Pancreatic Tumor Cells Research Articles

Isolation and culture of circulating pancreatic cancer tumor cells using a microfilter.

e16230 Background: The presence and number of circulating tumor cells (CTCs) are reportedly associated with prognosis but the rarity of CTCs in peripheral circulation limits the ability of conventional technologies to provide fully accurate analyses. The purpose of this study was thus to establish a novel approach to capture and culture the CTCs using a microfilter device. Methods: This was a prospective study in preoperative patients with pancreatic cancer at the University of Tsukuba Hospital after approved by the Institutional Review Board. Five ml of whole blood was collected from each patient into an EDTA-treated collection tube before pumping through an 8μm pore microfilter. Cells isolated on the filter were stained with PE-conjugated anti-EpCAM, FITC-conjugated anti-CD45 and Hoechst 33342. The filter was then transferred to a 35mm dish and cultured with 20%FBS-IMDM-F12 medium containing 2%Matrigel- EGF-FGF-B27 for 4-6 weeks. Cells were visualized with an automatic fluorescence microscope. A CTC was defined as an intact round oval cell with a visible nucleus, positive staining for EpCAM and negative staining for CD45. Results: A total of 9 patients [male/female; 4/5, median age;71 (39-78), Stage 0/IA/IIA/IIB; 1/3/3/2, preoperative chemotherapy/chemoradiation; 1/2] were enrolled. CTCs were captured in 8 out of 9 cases and cultured CTCs were kept viable for 4-6 weeks in 6 out of 9 cases. CTCs formed colonies by 4-6 weeks of culture, colonies were expanding and blood cells disappeared. No cultured CTCs were observed in the two cases who received preoperative chemotherapy. Conclusions: We successfully developed a novel culture method of patient CTC using a microfilter device. Our system supports the possibility of accurate detection and proliferation of CTCs on microfilters. Cultured CTCs could be used for patient-specific drug susceptibility testing and mutational profiles for future cancer patients.

Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia.

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.

Sesquiterpene Lactones as Promising Candidates for Cancer Therapy: Focus on Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease which confers to patients a poor prognosis at short term. PDAC is the fourth leading cause of death among cancers in the Western world. The rate of new cases of pancreatic cancer (incidence) is 10 per 100,000 but present a 5-year survival of less than 10%, highlighting the poor prognosis of this pathology. Furthermore, 90% of advanced PDAC tumor present KRAS mutations impacting in several oncogenic signaling pathways, many of them associated with cell proliferation and tumor progression. Different combinations of chemotherapeutic agents have been tested over the years without an improvement of significance in its treatment. PDAC remains as one the more challenging biomedical topics thus far. The lack of a proper early diagnosis, the notable mortality statistics and the poor outcome with the available therapies urge the entire scientific community to find novel approaches against PDAC with real improvements in patients’ survival and life quality. Natural compounds have played an important role in the process of discovery and development of new drugs. Among them, terpenoids, such as sesquiterpene lactones, stand out due to their biological activities and pharmacological potential as antitumor agents. In this review, we will describe the sesquiterpene lactones with in vitro and in vivo activity against pancreatic tumor cells. We will also discuss the mechanism of action of the compounds as well as the signaling pathways associated with their activity.

Synthetic cannabinoid WIN 55,212–2 inhibits growth and induces cell death of oral and pancreatic stem-like/poorly differentiated tumor cells

We report here that synthetic cannabinoid WIN 55,212–2 inhibits tumor cell proliferation and induces cell death of oral and pancreatic tumor cells, and the effect is much more pronounced on stem-like/poorly differentiated OSCSCs and MP2 cells when compared to well-differentiated OSCCs, and PL-12 tumor cells. In addition, WIN 55,212-2 decreases cell surface expression of CD44, CD54, MHC class I and PD-L1 on oral and pancreatic tumor cells with the exception of PD-L1 expression on well-differentiated PL-12 pancreatic tumor cells which exhibits an increase in the expression rather than a decrease. Overall, we demonstrate that WIN 55,212-2 has an increased targeting activity against cancer stem cells/poorly differentiated oral and pancreatic tumor cells when compared to well-differentiated tumor cells, and furthermore, such differences in function do not correlate with the levels of CB1 and CB2 receptor expression on tumor cells, suggesting it's function either through post-receptor mediated activation and/or yet-to-be identified novel receptors. Intraperitoneal (IP) delivery of WIN 55-212-2 in humanized BLT mice is found to impart an activating potential for NK cells demonstrating increased NK cell mediated cytotoxicity and secretion of IFN-γ in our preliminary experiments. These results not only suggest a direct targeting of CSCs/poorly differentiated tumors by WIN 55-212-2 but also by indirect targeting of such tumors through the activation and increased functions of NK cells.

