sara L. Seegers ◽  
Connor Frasier ◽  
Sarah Greene ◽  
Irina V. Nesmelova ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cell Lines ◽  
Genomic Stability ◽  
Oncolytic Viruses ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Virus Attachment ◽  
Vesicular Stomatitis ◽  
Moderately Resistant ◽  
Evolution Approach

ABSTRACT Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes. IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.

Investigating autophagy and glutamine metabolism as therapeutic targets for pancreatic cancer.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 381-381
Author(s):  
Mario Jardon ◽  
Steve Kalloger ◽  
Christina Iggulden ◽  
Nancy Erro Go ◽  
Paalini Sathiyaseelan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Targets ◽  
Glutamine Metabolism ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Multiple Cancer ◽  
Tissue Micro Array ◽  
Cancer Hallmarks ◽  
Functional Studies ◽  
New Therapeutic Approaches

381 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, for which new therapeutic approaches are urgently needed. We are developing novel combination therapy approaches based on the inhibition of glutamine metabolism and autophagy, to improve current treatments for PDAC. Since both processes are key mediators of multiple cancer hallmarks, and have inter-related but non-redundant roles, this combination may result in a more efficient disruption of cancer cell resistance to treatments. Methods: We interrogated the Pancreas Centre BC tissue micro-array, containing the epithelial component of 252 PDAC samples, for expression of three key glutamine-metabolizing proteins and two autophagy-related proteins: glutamine synthetase (GLUL), asparagine synthetase (ASNS), glutaminase C (GLS-GAC), microtubule-associated protein 1 light chain 3 beta (MAP1-LC3B or LC3B) and autophagy-related protein 4B (ATG4B). While previous efforts by other groups have focused on GLS-GAC, the role of GLUL in cancer has remained less well understood. We thus investigated the functional relevance of GLUL using a panel of PDAC cell lines. We are also investigating interactions between glutamine metabolism and autophagy, including the exploration of strategies to target GLUL and the evaluation of a new class of ATG4B inhibitors for the treatment of pancreatic cancers. Results: We found that GLUL, ASNS, GLS-GAC, LC3B and ATG4B were expressed in 31%, 58%, 99%, 51% and 73%, respectively, of PDAC samples. Furthermore, higher LC3B expression correlated with poor outcome. Our functional studies revealed that various PDAC cells express GLUL, which can be upregulated upon glutamine deprivation. In all cell lines tested, GLUL knockdown sensitized them to gemcitabine, as assessed by a long-term recovery assay. We also found that the candidate ATG4B inhibitors, shown to inhibit this target in cell-free assays, effectively inhibit PDAC cell proliferation at micromolar concentrations. Conclusions: Our study reveals that glutamine metabolism and autophagy are clinically relevant in PDAC and may have potential as therapeutic targets. Supported by Pancreas Centre BC, BC Cancer Foundation and VGH Foundation.

scholarly journals Feasibility and safety of electrochemotherapy (ECT) in the pancreas: a pre-clinical investigation

2015 ◽  
Vol 49 (2) ◽  
pp. 147-154 ◽  
Author(s):  
Roberto Girelli ◽  
Simona Prejanò ◽  
Ivana Cataldo ◽  
Vincenzo Corbo ◽  
Lucia Martini ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Lines ◽  
Clinical Investigation ◽  
Tumour Response ◽  
Chemotherapeutic Agents ◽  
Ductal Adenocarcinoma ◽  
Safe Procedure ◽  
Reversible Electroporation

