Pancreatic Stellate Cells Research Articles

Abstract 4809: A novel microfluidic platform for PDAC organoid culture and drug screening

Abstract Pancreatic Ductal Adenocarcinoma (PDAC), the most common pancreatic cancer type, will become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Current standard-of-care for patients with PDAC includes chemotherapeutic regimens and pancreatic cancer surgery. However, only 20% of the patients are eligible for surgery due to late diagnosis. Although chemotherapeutic regimens are the leading treatment to PDAC patients, these are still very limited and the development of resistance to treatment is often observed. PDAC tumors are characterized by high-density stroma and hypovascularization, therefore these present high interstitial pressure and hypoxia. These features potentially interfere with the efficiency of chemotherapeutic drugs and highlight the urgent need for novel PDAC screening platforms. Here, we describe the establishment of PDAC organoid cultures in the MIMETAS OrganoPlate®. The aim of this project was to determine the treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. To recapitulate in-vivo like conditions, the 2-lane OrganoPlate® from MIMETAS was used to study organoid growth and sensitivity to treatment. Several standard of care (chemo-)therapeutics were tested on PDAC organoids embedded within an extracellular matrix in the Organoplate® 2-lane. These were exposed to chemotherapeutic treatments for 72h. PDAC organoids showed an overall better survival when grown in co-culture with PSCs. Interestingly, organoids grown in co-culture showed a higher survival rate under hypoxic conditions. In contrast, when grown in monoculture cell viability was higher or similar under normoxic conditions than in hypoxia for the different chemotherapeutics. The OrganoPlate® 2-lane provides an excellent platform for (co-) cultivation and high-throughput phenotypic drug screening of PDAC organoids, thereby potentially enabling the development of novel in-vivo like model systems for efficient patient stratification and drug discovery. Citation Format: Marlene Geyer, Daniel Schreyer, Lisa-Marie Gaul, Désiréé Goubert, Susanne Pfeffer, Christian Pilarsky, Karla S. Queiroz. A novel microfluidic platform for PDAC organoid culture and drug screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4809.

Sono-promoted drug penetration and extracellular matrix modulation potentiate sonodynamic therapy of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) exhibits difficult penetration of most drugs, leading to a very poor therapeutic outcome with a quite low five-year survival rate. The foremost reason is the highly-dense extracellular matrix (ECM) with abundant collagen and fibronectin secreted by the activated pancreatic stellate cells (PSCs). Here, we constructed a sono-responsive polymeric perfluorohexane (PFH) nanodroplet to elicit a deep drug penetration in PDAC via the combination of exogenous ultrasonic (US) exposure and endogenous ECM modulation for potent sonodynamic therapy (SDT) of PDAC. Under US exposure, the rapid drug release and deep penetration in PDAC tissues were realized. The released and well penetrated all-trans retinoic acid (ATRA) as an inhibitor of activated PSCs successfully reduced the secretion of ECM components to form a non-dense matrix conducive to drug diffusion. Meanwhile, the sonosensitizer, manganese porphyrin (MnPpIX), was triggered to produce robust reactive oxygen species (ROS) to exert the SDT effect under US exposure. Furthermore, oxygen (O2) delivered by PFH nanodroplets alleviated tumor hypoxia and enhanced the eradication of cancer cells. Overall, the sono-responsive polymeric PFH nanodroplets were successfully developed as an efficient strategy for PDAC therapy. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma (PDAC) is a representative refractory cancer with a highly dense extracellular matrix (ECM), making it difficult for most drugs to penetrate the nearly impenetrable desmoplastic stroma. Seeking methods for deep drug penetration is an extremely pressing matter for the treatment of PDAC and many other solid tumors. Herein, we designed a fluoroalkane-modified polymer to prepare a sono-responsive polymeric perfluorohexane (PFH) nanodroplet for loading sonosensitizers, and inhibitors of activated PSCs and O2. Under ultrasonic exposure, the nanodroplet elicited deep drug penetration in PDAC via ultrasonic disturbance and stromal remodeling, inducing potent sonodynamic therapy (SDT) of PDAC. By combining exogenous ultrasonic exposure and endogenous ECM modulation, this work successfully alleviated the severe physiological barrier of PDAC and achieved a favourable treatment effect.