A phase II trial of the super-enhancer inhibitor Minnelide™ in advanced refractory adenosquamous carcinoma of the pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is a very rare and highly aggressive variant of pancreatic ductal adenocarcinoma, accounting for 0.5-4% of all pancreatic cancer cases in the USA. Current data indicate that epigenetic changes and MYC overexpression lead to squamous transdifferentiation of pancreatic tumor cells and development of ASCP. Minnelide™, an oral anti-super-enhancer drug that inhibits MYC expression in preclinical models of ASCP, has demonstrated safety in a phase I study. We describe the design for a phase II, open-label, single-arm trial of Minnelide in patients with advanced refractory ASCP.

Reciprocal interactions between fibroblast and pancreatic neuroendocrine tumor cells: putative impact of the tumor microenvironment

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abrogating regulatory T cells overcomes tumor-specific T cell exhaustion and prevents metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths and has a dismal 5-year survival rate of 10 percent. PDA lethality is attributed to late diagnosis, early metastasis, and therapeutic resistance. Metastasis can occur before the development of histologically detectable tumors and is a leading cause of cancer-related deaths. We identified that pancreatic tumor cells derived from mice that resist immunotherapy (e.g., tumor escape variants, TEV) and re-implanted into the pancreas of syngeneic and immunocompetent mice rapidly metastasize, reflecting the pathogenesis of human PDA. We show that TEVs retain the targeted tumor antigen, and despite a defect in IFNgamma-inducible MHC class I upregulation, TEVs remain sensitive to tumor antigen specific T cell-mediated lysis in vitro, suggesting TEVs may confer unique qualities in vivo to resist T cell killing. Using a peptide:MHC tetramer to identify the tumor-specific CD8 T cells, we identified that intratumoral T cells in EV tumors have increased Granzyme B production and a reduction in prototypical exhaustion markers PD1, Lag3, and Tox, perhaps due to reduced MHC class I signaling. Notably, primary tumors from TEVs were significantly enriched for Foxp3+ Tregs as compared to parental tumors. Using a genetic model, we identified that Treg depletion resulted in a drastic reduction in tumor burden and metastasis and improved tumor-specific T cell function in TEV tumors. Tumor-cell intrinsic changes driving Treg accumulation in TEVs will be discussed. In summary, Tregs are key drivers of both T cell exhaustion and immunosuppression in pancreatic cancer and may prove a valuable clinical target for tumors that evade immune checkpoint blockade. Supported by grants from NIH (R01 CA249393)

Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and the patients are generally diagnosed with distant metastasis. Liver is one of the preferred organs of distant metastasis, and liver metastasis is the leading cause of death in PDAC. Diet-induced obesity (DIO) is a risk factor for PDAC, and it remains unclear whether and how DIO contributes to liver metastasis of PDAC. In our study, we found that DIO significantly promoted PDAC liver metastasis compared with normal diet (ND) in intrasplenic injection mouse model. RNA-seq analysis for liver metastasis nodules showed that the various chemokines and several chemokine receptors were altered between ND and DIO samples. The expression levels of CX3CL1 and CX3CR1 were significantly upregulated in DIO-induced liver metastasis of PDAC compared to ND. Increased CX3CL1 promoted the recruitment of CX3CR1-expressing pancreatic tumor cells. Taken together, our data demonstrated that DIO promoted PDAC liver metastasis via CX3CL1/CX3CR1 axis.