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease generally refractory to standard chemotherapeutic agents; therefore improvements in anticancer therapies are mandatory. A major determinant of therapeutic resistance in PDAC is the poor drug delivery to neoplastic cells, mainly due to an extensive fibrotic reaction. Electroporation can be used in vivo to increase cancer cells’ local uptake of chemotherapeutics (electrochemotherapy, ECT), thus leading to an enhanced tumour response rate. In the present study, we evaluated the in vivo effects of reversible electroporation in normal pancreas in a rabbit experimental model. We also tested the effect of electroporation on pancreatic cancer cell lines in order to evaluate their increased sensitivity to chemotherapeutic agents. Materials and methods. The application in vivo of the European Standard Operating Procedure of Electrochemotherapy (ESOPE) pulse protocol (1000 V/cm, 8 pulses, 100 μs, 5 KHz) was tested on the pancreas of normal New Zealand White Rabbits and short and long-term toxicity were assessed. PANC1 and MiaPaCa2 cell lines were tested for in vitro electrochemotherapy experiments with and without electroporation. Levels of cell permeabilization were determined by flow cytometry, whereas cell viability and drug (cisplatin and bleomycin) sensitivity of pulsed cells were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Results. In healthy rabbits, neither systemic nor local toxic effects due to the electroporation procedure were observed, demonstrating the safety of the optimized electric parameters in the treatment of the pancreas in vivo. In parallel, we established an optimized protocol for ECT in vitro that determined an enhanced anti-cancer effect of bleomycin and cisplatin with respect to treatment without electroporation. Conclusions. Our data suggest that electroporation is a safe procedure in the treatment of PDAC because it does not affect normal pancreatic parenchyma, but has a potentiating effect on cytotoxicity of bleomycin in pancreatic tumour cell lines. Therefore, ECT could be considered as a valid alternative for the local control of non-resectable pancreatic cancer.

scholarly journals Robo2 Predicts a Better Prognosis and Inhibits Malignant Behavior in Vivo in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Author(s):  
Cheng Ding ◽  
Yatong Li ◽  
Shunda Wang ◽  
Cheng Xing ◽  
Lixin Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Ductal Adenocarcinoma ◽  
Migration And Invasion ◽  
Pdac Cell ◽  
Pancreatic Cell

Abstract BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an extremely poor prognosis and a high mortality rate. Genome-wide studies have shown that the SLIT/ROBO signaling pathway plays an important role in pancreatic tumor development and progression. However, the effect and mechanism of ROBO2 in the progression of pancreatic cancer remains largely unknown.MethodsIn this study, real-time polymerase chain reaction (RT-PCR) and western blot analyses were adopted to evaluate the expression level of ROBO2 and proteins in pancreatic cell lines. Cell migration and invasion and cell proliferation were conducted in AsPC-1 and MIA PaCa-2 cell lines. RNA sequencing and western blot were undertaken to explore the mechanisms and potential targeted molecules. ROBO2 expression in tumor tissues was evaluated by immunohistochemistry in 95 patients.ResultsROBO2 expression was downregulated in PDAC cell lines and tissue samples. A high level of ROBO2 was associated with good overall survival. Upregulation of ROBO2 inhibited PDAC cell proliferation, migration, and invasion, whereas the opposite results were found in the ROBO2 downregulation group. In addition, xenograft animal models further confirmed the effect of ROBO2 on proliferation. Finally, the RNA sequencing results indicated that ROBO2 facilitates anti-tumorigenicity partly via inhibiting ECM1 in PDAC. ConclusionsOur work suggests that ROBO2 inhibits tumor progression in PDAC and may serve as a predictive biomarker and therapeutic target in PDAC.

scholarly journals PEPT1 is essential for the growth of pancreatic cancer cells: A viable drug target

2021 ◽  
Author(s):  
Bradley Schniers ◽  
Devaraja Rajasekaran ◽  
Ksenija Korac ◽  
Tyler Sniegowski ◽  
Vadivel Ganapathy ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cell Lines ◽  
Intracellular Localization ◽  
Peptide Transporter ◽  
Pdac Cell ◽  
Functional Link ◽  
Pancreatic Cancer Cells