"The Role of Pancreatic Stellate Cells in the Repair of Pancreatic Injury"

"The Role of Pancreatic Stellate Cells in the Repair of Pancreatic Injury"

Tcf21 Alleviates Pancreatic Fibrosis by Regulating the Epithelial-Mesenchymal Transformation of Pancreatic Stellate Cells.

The activation of pancreatic stellate cells (PSCs) plays a key role in the occurrence and development of chronic pancreatitis (CP) and pancreatic fibrosis, which is related to the process of epithelial-mesenchymal transition (EMT). This study was designed to investigate the effect and mechanism of Tcf21 (one of tumor suppressor genes) on pancreatic inflammation and fibrosis in vivo and in vitro. C57BL/6 male mice were intraperitoneally injected with caerulein for 6weeks to establish CP animal model. Fixed pancreatic tissue paraffin-embedded sections were used for immunohistochemistry staining of Tcf21, fibrosis-related markers (α-SMA), interstitial markers (Vimentin) and epithelial markers (E-cadherin). Western blotting and qRT-PCR assay were performed to analyze the change of expression of the above markers after stimulation of TGF-β1 or overexpressed Tcf21 lentivirus transfection in human pancreatic stellate cells (HPSCs). The pancreatic expression of α-SMA and Vimentin of CP mice significantly increased, while the expression of Tcf21 and E-cadherin significantly decreased. TGF-β1 could promote activation and EMT process of HPSCs, and inhibited the expression of Tcf21. Overexpression of Tcf21 could significantly down-regulate the expression of α-SMA, Fibronectin and Vimentin, and up-regulated the expression of ZO-1 of HPSCs. Cell Counting Kit-8 assay and scratch wound-healing assay results showed that overexpression of Tcf21 could significantly inhibit the cell migration and proliferation of HPSCs. Overexpression of Tcf21 could significantly alleviate the activation, proliferation, migration of PSCs by regulating the EMT process. Tcf21 had a potential prospect of a new target for CP therapy.

Cholecystokinin receptor antagonist induces pancreatic stellate cell plasticity rendering the tumor microenvironment less oncogenic

Cholecystokinin receptor antagonist induces pancreatic stellate cell plasticity rendering the tumor microenvironment less oncogenic

Quantifying heterogeneity to drug response in cancer–stroma kinetics

A crucial challenge in medicine is choosing which drug (or combination) will be the most advantageous for a particular patient. Usually, drug response rates differ substantially, and the reasons for this response unpredictability remain ambiguous. Consequently, it is central to classify features that contribute to the observed drug response variability. Pancreatic cancer is one of the deadliest cancers with limited therapeutic achievements due to the massive presence of stroma that generates an environment that enables tumor growth, metastasis, and drug resistance. To understand the cancer-stroma cross talk within the tumor microenvironment and to develop personalized adjuvant therapies, there is a necessity for effective approaches that offer measurable data to monitor the effect of drugs at the single-cell level. Here, we develop a computational approach, based on cell imaging, that quantifies the cellular cross talk between pancreatic tumor cells (L3.6pl or AsPC1) and pancreatic stellate cells (PSCs), coordinating their kinetics in presence of the chemotherapeutic agent gemcitabine. We report significant heterogeneity in the organization of cellular interactions in response to the drug. For L3.6pl cells, gemcitabine sensibly decreases stroma-stroma interactions but increases stroma-cancer interactions, overall enhancing motility and crowding. In the AsPC1 case, gemcitabine promotes the interactions among tumor cells, but it does not affect stroma-cancer interplay, possibly suggesting a milder effect of the drug on cell dynamics.

Immune cells and immune cell-targeted therapy in chronic pancreatitis.