The NF-κB/miR-488/ERBB2 axis modulates pancreatic cancer cell malignancy and tumor growth through cell cycle signaling

ABSTRACT Pancreatic cancer is one of the malignancies having the poorest prognosis due to late diagnoses and lack of efficient treatment regimens. The identification of potential miRNA-targeted gene axes could act as targets for developing novel treatment strategies. Herein, it was assessed that miR-488 expression was markedly downregulated within pancreatic carcinoma. Higher expression of miR-488 was shown to be linked to better prognosis rates of pancreatic carcinoma as per online data. Within two pancreatic tumor cells, MIA PaCa-2 and PANC-1, miR-488 overexpression significantly suppressed malignant cytological behavior by inhibiting cell viability, enhancing cell apoptosis, and inducing cell cycle G2/M-phase arrest. Moreover, miR-488 overexpression also decreased the protein levels of cell cycle regulators, including cyclin A, cyclin B, CDK1, and CDK2. miR-488 directly targets ERBB2 (receptor tyrosine-protein kinase2) to suppress the expression of ERBB2 by targeting its 3ʹUTR. ERBB2 knockdown in MIA PaCa-2 and PANC-1 cell lines suppressed, but miR-488 inhibition enhanced the cancer cell biological malignant behavior; the effects of miR-488 inhibition on pancreatic cancer cells were significantly reversed by ERBB2 knockdown. NF-κB suppressed the expression of miR-488 transcriptionally via targeting its promoter region, consequentially repressing the tumor-suppressive effects of miR-488 upon pancreatic tumor cells. Thus, an NF-κB/miR-488/ERBB2 axis modulating pancreatic cancer cell malignancy and tumor growth through cell cycle signaling was conclusively demonstrated.

Nickel's Role in Pancreatic Ductal Adenocarcinoma: Potential Involvement of microRNAs.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types with a limited overall survival rate due to the asymptomatic progression of symptoms in metastatic stages of the malignancy and the lack of an early reliable diagnostic biomarker. MicroRNAs (miRs/miRNAs) are small (~18–24 nucleotides), endogenous, non-coding RNAs, which are closely linked to the development of numerous malignancies comprising PDAC. Recent studies have described the role of environmental pollutants such as nickel (Ni) in PDAC, but the mechanisms of Ni-mediated toxicity in cancer are still not completely understood. Specifically, Ni has been found to alter the expression and function of miRs in several malignancies, leading to changes in target gene expression. In this study, we found that levels of Ni were significantly higher in cancerous tissue, thus implicating Ni in pancreatic carcinogenesis. Hence, in vitro studies followed by using both normal and pancreatic tumor cell lines and increasing Ni concentration increased lethality. Comparing LC50 values, Ni-acetate groups demonstrated lower values needed than in NiCl2 groups, suggesting greater Ni-acetate. Panc-10.05 cell line appeared the most sensitive to Ni compounds. Exposure to Ni-acetate resulted in an increased phospho-AKT, and decreased FOXO1 expression in Panc-10.05 cells, while NiCl2 also increased PTEN expression in Panc-10.05 cells. Specifically, following NiCl2 exposure to PDAC cells, the expression levels of miR-221 and miR-155 were significantly upregulated, while the expression levels of miR-126 were significantly decreased. Hence, our study has suggested pilot insights to indicate that the environmental pollutant Ni plays an important role in the progression of PDAC by promoting an association between miRs and Ni exposure during PDAC pathogenesis.

Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with 3D-Architectured Scaffolds.

