Primary Pancreatic Cancer Cells Research Articles

Identification of 2 Chinese Primary Pancreatic Ductal Adenocarcinoma Cancer Cell Lines and Their Phenotypes.

Pancreatic cancer (PC) is one of the most lethal cancers. Currently, the most commonly used PC cell lines were all derived from White individuals, and few models can be used to study in Asian populations. In China, the incidence of PC is increasing every year, highlighting the need to develop new Chinese PC cell lines for cancer research. A total of 203 patients diagnosed with PC, enrolled from January 2012 to December 2017 in our hospital (Department of Hepatobiliary and Pancreatic Surgery), had received surgery. Primary PC patient tumor samples were harvested sterilely from surgery, minced, and digested in collagenase. Cells were cultured in plates precoated with l-polylysine. Two primary PC cell lines, Si-Liang 187 and Si-Liang 188, were established, both of which grew as adherent monolayers and demonstrated distinguished phenotypes. Si-Liang 187 shows the typical mesenchymal pattern, whereas Si-Liang 188 shows epithelial phenotype. Moreover, they carry different genetic backgrounds according to whole exome sequencing. Two new Chinese PC cell lines were established, both of which were characterized and confirmed with high tumorigenicity. They may serve as useful tools for pathogenesis research when evaluating new treatment strategies in Asian patients.

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

Elevated expression of nuclear receptor-binding SET domain 3 promotes pancreatic cancer cell growth

The nuclear receptor-binding SET domain 3 (NSD3) catalyzes methylation of histone H3 at lysine 36 (H3K36), and promotes malignant transformation and progression of human cancer. Its expression, potential functions and underlying mechanisms in pancreatic cancer are studied. Bioinformatics studies and results from local human tissues show that NSD3 is upregulated in human pancreatic cancer tissues, which is correlated with poor overall survival. In primary and established pancreatic cancer cells, NSD3 silencing (by shRNAs) or CRISPR/Cas9-induced NSD3 knockout potently inhibited cell proliferation, migration and invasion, while provoking cell cycle arrest and apoptosis. Conversely, ectopic expression of NSD3-T1232A mutation significantly accelerated proliferation, migration, and invasion of pancreatic cancer cells. H3K36 dimethylation, expression of NSD3-dependent genes (Prkaa2, Myc, Irgm1, Adam12, and Notch3), and mTOR activation (S6K1 phosphorylation) were largely inhibited by NSD3 silencing or knockout. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD3 shRNA potently inhibited pancreatic cancer xenograft growth in nude mice. These results suggest that elevated NSD3 could be an important driver for the malignant progression of pancreatic cancer.

Dendrimeric nanosystem consistently circumvents heterogeneous drug response and resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with no efficacious treatment. The application of nanomedicine is expected to bring new hope to PDAC treatment. In this study, we report a novel supramolecular dendrimeric nanosystem carrying the anticancer drug doxorubicin, which demonstrated potent anticancer activity, markedly overcoming the heterogeneity of drug response and resistance of primary cultured tumor cells derived from PDAC patients. This dendrimer nanodrug was constructed with a fluorinated amphiphilic dendrimer, which self-assembled into micelles nanostructure and encapsulated doxorubicin with high loading. Because of the fine nanosize, stable formulation and acid-promoted drug release, this dendrimeric nanosystem effectively accumulated in tumor, with deep penetration in tumor tissue and rapid drug uptake/release profile in cells, ultimately resulting in potent anticancer activity and complete suppression of tumor growth in patient-derived xenografts. Most importantly, this dendrimer nanodrug generated uniform and effective response when treating 35 primary pancreatic cancer cell lines issued from patient samples as a robust platform for preclinical drug efficacy testing. In addition, this dendrimer nanodrug formulation was devoid of adverse effects and showed excellent tolerability. Given all these uniquely advantageous features, this simple and convenient dendrimer nanodrug holds great promise as a potential candidate to treat the deadly PDAC.

Telomerase and Pluripotency Factors Jointly Regulate Stemness in Pancreatic Cancer Stem Cells.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with very limited therapeutic options. Cancer stem cells (CSCs) are essential for propagation of PDAC, but also for its metastatic activity and chemoresistance. To date, it is still unclear how cancer stem cells (CSCs) regulate their ‘stemness’ and self-renewal properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. Here, we investigate how telomerase regulation affects CSC biology in PDAC, and delineate the mechanisms by which telomerase activity and CSC properties are linked.To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the “stemness” maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

Nanomechanical Insight of Pancreatic Cancer Cell Membrane during Receptor Mediated Endocytosis of Targeted Gold Nanoparticles.