PEPT1 is a proton-coupled peptide transporter that is upregulated in PDAC cell lines and PDXs, with little expression in normal pancreas. However, the relevance of this upregulation to cancer progression and the mechanism of upregulation have not been investigated. Herein, we show that PEPT1 is not just upregulated in a large panel of PDAC cell lines and PDXs but is also functional and transport-competent. PEPT2, another proton-coupled peptide transporter, is also overexpressed in PDAC cell lines and PDXs, but is not functional due to its intracellular localization. Using glibenclamide as a pharmacological inhibitor of PEPT1, we demonstrate in cell lines in vitro and mouse xenografts in vivothat inh­­ibition of PEPT1 reduces the proliferation of the cancer cells. These findings are supported by genetic knockdown of PEPT1 with shRNA, wherein the absence of the transporter significantly attenuates the growth of cancer cells, both in vitro and in vivo, suggesting that PEPT1 is critical for the survival of cancer cells. We also establish that the tumor-derived lactic acid (Warburg effect) in the tumor microenvironment supports the transport function of PEPT1 in the maintenance of amino acid nutrition in cancer cells by inducing MMPs and DPPIV to generate peptide substrates for PEPT1 and by generating a H+ gradient across the plasma membrane to energize PEPT1. Taken collectively, these studies demonstrate a functional link between PEPT1 and extracellular protein breakdown in the tumor microenvironment as a key determinant of pancreatic cancer growth, thus identifying PEPT1 as a potential therapeutic target for PDAC.

scholarly journals 904 Repurposing CD26 (DPP4) inhibitors to enhance immunotherapy response in pancreatic ductal adenocarcinoma

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A948-A949
Author(s):  
Maggie Phillips ◽  
Michael Ware ◽  
Cameron Herting ◽  
Thomas Mace ◽  
Shishir Maithel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Lines ◽  
Tumor Volume ◽  
Fold Change ◽  
Ductal Adenocarcinoma ◽  
Murine Models ◽  
Pdac Cell ◽  
Cd26 Expression

BackgroundPancreatic ductal adenocarcinoma (PDAC) is refractory to immunotherapy due in part to cellular cross-talk with cancer associated fibroblasts (CAFs). These interactions shape the microenvironment in a manner that is profoundly immunosuppressive. Our group is identifying novel targets in the PDAC stroma that can be manipulated to enhance immunotherapy efficacy. We hypothesize dysregulation of the serine protease, CD26/DPP4 in PDAC contributes to the limited efficacy of immunotherapy. Further, we posit targeting CD26 enzymatic activity using inhibitors that are FDA-approved for adult patients with Type 2 Diabetes Mellitus can enhance the efficacy of immunotherapy in PDAC.MethodsPrimary CAFs isolated from patient PDAC resection specimens under an IRB-approved protocol, were subject to NanoString analysis.1 CD26 protein expression was measured in primary and immortalized CAFs and PDAC cells by immunoblot, flow cytometry and immunofluorescence, while ELISA detected soluble CD26. For in vivo efficacy, luciferase-expressing KPC-tumor cells were implanted orthotopically in the pancreas of immune-competent C57BL/6 mice. Bioluminescence imaging (BLI) confirmed established tumors and mice were randomized to sitagliptin (75 mg/kg in drinking water, CD26/DPP4 inhibitor), anti-PD-L1 Ab (200 ug 2x/week), or both combined for 3 weeks. Controls received vehicle or isotype control Ab. BLI utilized to track tumor progression and tissues harvested for analysis at study endpoint (day 18 of treatment).ResultsNanoString analysis identified CD26/DPP4 as significantly upregulated in RNA transcripts from primary CAFs vs. fibroblasts from normal pancreas (figure 1). We confirmed abundant CD26 expression on patient-derived CAFs and immortalized CAF cell lines, however, lower CD26 expression was observed on human PDAC cell lines (HPAC, PANC-1) by immunoblot, flow cytometry and immunofluorescence (figure 5).Abstract 904 Figure 1(A) Schema for analysis of transcript from n=10 primary CAFs (PSC) from PDAC patients vs. normal human pancreatic fibroblasts (HPPFC) via NanoString nCounter PanCancer Immune Profiling Panel. (B) Heat map of gene expression with upregulate DPP4 or CD26 transcript detected. Adapted from Mace et al., 2016.Abstract 904 Figure 2Validation of CD26 protein expression in human PDAC-derived CAF and PDAC cell lines by immunoblot analysisAbstract 904 Figure 3Analysis of surface human CD26 expression in PBMCs, PDAC-derived CAFs (h-iPSC-PDAC-1), and PDAC cells (PANC-1) by flow cytometry. Histograms representing human surface CD26 expressionAbstract 904 Figure 4Immunofluorescence analysis of CD26/DPP4 cellular localization in a human PDAC-derived CAF cell lineAbstract 904 Figure 5Combined Sitagliptin and PD-L1 blockade in a murine orthotopic model of PDAC. Fold change in tumor volume, determined by BLI, comparing baseline (Day 0 of treatment) to Day 18 of treatment. Each bar represents fold change in BLI determined tumor volume for each animalConclusionsOur results are the first to describe CD26 expression on PDAC-derived CAFs and indicate that sitagliptin augments anti-tumor activity of anti-PD-L1 in PDAC tumor-bearing mice. Our ongoing work will provide insight into specific immune cell populations responsible for efficacy of immunotherapy in murine models of PDAC, and the role of CD26 in various cellular compartments within the PDAC microenvironment.ReferencesMace TA, Shakya R, Pitarresi JR, Swanson B, McQuinn CW, Loftus S, Nordquist E, Cruz-Monserrate Z, Yu L, Young G, Zhong X, Zimmers TA, Ostrowski MC, Ludwig T, Bloomston M, Bekaii-Saab T, Lesinski GB. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut 2018;67(2):320–32.