Two-photon polymerization has recently emerged as a promising technique to fabricate scaffolds for three-dimensional (3D) cell culture and tissue engineering. Here, we combined 3D-printed microscale scaffolds fabricated using two-photon polymerization with a bioactive layer-by-layer film coating. This bioactive coating consists of hyaluronic acid and poly(l-lysine) of controlled stiffness, loaded with fibronectin and bone morphogenic proteins 2 and 4 (BMP2 and BMP4) as matrix-bound proteins. Planar films were prepared using a liquid handling robot directly in 96-well plates to perform high-content studies of cellular processes, especially cell adhesion, proliferation, and BMP-induced signaling. The behaviors of two human pancreatic cell lines PANC1 (immortalized) and PAN092 (patient-derived cell line) were systematically compared and revealed important context-specific cell responses, notably in response to film stiffness and matrix-bound BMPs (bBMPs). Fibronectin significantly increased cell adhesion, spreading, and proliferation for both cell types on soft and stiff films; BMP2 increased cell adhesion and inhibited proliferation of PANC1 cells and PAN092 on soft films. BMP4 enhanced cell adhesion and proliferation of PANC1 and showed a bipolar effect on PAN092. Importantly, PANC1 exhibited a strong dose-dependent BMP response, notably for bBMP2, while PAN092 was insensitive to BMPs. Finally, we proved that it is possible to combine a microscale 3D Ormocomp scaffold fabricated using the two-photon polymerization technique with the bioactive film coating to form a microscale tumor tissue and mimic the early stages of metastatic cancer.

Overexpressed integrin alpha 2 inhibits the activation of the transforming growth factor \u03b2 pathway in pancreatic cancer via the TFCP2-SMAD2 axis

BackgroundIntegrin alpha 2 (ITGA2) has been recently reported to be an oncogene and to play crucial roles in tumor cell proliferation, invasion, metastasis, and angiogenesis. Our previous study showed that ITGA2 was overexpressed in pancreatic cancer and promoted its progression. However, the mechanism of ITGA2 overexpression and other mechanisms for promoting the progression of pancreatic cancer are still unclear.MethodsThe GEPIA database was used to confirm the expression of ITGA2 in pancreatic cancer. To verify the influence of ITGA2 and TGF-β on the morphological changes of pancreatic cancer and tumor cell progression, we conduct CCK8 test, plate cloning, flow cytometry experiments and animal experiments. Then we conduct Western blot, RT-qPCR to explore the relationship between ITGA2 and TGF-β, and then find the key molecules which can regulate them by immunoprecipitation, Western blot, RT-qPCR, CHIP, nuclear and cytoplasmic separation test.ResultsThe results of the present study show that the abnormal activation of KRAS induced the overexpression of ITGA2 in pancreatic cancer. Moreover, ITGA2 expression significantly suppressed the activation of the TGF-β pathway. ITGA2 silencing enhanced the anti-pancreatic cancer proliferation and tumor growth effects of TGF-β. Mechanistically, ITGA2 expression suppressed the activation of the TGF-β pathway by inhibiting the SMAD2 expression transcriptionally. In addition, it interacted with and inhibited the nuclear translocation of TFCP2, which induced the SMAD2 expression as a transcription factor. Furthermore, TFCP2 also induced ITGA2 expression as a transcription factor, and the TFCP2 feedback regulated the ITGA2-TFCP2-SMAD2 pathway.ConclusionsTaken together, these results indicated that ITGA2 expression could inhibit the activation of the TGF-β signaling pathway in pancreatic cancer via the TFCP2-SMAD2 axis. Therefore, ITGA2, by effectively enhancing the anti-cancer effects of TGF- β, might be a potential clinical therapeutic target for pancreatic cancer.