Nanoscale alterations in the cellular membrane transpire during cellular interactions with the extracellular environment through the endocytosis processes. Although the biological innuendos as well as alterations in cellular morphology during endocytosis are well-known, nanomechanical amendments in the cellular membrane are poorly understood. In this manuscript, atomic force microscope is employed to demonstrate the nanomechanical alterations in membrane dynamics during receptor mediated endocytosis of gold nanoparticles conjugated with either plectin-1 targeted peptide (PTP-GNP) or scrambled peptide (sPEP-GNP). Plectin-1 is aberrantly overexpressed at cell membrane of pancreatic cancer cells and is known to provide and maintain cellular mechanical integrity. During receptor mediated endocytosis of nanoparticles, we demonstrate temporal nanomechanical changes of cell membrane in both immortal pancreatic cancer Panc1 cells and patient derived primary pancreatic cancer cell, 4911. We further confirm the alterations of plectin-1 expression in Panc1 cell membrane during the receptor mediated endocytosis using classical streptavidin-biotin reaction and establish its association with nanomechanical alteration in membrane dynamics. Withdrawal of PTP-GNPs from the cell culture restores the plectin-1 expression at the membrane and reverses the mechanical properties of Panc1. We also show a distinctly opposite trend in nanomechanical behavior in cancer and endothelial cells when treated with sPEP-GNP and PTP-GNP, respectively, signifying receptor independent endocytosis process. This study illustrates the nanomechanical perspective of cell membrane in receptor mediated endocytosis of nanoparticles designed for organ specific drug delivery.

Authentication of Primary Murine Cell Lines by a Microfluidics-Based Lab-On-Chip System.

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.

Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC, also known as pancreatic cancer) is one of the deadliest tumor types, characterized by poor prognosis, profound chemoresistance and overall low survival. Gemcitabine remains the standard of care for all stages of PDAC, however, with poor clinical benefits which is considered to be due to reduced drug availability in tumor cells. Gemcitabine-induced cytotoxicity depends upon sufficient drug uptake followed by intracellular activation. Pancreatic stellate cells (PSCs), a major stromal component of PDAC, were recently reported to scavenge active metabolites of gemcitabine, thereby making it unavailable for cancer cells. Gemcitabine uptake and processing in both tumor cells and PSCs, as well as expression analysis of its molecular metabolic regulators, was investigated in this study. We observed heterogeneous gemcitabine-induced cytotoxicity in different pancreatic cancer cells whereas it was absent in PSCs. The gemcitabine-induced cytotoxicity in pancreatic cancer cells was driven by differential expression of its molecular regulators.Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

Sodium cantharidinate, a novel anti-pancreatic cancer agent that activates functional p53.

Despite the use of many types of chemotherapies for pancreatic cancer, optimal efficacy has not been obtained so far. Pancreatic cancer shows a high incidence of TP53 mutations, inactivating its tumor suppressor activity. In this study, we identified sodium cantharidinate as a novel, potential anti-pancreatic cancer agent that activates p53 function. Sodium cantharidinate reduced the viability of pancreatic cancer cells, including the human primary pancreatic cancer cells, PANC-1, AsPC-1, SW1990 and BXPC-3, in a dose-dependent manner. Sodium cantharidinate induced apoptosis and DNA damage of pancreatic cancer cells. Furthermore, proteome-wide sequencing analysis detected a marked perturbation in p53 signaling pathway on PANC-1 cells upon sodium cantharidinate. Consistent with the previous results, sodium cantharidinate treatment decreased Bcl-2 and mitochondrial cytochrome-c protein expression, as well as phosphorylation of MDM2; meanwhile, it increased the levels of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax, and phosphorylated p53, thus inducing the apoptosis of pancreatic cancer cells. The p53-activating effect of sodium cantharidinate was strongly abrogated by treatment with TP53-targeting shRNA. Moreover, sodium cantharidinate inhibited neoplasm growth via the JAK2-STAT3 pathway, which was inhibited by shRNA-TP53 and triggered by combination with gemcitabine. Combination therapy indicated that sodium cantharidinate and gemcitabine synergistically reduced ex vivo and in vivo growth of pancreatic neoplasm. Further docking studies revealed the different binding fates of sodium cantharidinate to activate wild-type p53 function. Thus, sodium cantharidinate could be a potential agent with promising anti-pancreatic cancer efficacy.