In recent years, studies have attempted to understand the immune cells and mechanisms underlying the pathogenesis of chronic pancreatitis (CP) by constructing a model of CP. Based on these studies, the innate immune response is a key factor in disease pathogenesis and inflammation severity. Novel mechanisms of crosstalk between immune and non-immune pancreatic cells, such as pancreatic stellate cells (PSC), have also been explored. Immune cells, immune responses, and signaling pathways in CP are important factors in the development and progression of pancreatitis. Based on these mechanisms, targeted therapy may provide a feasible scheme to stop or reverse the progression of the disease in the future and provide a new direction for the treatment of CP. This review summarizes the recent advances in research on immune mechanisms in CP and the new advances in treatment based on these mechanisms.

Effects of functional foods, nutraceuticals, and herbal products on pancreas?

To the Editor: Today functional foods, nutraceuticals, and herbal products are becoming a part of human daily diet. The patient's interest and demand for them are rising. Globally, healthcare systems and people have shown an increased desire to prevent some diseases and to improve their performances. Published data about the properties and side effects of these products on the pancreas are still lacking, and more studies are required to provide a definitive opinion on their clinical benefits and safeties. The industry of functional foods, nutraceuticals, and herbal products is gaining popularity. Functional food is food with chemical or biological additives that can modify human physiological systems such as digestive, endocrine, nervous, immune, and cardiovascular. The term first appeared in Japanese literature in 1984.[1] Nutraceutical supplements are food or ingredients with medical or health benefits for the prevention and treatment of diseases. This term was introduced by Stephen L. De Felice in 1989. Nutritional supplements are not essential to the human diet but are considered safe and able to prevent and manage some human diseases.[1] Finally, herbal products consist of whole plants or fragmented/plants, algae, fungi, and lichens containing vitamins or minerals with well-documented evidence for safety.[2] Diet plays a key role in pancreatic health and diseases. The pancreas, in fact, is involved in nutrition, digestion, and absorption of the ingested food. Published data about functional foods’ effects on the pancreas are often contradictory. Specifically, probiotics and prebiotics are much prescribed today for their potential health effects on humans.[3] Probiotics are living microorganisms, non-pathogenic bacteria, or yeasts, such as Lactobacillus, Streptococcus, Saccharomyces, Bifidobacterium, etc. Prebiotics are constituents of food and dietary supplements, which are not absorbed during digestion, but used selectively by the microorganisms of the human gut “microbiota.” They are oligosaccharides such as inulin, fructo-oligosaccharides galacto-oligosaccharides, non-digestible carbohydrates, etc, or other substances such as lactulose. Probiotics and prebiotics can modulate the compositions, the activities, and the balance of the intestinal microbiota with a “believed” positive impact on other systems. As regards the pancreas, a multicenter and double-blind trial on patients with acute necrotizing pancreatitis, registered a high mortality in the group treated with probiotics (24 of 152 patients died vs. nine of 144 controls).[3] This was explained with an inappropriate combination of probiotics and an incorrect timing of their administration (probiotics were not administered at the beginning of the symptoms).[3] On the other hand, another study on rats with acute pancreatitis (AP) has had positive outcomes after treatment with probiotics (Lactobacilli strains).[4] Probiotics were administered 4 days before the induction of pancreatitis.[4] Other functional foods, such as curcumin, resveratrol, Ginkgo biloba, garcinol, and biochanin A, have been studied for their beneficial and protective effects against AP and in the prevention of pancreatic cancer (PC).[5] These nutraceuticals can regulate inflammation, showing potent antioxidant properties against hydrogen peroxide/free radicals and oxidative damages derived by lipid oxidations, and downregulate pro-inflammatory cytokines. They have inhibitory activity against the nuclear factor kappa-light-chain-enhancer of activated B cells and Janus kinase/signal transducers and activators of transcription pathways, upregulating pro-inflammatory cytokines as the interleukin (IL)-6 and IL-17, and promoting the attenuation of the immune cell migrations, lowering chemokines levels.[4,5] Moreover, published data underlined that they can modify cell apoptosis processes, stop calcium overload, reduce pancreatic and extra-pancreatic inflammation, and inhibit the development and progression of PC acting on different signaling pathways, such as NF-κB, COX-2, and EGFR, that are aberrant in case of malignancy.[5] Furthermore, nutraceuticals also modulate epigenetic processes through the down-regulation of coding genes and microRNAs, influencing pancreatic stellate cells and tumor microenvironment.[5] They are considered promising agents for the prevention of PC. More importantly, nutraceuticals could also be used as an adjuvant therapy to enhance the effect of standard chemotherapy for better patients’ outcomes. However, more clinical trials and in vivo and in vitro studies are warranted to explore this field.[5] In conclusion, diet is considered one of the most important elements to maintain and promote good health and to prevent chronic diseases. Nutrition is a major modifiable determinant. Daily diet can have an influence (positive or negative, based on ingested foods and nutrients) on pancreatic health and on overall health. The global use of functional foods as nutraceutical supplements, probiotics, prebiotics, and herbal products has been rising, sometimes out of medical control. These products attract patients’ interest and healthcare systems to manage both acute and chronic disorders. But today the market is still very huge and more studies are required to provide a definitive opinion on the clinical benefits and safety of regular consumption of these products for the pancreas. Conflicts of interest None.