The Immune Landscape of Human Pancreatic Ductal Carcinoma: Key Players, Clinical Implications, and Challenges.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancer worldwide, with an overall survival (OS) rate, all stages combined, of still <10% at 5 years. For most patients with unresectable or recurrent PDAC, few therapeutic options are available with limited efficacy (median OS ≤ 12 months). Moreover, immune checkpoint inhibitors that have been tested so far in PDAC failed to improve patient survival. This resistance of PDAC to therapies is, in part, related to the dense tumor stroma, which creates a mechanical barrier around the tumor cells and generates high interstitial pressure, preventing appropriate vascularization and thus limiting exposure to treatments. In that PDAC tumoral microenvironment, the inflammatory cell infiltrates are unbalanced toward an immunosuppressive over pro-inflammatory phenotype. Therefore, there is an urgent need to better define the composition of PDAC stroma and key immune players in order to identify new therapeutic targets and strategies.Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancer worldwide with an overall survival rate, all stages combined, of still <10% at 5 years. The poor prognosis is attributed to challenges in early detection, a low opportunity for radical resection, limited response to chemotherapy, radiotherapy, and resistance to immune therapy. Moreover, pancreatic tumoral cells are surrounded by an abundant desmoplastic stroma, which is responsible for creating a mechanical barrier, preventing appropriate vascularization and leading to poor immune cell infiltration. Accumulated evidence suggests that PDAC is impaired with multiple “immune defects”, including a lack of high-quality effector cells (CD4, CD8 T cells, dendritic cells), barriers to effector cell infiltration due to that desmoplastic reaction, and a dominance of immune cells such as regulatory T cells, myeloid-derived suppressor cells, and M2 macrophages, resulting in an immunosuppressive tumor microenvironment (TME). Although recent studies have brought new insights into PDAC immune TME, its understanding remains not fully elucidated. Further studies are required for a better understanding of human PDAC immune TME, which might help to develop potent new therapeutic strategies by correcting these immune defects with the hope to unlock the resistance to (immune) therapy. In this review, we describe the main effector immune cells and immunosuppressive actors involved in human PDAC TME, as well as their implications as potential biomarkers and therapeutic targets.

The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies.

Simple SummaryA limitation to successful therapeutic outcomes for breast and other cancer patients is the ability of small subsets of tumor cells to resist the apoptotic cell death provoked by currently employed therapeutic agents. These therapy-resistant cancer stem cell populations can then seed recurrent tumors and metastatic lesions, compromising the efficacy of the treatment regimen. The aim of our study was to assess the hypothesis that cationic amphiphilic drugs (CADs), which induce tumor cell death via the unrelated programmed necrotic mechanism, exhibit efficacy toward cancer stem cell populations that are resistant to currently employed therapeutics. We found that the therapy-resistant stem-like subpopulation of cells from a variety of breast cancer models are as sensitive to CADs as the bulk population. Our observations imply that the incorporation of cationic amphiphilic anticancer agents into existing therapeutic regimens could ultimately improve breast cancer patient outcomes by minimizing tumor recurrence and metastatic outgrowth.The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

SOX9 Triggers Different Epithelial to Mesenchymal Transition States to Promote Pancreatic Cancer Progression.

Simple SummaryPancreatic cancers are lethal types of cancer. A majority of patients progress to an advanced and metastatic disease, which remains a major clinical problem. Therefore, it is crucial to identify critical regulators to help predict the disease progression and to develop more efficacious therapeutic approaches. In this work we found that an increased expression of the developmental factor SOX9 is associated with metastasis, a poor prognosis and resistance to therapy in pancreatic ductal adenocarcinoma patients and in cell cultures. We also found that this effect is at least in part due to the ability of SOX9 to regulate the activity of stem cell factors, such as BMI1, in addition to those involved in EMT and metastasis. Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers mainly due to spatial obstacles to complete resection, early metastasis and therapy resistance. The molecular events accompanying PDAC progression remain poorly understood. SOX9 is required for maintaining the pancreatic ductal identity and it is involved in the initiation of pancreatic cancer. In addition, SOX9 is a transcription factor linked to stem cell activity and is commonly overexpressed in solid cancers. It cooperates with Snail/Slug to induce epithelial-mesenchymal transition (EMT) during neural development and in diseases such as organ fibrosis or different types of cancer. Methods: We investigated the roles of SOX9 in pancreatic tumor cell plasticity, metastatic dissemination and chemoresistance using pancreatic cancer cell lines as well as mouse embryo fibroblasts. In addition, we characterized the clinical relevance of SOX9 in pancreatic cancer using human biopsies. Results: Gain- and loss-of-function of SOX9 in PDAC cells revealed that high levels of SOX9 increased migration and invasion, and promoted EMT and metastatic dissemination, whilst SOX9 silencing resulted in metastasis inhibition, along with a phenotypic reversion to epithelial features and loss of stemness potential. In both contexts, EMT factors were not altered. Moreover, high levels of SOX9 promoted resistance to gemcitabine. In contrast, overexpression of SOX9 was sufficient to promote metastatic potential in K-Ras transformed MEFs, triggering EMT associated with Snail/Slug activity. In clinical samples, SOX9 expression was analyzed in 198 PDAC cases by immunohistochemistry and in 53 patient derived xenografts (PDXs). SOX9 was overexpressed in primary adenocarcinomas and particularly in metastases. Notably, SOX9 expression correlated with high vimentin and low E-cadherin expression. Conclusions: Our results indicate that SOX9 facilitates PDAC progression and metastasis by triggering stemness and EMT.