E-cadherin might be a stage-dependent modulator in aggressiveness in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) pathology is known for its uncontrollable progress due to highly invasive characteristics and refractory behavior against existing chemotherapies. The aberrant expression of CDH1 (expresses the protein E-cadherin) is associated with increased overall survival in various cancers, however, E-cadherin expression in PDAC progression has remained elusive. We investigated the impact of exogenously elevated E-cadherin levels on the tumorigenicity of transduced low grade and metastatic PDAC cell lines, Panc-1 and AsPC-1, respectively. Constitutive expression of E-cadherin promoted a more hybrid E/M state in AsPC-1 cells, while it was associated with the acquisition of a more epithelial-like state in Panc1 cells. Our study suggests that E-cadherin may play differential roles in determining the metastatic characteristics of primary and metastatic pancreatic cancer cells.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Abstract D050: Enhancing efficacy of modified-gemcitabine nanoparticles in pancreatic PDX model

Abstract Gemcitabine (Gem) is preferred anticancer drug for the treatment of pancreatic cancer (PCa) either alone in debilitated patients or in combination with other drugs in healthy patients, however; the therapeutic concentration of Gem is severally reduced due to rapid metabolism. Due to its short stay in the blood, the maintenance of therapeutic concentrations of Gem requires a continuous parenteral administration leading to severe side effects such as renal and hematological toxicities. This inherent drawback has necessitated novel approach of delivering Gem to improve stability. The objective of this study was to chemically modify Gem and evaluate its anticancer activity against pancreatic cancer cells. Gem was modified by linking 4-amino group of Gem and stearoyl linear acyl derivative to form 4-(N)-stearoyl-gemcitabine (Gem-stearate). Gem-stearate nanoparticle (GSN) was further prepared by mixing lecithin and labrasol solutions until homogenous mixture was formed. Gem-stearate was then added to the mixture and vortexed intermittently until homogenous solution was achieved. The bond between 4-amino group of Gem and stearoyl derivative was confirmed by Nuclear Magnetic Resonance (NMR) and micro-elemental analysis. The particle size of GSN was determined by using a Particle Size Analyze. Patient-derived primary pancreatic cancer cells (CMZ and G46Ca) and MiaPaCa-2 cells were treated with blank nanoparticles and different concentrations of free Gem and GSN for 48 hours and determined the viability by using Resazurin assay. Mice with pre-established tumors (patient-derived xenografts (PDX)) of pancreatic model (G46Ca) were treated with Gem and GSN. Results: Analysis of the H-NMR spectra displayed amide bond single peak of interest was at 11ppm suggesting a bond formation between 4-amino group of Gem and stearoyl derivative. The mass fractions of elements of GSN were found to be (Theory (T) and Found (F)): i) Carbon: 61.23% (T) and 60.97±0.07% (F), ii) Hydrogen: 8.56% (T) and 8.59±0.08% (F), iii) Nitrogen: 7.93 % (T) and 7.52±0.01% (F) and iv) Fluorine:7.17% (T) and 6.94±0.02% (F)). Growth inhibition of GSN-treated CMZ culture (IC50 =21±5μM) was remarkably higher than free Gem treated CMZ culture (IC50 = 62±3 μM). Similar trend of higher GSN inhibitions in G46Ca and MiaPaCa-2 cultures were found (IC50 =46±16 μM; IC50 =27±4 μM) respectively compared with free Gem treated G46Ca and Mia-PaCa-2 culture (IC50 =68±26 μM; IC50 =54±52 μM) respectively. Put together, the anticancer activity of GSN nanoparticle was significantly more effective than free Gem in CMZ, G46Ca and MiaPaCa-2 cultures compared with their corresponding free Gem treated cultures. For the tumor efficacy studies, GSN exhibited significant tumor growth inhibition compared with molar equivalent dose of free Gem. Immunohistostaining showed that GSNs have significant antiproliferative activity in G46Ca tumors. Conclusion: This study reveals that GSN may be a novel approach in delivering an effective and stable Gem to treat pancreatic cancer. Citation Format: Tiara King, Taylor Smith, Kevin Affram, Jose Trevino, Bo Han, Edward Agyare, Andriana Inkoom. Enhancing efficacy of modified-gemcitabine nanoparticles in pancreatic PDX model [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D050.