Purinergic P2X7R as a potential target for pancreatic cancer.

Pancreatic cancer is one of the deadliest types of cancer, with a death rate nearly equal to the incidence. The P2X7 receptor (P2X7R) is a kind of extracellular adenosine triphosphate (ATP)-gated ion channel with special permeability, which exists in most tissues of human body and mediates inflammation-related signaling pathways and immune signal transduction after activation. P2X7R is also present on the surface of several tumor cells and is involved in tumor growth and progression. P2X7R expression in pancreatic cancer has also been identified in recent studies. Activation of P2X7R in pancreatic cancer can support the proliferation of pancreatic stellate cells, participate in protein interactions, and mediate ERK1/2, IL-6/STAT3, hCAP-18/LL-37, PI3K/AKT signaling pathways to promote pancreatic cancer progression. Inhibitors targeting P2X7R can inhibit the development of pancreatic cancer and are expected to be used in clinical therapy. Therefore, P2X7R is promising as a potential therapeutic target for pancreatic cancer. This article reviews the progress of research on P2X7R in pancreatic cancer.

ASIC1a involves the acid-mediated activation of pancreatic stellate cells associated with autophagy induction.

The acidic tumor microenvironment (TME) of pancreatic cancer affects the physiological function of pancreatic stellate cells (PSCs), which in turn promotes cancer progression. Acid-sensing ion channel 1a (ASIC1a) is responsible for acidosis-related physiopathological processes. In this study, we investigated the effect of acid exposure on the activation and autophagy of PSCs, and the role of ASIC1a in these events. The results showed that acidic medium upregulated the expression of ASIC1a, induced PSCs activation and autophagy, which can be suppressed by inhibiting ASIC1a using PcTx1 or ASIC1a knockdown, suggesting that ASIC1a involves these two processes. In addition, the acid-induced activation of PSCs was impaired after the application of autophagy inhibitor alone or in combination with ASIC1a siRNA, meaning a connection between autophagy and activation. Collectively, our study provides evidence for the involvement of ASIC1a in the acid-caused PSCs activation, which may be associated with autophagy induction.

5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFβ1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFβ1 treatment exerted a more substantial effect than TGFβ1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts

ObjectivePancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity...

A microfluidic-based PDAC organoid system reveals the impact of hypoxia in response to treatment

Pancreatic Ductal Adenocarcinoma (PDAC) is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Standard of care chemotherapeutic treatment only prolongs the survival of patients for a short timeframe. Therefore, it is important to understand events driving treatment failure in PDAC as well as identify potential more effective treatment opportunities. PDAC is characterized by a high-density stroma, high interstitial pressure and very low oxygen tension. The aim of this study was to establish a PDAC platform that supported the understanding of treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. Cultures were exposed to Gemcitabine in combination with molecules targeting relevant molecular programs that could explain treatment specific responses under different oxygen pressure conditions. Two groups of treatment responses were identified, showing either a better effect in monoculture or co-culture. Moreover, treatment response also differed between normoxia and hypoxia. Modulation of response to Gemcitabine was also observed in presence of a Hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) inhibitor and HIF inhibitors. Altogether this highlights the importance of adjusting experimental conditions to include relevant oxygen levels in drug response studies in PDAC.