Poor outcome after systemic therapy in secondary high-grade pancreatic neuroendocrine tumors.

Longitudinal changes in pancreatic neuroendocrine tumor (panNET) cell proliferation correlate with fast disease progression and poor prognosis. The optimal treatment strategy for secondary panNET grade (G)3 that has progressed from a previous low- or intermediate-grade to high-grade panNET G3 is currently unknown. This was a single-center retrospective cohort study aimed to characterize treatment patterns and outcomes among patients with secondary panNET-G3. Radiological responses were assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. A total of 22 patients were included and received a median of 2 (range, 1–4) treatment lines in 14 different combinations. Median overall survival (OS) was 9 months (interquartile range (IQR): 4.25–17.5). For the 15 patients who received platinum–etoposide chemotherapy, median OS was 7.5 months (IQR: 3.75–10) and median progression-free survival (PFS) was 4 months (IQR: 2.5–5.5). The 15 patients who received conventional panNET therapies achieved a median OS of 8 months (IQR: 5–16.75) and median PFS was 5.5 months (IQR: 2.75–8.25). We observed one partial response on 177Lu DOTA-TATE therapy. In conclusion, this hypothesis-generating study failed to identify any promising treatment alternatives for patients with secondary panNET-G3. This demonstrates the need for both improved biological understanding of this particular NET entity and for designing prospective studies to further assess its treatment in larger patient cohorts.

Peptides against Low Density Lipoprotein (LDL) Aggregation Inhibit Intracellular Cholesteryl Ester Loading and Proliferation of Pancreatic Tumor Cells.

Simple SummaryDyslipidemia is a modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers. A key component of dyslipidemia is a high level of small and dense low-density lipoproteins (LDLs). These LDLs have a high probability to be entrapped and modified (aggregated) in the extracellular matrix (ECM), becoming a source of cholesterol for tumor cells. However, the effect of aggregated LDLs on tumor progression has been unexplored. The aim of this work was to determine the effect of modified LDLs on intracellular cholesteryl ester/free cholesterol ratio (CE/FC) and cancer cell growth, and the efficacy of peptides designed to inhibit LDL aggregation on these processes. Our results show that aggregated LDL upregulates the intracellular CE/FC ratio and cell growth in pancreatic cancer and that these upregulatory effects were blocked by peptides against LDL aggregation. We propose that anti-LDL aggregation peptides deserve to be further investigated as anti-tumoral strategies.Dyslipidemia, metabolic disorders and/or obesity are postulated as risk factors for pancreatic ductal adenocarcinoma (PDAC). The majority of patients with these metabolic alterations have low density lipoproteins (LDLs) with increased susceptibility to become aggregated in the extracellular matrix (ECM). LDL aggregation can be efficiently inhibited by low-density lipoprotein receptor-related protein 1 (LRP1)-based peptides. The objectives of this work were: (i) to determine if aggregated LDLs affect the intracellular cholesteryl ester (CE)/free cholesterol (FC) ratio and/or the tumor pancreatic cell proliferation, using sphingomyelinase-modified LDL particles (Aggregated LDL, AgLDL); and (ii) to test whether LRP1-based peptides, highly efficient against LDL aggregation, can interfere in these processes. For this, we exposed human pancreatic cancer cell lines (PANC-1, RWP-1 and Capan-1) to native (nLDL) or AgLDLs in the absence or presence of LRP1-based peptides (DP3) or irrelevant peptides (IP321). Results of thin-layer chromatography (TLC) following lipid extraction indicate that AgLDLs induce a higher intracellular CE/FC ratio than nLDL, and that DP3 but not IP321 counteracts this effect. AgLDLs also increase PANC-1 cell proliferation, which is inhibited by the DP3 peptide. Our results indicate that AgLDL-induced intracellular CE accumulation plays a crucial role in the proliferation of pancreatic tumor cell lines. Peptides with anti-LDL aggregation properties may thus exhibit anti-tumor effects.