TGF-\u03b21 secreted by pancreatic stellate cells promotes stemness and tumourigenicity in pancreatic cancer cells through L1CAM downregulation

Pancreatic stellate cells (PSCs) secrete high levels of transforming growth factor-β1 (TGF-β1) that contributes to the development of pancreatic ductal adenocarcinoma (PDAC). TGF-β1 modulates the expression of L1 cell adhesion molecule (L1CAM), but its role in tumour progression still remains controversial. To clarify L1 function in PDAC and cellular phenotypes, we performed L1CAM cell sorting, silencing and overexpression in several primary pancreatic cancer cells. PSCs silenced for TGF-β1 were used for crosstalk experiments. We found that TGF-β1 secreted by PSCs negatively regulates L1CAM expression, through canonical TGF-β-Smad2/3 signalling, leading to a more aggressive PDAC phenotype. Cells with reduced expression of L1CAM harboured enhanced stemness potential and tumourigenicity. Inactivation of TGF-β1 signalling in PSCs strongly reduced the aggressiveness of PDAC cells. Our data provide functional proof and mechanistic insights for the tumour-suppressive function of L1CAM via reducing stemness. Rescuing L1CAM expression in cancer cells through targeting of TGF-β1 reverses stemness and bears the potential to improve the still miserable prognosis of PDAC patients.

Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary and gemcitabine-resistant pancreatic cancer cells

A new series of eighteen imidazo [2,1-b] [1,3,4]thiadiazole derivatives was efficiently synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably cytotoxic activity with the half maximal inhibitory concentration values (IC50) ranging from 0.23 to 11.4 μM, and 0.29–12.2 μM, respectively. However, two additional compounds, 12b and 13g, displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma (PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and gemcitabine-resistant (Panc-1R), eliciting IC50 values ranging from micromolar to sub-micromolar level, associated with significant reduction of cell-migration and spheroid shrinkage. These remarkable results might be explained by modulation of key regulators of epithelial-to-mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one of the possible mechanisms of action, using a specific ELISA. In conclusion, novel imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and PTK2/FAK.

Primary and Metastatic Pancreatic Cancer Cells Exhibit Differential Migratory Potentials.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastatic spread in more than 50% of patients. In this study, we sought to understand the migratory properties of (non)metastatic PDAC cells and determine whether the migration of cancer stem cell (CSC) populations accounts for the aggressive nature of this disease. The migratory abilities of primary and metastatic PDAC cell lines were investigated using a microfluidic device and time-lapse photography. The velocity, time of delay of mobilization, and number of migratory cells were analyzed. Cancer stem cell subpopulations were isolated by fluorescence-activated cell sorting and their migratory properties compared with their non-CSC counterparts. Primary cancer cells exhibited higher velocities, greater number of migratory cells, and a shorter time of delay of mobilization in comparison to metastatic cell lines. Characterization of CSC populations revealed primary PDAC cell lines were composed of fewer CD133 and CD24CD44 CSC subpopulations than metastatic cells. Moreover, migratory analysis of CSC subpopulations revealed lower velocities, fewer migratory cells, and a greater time of delay of mobilization than non-CSC. Primary cancer cells demonstrate enhanced migratory abilities in comparison to metastatic PDAC cells. Those differences may result from lower CSC subpopulations in primary cells because CSC populations demonstrated impaired migratory abilities in contrast to non-CSC.

Abstract A31: Investigating the effect of myeloid Arg1 deletion on tumor growth and CD8+ T-cell infiltration and activation in pancreatic cancer

Abstract PDA is characterized by an extensive fibroinflammatory stroma. This fibroinflammatory stroma is mainly composed of fibroblasts and tumor-infiltrating immune cells. The most abundant infiltrating immune cells are myeloid cells. Myeloid cells including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and granulocytes are required for PDA tumor growth and maintenance. Myeloid cells have the ability to suppress antitumor T-cell responses in PDA, as depletion of myeloid cells restores CD8+ T-cell immunity. Our previous characterization of myeloid cells infiltrating the neoplastic pancreas has revealed that myeloid cells express high levels of Arginase 1 (Arg1), an enzyme that depletes the amino acid L-arginine from the microenvironment and a signature marker of immunosuppressive macrophages and TAMs. In turn, L-arginine is required for CD8+ T-cell activation. An increase in Arginase levels has been reported also in other cancers, including lung, gastrointestinal, and bladder cancer. Thus, Arg1 expression in myeloid cells might be a key mediator of immune suppression, although this possibility has not been investigated directly in pancreatic cancer. Based on these observations, we test the hypothesis that myeloid cell polarization in the tumor microenvironment mediates immune suppression in PDA through expression of Arginase 1. The objective of this study is to provide novel insights into the role of Arg1 in pancreatic cancer, and the overall goal is to identify new therapeutic targets for combination therapy in pancreatic cancer. We used a genetically engineered mouse model (LysM-Cre;Arg1f/f) to delete the Arg1 gene, specifically from the myeloid cell compartment (LysM+ cells), including macrophages and neutrophils. We orthotopically implanted primary mouse pancreatic cancer cell lines into C57BL/6 LysM-Cre;Arg1f/f and wild-type (WT) mice. We evaluated tumor growth and CD8+ T-cell infiltration and activation between LysM-Cre;Arg1f/f and WT control mice. We used MRI imaging to determine tumor volume, and we used immunofluorescence and mass cytometry analysis to investigate changes in immune cell infiltration. We confirmed Arg1 depletion in LysM-Cre;Arg1f/f mice by Western blotting, co-immunofluorescence, and mass cytometry. We observed a decreasing trend in tumor growth and volume in LysM-Cre;Arg1f/f mice, an increase in iNOS expression (a marker of inflammatory macrophages), and an increase in CD8+ T-cell number and activity compared to the WT. These results support the notion that Arg1 might be a moderator of immune suppression in pancreatic cancer. Citation Format: Rosa E. Menjivar, Christopher Halbrook, Ashley Velez, Fatima Lima, Carlos Espinoza, Stefanie Galban, Yaqing Zhang, Costas Lyssiotis, Marina Pasca di Magliano. Investigating the effect of myeloid Arg1 deletion on tumor growth and CD8+ T-cell infiltration and activation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A31.