Targeting Periostin Expression Makes Pancreatic Cancer Spheroids More Vulnerable to Natural Killer Cells

Pancreatic cancer (PaCa) characteristically has a dense tumor microenvironment, which results in poor patient prognosis. Pancreatic stellate cells (PSCs) are the most abundant cells in the PaCa microenvironment and the principal source of collagen. Periostin, a matricellular protein, is produced specifically by PSCs and promotes the aggressiveness of PaCa cells by facilitating extracellular collagen assembly. Here, we aimed to decrease extracellular collagen assembly by suppressing periostin, thereby increasing the cytotoxic activity of natural killer (NK) cells. Periostin expression was suppressed in PSCs (called PSC-P) using CRISPR-Cas9. PaCa cells (BxPC-3) were co-cultured with PSC and PSC-P cells in a 3D environment to form tumor spheroids mimicking the tumor microenvironment. The extracellular collagen production of spheroids was evaluated by Masson's trichrome staining. The cytotoxic activity of NK-92 cells was analyzed by flow cytometry and confocal microscopy via CD107a staining. Cell death in BxPC-3 cells was evaluated by measuring Annexin-V and PI positivity using flow cytometry. As a result, periostin suppression decreased extracellular collagen and increased the infiltration of NK-92 cells into spheroids, and induced cell death in PaCa cells. In conclusion, we suggest that periostin might be a therapeutic target for PaCa and further analysis is warranted using in vivo models for proof-of-concept.

Pancreatic stellate cell-induced gemcitabine resistance in pancreatic cancer is associated with LDHA- and MCT4-mediated enhanced glycolysis

BackgroundProfound resistance to chemotherapy remains a major challenge in achieving better clinical outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). Recent studies indicate that gemcitabine (GEM) resistance is promoted both by pancreatic stellate cells (PSCs) and through increased glycolysis. However, it remains unknown whether PSCs affect GEM sensitivity via glycolytic regulation.MethodsHuman pancreatic cancer cell (PCC) lines (BxPC-3, Capan-2, HPAF-II, Mia PaCa-2, Panc-1, SW-1990) were exposed to three different PSC-conditioned media (PSC-CM; PSC-1, PSC-2, HPaSteC), following either pre-treatment with glycolysis inhibitor NV-5440 or transfection for transient silencing of key glycolytic regulators (LDHA and MCT4). Proliferation, glucose transport, extracellular lactate, and GEM sensitivity were assessed. Protein expression was determined by Western blot and immunostaining. Moreover, secreted proteins in PSC-CMs were profiled by mass spectrometry (MS).ResultsWhile exposure to PSC-CMs did not affect glucose transport in PCCs, it increased their lactate release and proliferation, and reduced the sensitivity for GEM. Both NV-5440 treatment and transient silencing of LDHA and MCT4 inhibited these PSC-induced changes in PCCs. MS analysis identified 688 unique proteins with differential expression, of which only 87 were common to the three PSC-CMs. Most PSC-secreted proteins were extracellular matrix-related, including SPARC, fibronectin, and collagens. Moreover, exposure to PSC-CMs increased the phosphorylation of ERK in PCCs, but the treatment of PCCs with the MEK/ERK inhibitor PD98059 resulted in a reduction of PSC-CM-induced glycolysis and improved GEM sensitivity.ConclusionsThe study findings suggest that PSC-secreted factors promote both glycolysis and GEM resistance in PCCs, and that glycolysis inhibition by NV-5440 and blocking of ERK phosphorylation by PD98059 protect PCCs from PSC-CM-induced loss of GEM sensitivity. Taken together, PSCs appear to promote GEM resistance in PDAC via glycolysis. Thus, targeting glycolysis may improve the effect of chemotherapy in PDAC.