scholarly journals Contribution of nuclear BCL10 expression to tumor progression and poor prognosis of advanced and/or metastatic pancreatic ductal adenocarcinoma by activating NF-κB-related signaling

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sung-Hsin Kuo ◽  
Shih-Hung Yang ◽  
Ming-Feng Wei ◽  
Hsiao-Wei Lee ◽  
Yu-Wen Tien ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Progression ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Cyclin B1 ◽  
Ductal Adenocarcinoma ◽  
Clinical Samples ◽  
Wild Type ◽  
Pdac Cell

Abstract Background We previously demonstrated that nuclear BCL10 translocation participates in the instigation of NF-κB in breast cancer and lymphoma cell lines. In this study, we assessed whether nuclear BCL10 translocation is clinically significant in advanced and metastatic pancreatic ductal adenocarcinoma (PDAC). Method and materials We analyzed the expression of BCL10-, cell cycle-, and NF-κB- related signaling molecules, and the DNA-binding activity of NF-κB in three PDAC cell lines (mutant KRAS lines: PANC-1 and AsPC-1; wild-type KRAS line: BxPC-3) using BCL10 short hairpin RNA (shBCL10). To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. We assessed the expression patterns of BCL10 and NF-κB in tumor cells in 136 patients with recurrent, advanced, and metastatic PDAC using immunohistochemical staining. Results We revealed that shBCL10 transfection caused cytoplasmic translocation of BCL10 from the nuclei, inhibited cell viability, and enhanced the cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Inhibition of BCL10 differentially blocked cell cycle progression in PDAC cell lines. Arrest at G1 phase was noted in wild-type KRAS cell lines; and arrest at G2/M phase was noted in mutant KRAS cell lines. Furthermore, shBCL10 transfection downregulated the expression of phospho-CDC2, phospho-CDC25C, Cyclin B1 (PANC-1), Cyclins A, D1, and E, CDK2, and CDK4 (BxPC-3), p-IκBα, nuclear expression of BCL10, BCL3, and NF-κB (p65), and attenuated the NF-κB pathway activation and its downstream molecule, c-Myc, while inhibition of BCL10 upregulated expression of p21, and p27 in both PANC-1 and BxPC-3 cells. In a PANC-1-xenograft mouse model, inhibition of BCL10 expression also attenuated the tumor growth of PDAC. In clinical samples, nuclear BCL10 expression was closely associated with nuclear NF-κB expression (p < 0.001), and patients with nuclear BCL10 expression had the worse median overall survival than those without nuclear BCL10 expression (6.90 months versus 9.53 months, p = 0.019). Conclusion Nuclear BCL10 translocation activates NF-κB signaling and contributes to tumor progression and poor prognosis of advanced/metastatic PDAC.

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