Cytotoxic effects of gemcitabine-loaded solid lipid nanoparticles in pancreatic cancer cells

This study investigated the cytotoxic effects of gemcitabine-loaded solid lipid nanoparticle (Gem-SLN) on the patient-derived primary pancreatic cancer cell lines (PPCL-46) and MiaPaCa-2 pancreatic cancer cells. Different SLN formulations were prepared from glyceryl monostearate (GMS), polysorbate 80 (Tween® 80) and poloxamer 188 (Pol 188) as surfactants using a cold homogenization method. Gem-SLN was characterized for particle size and charge distribution, entrapment efficiency and loading capacity. Fourier Transform Infra-Red (FTIR) spectroscopy was used to verify Gem and SLN interaction while differential scanning calorimetry (DSC) was used to acquire thermodynamic information on Gem-SLN. Cytotoxicity studies was conducted on PPCL-46 cells and Mia-PaCa-2 cells. Among the different Gem-SLN formulations prepared, Gem-SLN15 was selected based on entrapment efficiency (EE) of Gem, loading efficiency of Gem, cytotoxicity and rate of Gem release. Cytotoxic effect of Gem-SLN15-treated PPCL-46 culture (IC50 (2D) = 27 ± 5 μM; IC50 (3D) = 66 ± 2 μM) was remarkably higher than gemcitabine hydrochloride (GemHCl)-treated PPCL-46 culture (IC50 (2D) = 126 ± 3 μM; IC50 (3D) = 241 ± 3 μM). Similar trend of higher Gem-SLN15 inhibition in MiaPaCa-2 culture was found (IC50 (2D) = 56 ± 16 μM; IC50 (3D) = 127 ± 4 μM) compared with GemHCl-treated Mia-PaCa-2 culture (IC50 (2D) = 188 ± 46 μM; IC50 (3D) = 254 ± 52 μM). Overall, Gem-SLN15 proved to be more effective against PPCL_46 and Mia-PaCa-2 cells than GemHCl treated PPCL-46 and Mia-PaCa-2 cancer cells.

Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity

Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques – (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells’ enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.

MEK Inhibition Targets Cancer Stem Cells and Impedes Migration of Pancreatic Cancer Cells In Vitro and In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with a very poor prognosis. At the same time, its incidence is on the rise, and PDAC is expected to become the second leading cause of cancer-related death by 2030. Despite extensive work on new therapeutic approaches, the median overall survival is only 6-12 months after diagnosis and the 5-year survival is less than 7%. While pancreatic cancer is particularly difficult to treat, patients usually succumb not to the growth of the primary tumor, but to extensive metastasis; therefore, strategies to reduce the migratory and metastatic capacity of pancreatic cancer cells merit close attention. The vast majority of pancreatic cancers harbor RAS mutations. The outstanding relevance of the RAS/MEK/ERK pathway in pancreatic cancer biology has been extensively shown previously. Due to their high dependency on Ras mutations, pancreatic cancers might be particularly sensitive to inhibitors acting downstream of Ras. Herein, we use a genetically engineered mouse model of pancreatic cancer and primary pancreatic cancer cells were derived from this model to demonstrate that small-molecule MEK inhibitors functionally abrogate cancer stem cell populations as demonstrated by reduced sphere and organoid formation capacity. Furthermore, we demonstrate that MEK inhibition suppresses TGFβ-induced epithelial-to-mesenchymal transition and migration in vitro and ultimately results in a highly significant reduction in circulating tumor cells in mice.

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for “stage specific embryonic antigen-4” (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC–PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.