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC50) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

The induction of peripheral trained immunity in the pancreas incites anti-tumor activity to control pancreatic cancer progression

Despite the remarkable success of immunotherapy in many types of cancer, pancreatic ductal adenocarcinoma has yet to benefit. Innate immune cells are critical to anti-tumor immunosurveillance and recent studies have revealed that these populations possess a form of memory, termed trained innate immunity, which occurs through transcriptomic, epigenetic, and metabolic reprograming. Here we demonstrate that yeast-derived particulate β-glucan, an inducer of trained immunity, traffics to the pancreas, which causes a CCR2-dependent influx of monocytes/macrophages to the pancreas that display features of trained immunity. These cells can be activated upon exposure to tumor cells and tumor-derived factors, and show enhanced cytotoxicity against pancreatic tumor cells. In orthotopic models of pancreatic ductal adenocarcinoma, β-glucan treated mice show significantly reduced tumor burden and prolonged survival, which is further enhanced when combined with immunotherapy. These findings characterize the dynamic mechanisms and localization of peripheral trained immunity and identify an application of trained immunity to cancer.

Secretory leukocyte proteinase inhibitor: A key player in the dialogue between the tumor and its microenvironment in pancreatic cancer patients.

592 Background: Secretory leukocyte proteinase inhibitor (SLPI) is a non-glycosylated pleiotropic protein with anti-protease, microbicidal, healing, anti-inflammatory and immunomodulatory activity. Previous studies have shown that SLPI is associated with pancreatic tumor progression by promoting cancer cell survival and proliferation. However, SLPI can also act on the tumor microenvironment to affect the local immune response. In the present work we evaluate the role of SLPI in patients with pancreatic cancer and its activity as a tumor escape factor from the immune response. Methods: Serum levels of SLPI was measured in pancreatic cancer patients by sandwich ELISA. RNA-seq was performed on 76 patient-derived xenografts (PDX) and we computed independent component analysis focusing the study on the component that best correlated with SLPI gene. Secretome analysis was performed on 38 different cell lines cells culture derived from PDX. Human monocytes were isolated and differentiated into immature dendritic cells in the presence or absence of SLPI or in the presence of a pancreatic tumor cell line that did or did not express SLPI. Results: The frequency distribution of serum SLPI values showed patients with low and high SLPI value (cut-off value 61.5 ng/ml). Most patients with high serum SLPI concentration had unresectable tumors. There was an indirect association between serum SLPI levels and the time of disease recurrence. The transcriptome analysis showed that expression of SLPI is associated with immune and cancer functional clusters. The analysis of the secretome showed that SLPI was present in all cell lines from PDX. The heat map of the secretome exhibited that SLPI was directly associated with factors described in tumor microenvironment and related with tumor immune evasion mechanisms, such as CSF1, SECTM1, LGALS3, IL1RN, CD59, CD55, CD46, Fas, PVR, PVRL2. Remarkably, the closest group associated with SLPI was CSF1. The latter contributes to the depressed function of antigen-presenting cells, as a result of skewed differentiation of monocytes towards macrophage-like cells rather than dendritic cells. Furthermore, in vitro experiments demonstrated that SLPI or SLPI-producing pancreatic tumor cell lines impaired the differentiation of human monocytes towards dendritic cells and their immunostimulatory capacity. Conclusions: These results suggest that SLPI may contribute to the immunosuppressive microenvironment of pancreatic cancer by acting as a tumor immune evasion factor. Clinically, this SLPI activity could be reflected in the association of the serum SLPI value with disease recurrence or progression.