Cancer-Associated Fibroblasts: Lessons from Pancreatic Cancer

Cancer-associated fibroblasts (CAFs) are present in all malignancies. Arguably, in none are they as prevalent as they are in pancreatic ductal adenocarcinoma (PDAC), where they often outnumber cancer cells. The origin and function of CAFs are still not completely understood, and attempts to target this cell population as a component of combination therapy have so far not succeeded. Our understanding of pancreatic CAFs is in rapid evolution. Heterogeneity of CAFs is the key concept to understand this cell population. We discuss heterogeneity of origin, with recent findings challenging the notion that CAFs uniformly derive from pancreatic stellate cells, and instead suggesting that multiple types of resident fibroblasts contribute to CAF expansion. Heterogeneity in gene expression divides CAFs in different subpopulations. Most importantly, heterogeneity in function underlies the complexity of CAFs. CAFs deposit components of the extracellular matrix, contributing to the high interstitial pressure in pancreatic cancer. CAFs serve as “feeder” cells for cancer cells by providing metabolites, thus mitigating the effect of the low-nutrient environment of PDAC. At the same time, CAFs regulate the function of the immune system, inhibiting antitumor immune responses. Understanding the functional role of different CAF populations and the drivers of each of their functional roles is key to devising new ways to target this cell population in PDAC.

Abstract B034: CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

Abstract B035: CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas

Abstract Pancreatic cancer is a fatal disease and is one of the most aggressive and metastatic malignancies worldwide. The dissemination of tumor cells is the prerequisite of metastases and correlates with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumor progression. Migrating cancer stem cells (miCSCs) characterized by CD133+ and CXCR4+ expression play a pivotal role in malignant tumor formation, have been reported to form the invasive front of the metastasis and in silico analysis showed that both CSCs and miCSCs are significantly upregulated in PDAC. However, the regulatory pattern maintaining these CSCs and especially miCSCs in PDAC remains widely elusive. Thus, this study further helps to unravel pathways responsible for maintenance of CSC (CD133+) and miCSC (CD133+ CXCR4+) population. To identify key signaling pathways responsible for both CSCs and miCSCs maintenance, we first generated a protein-protein interaction network using STRING database. Afterwards, we validated these signaling pathway(s) involved in aiding CSC and miCSC population by performing shRNA mediated knockdown of key signaling proteins in different patient-derived pancreatic cancer cell lines. Moreover, we interrogated the involvement of the tumor-stroma crosstalk in the regulation of these pathways by co-culturing tumor cells with pancreatic stellate cells. Protein-protein interaction network incorporating relevant factors involved in EMT, stemness, as well as SHH, NF-kB and AKT signaling pathway identified a strong link between the CXCL12/CXCR4 signaling axis and BMI1. Migration assay, sphere formation assay and western blot upon shRNA mediated knockdown of either CXCR4 and/or BMI1 ascertained BMI1 as a key player downstream of the CXCL12/CXCR4 axis to mechanistically effect both EMT and stemness. Pathway focused gene expression analysis as well as ELISA and immunofluorescence for CXCL12 and actin filaments, respectively, revealed the indispensable nature of tumor-stroma crosstalk on promoting CSC and miCSC population through the chemokine CXCL12. In addition, co-culture systems revealed that particularly pancreatic stellate cells play a significant role in maintaining both CSCs and miCSCs population as well as their characteristic phenotype including but not limited to chemotherapy resistance. Taken together, our results obtained in this study established mechanistically that the CXCL12/CXCR4 signaling pathway driven by tumor-stroma crosstalk not only enhances but also maintains both CSCs and miCSCs in pancreatic ductal adenocarcinomas through BMI1 ultimately promoting metastases and therapeutic resistance. Citation Format: Kanishka Tiwary, Anton Lahusen, Syeda Inaas, Stefanie Hauff, Karolin Walter, Alexander Kleger, Thomas Seufferlein, Bruno Sainz Jr., Patrick Christian Hermann. CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B035.

The Role of MicroRNAs in Pancreatitis Development and Progression.

Pancreatitis (acute and chronic) is an inflammatory disease associated with significant morbidity, including a high rate of hospitalization and mortality. MicroRNAs (miRs) are essential post-transcriptional modulators of gene expression. They are crucial in many diseases' development and progression. Recent studies have demonstrated aberrant miRs expression patterns in pancreatic tissues obtained from patients experiencing acute and chronic pancreatitis compared to tissues from unaffected individuals. Increasing evidence showed that miRs regulate multiple aspects of pancreatic acinar biology, such as autophagy, mitophagy, and migration, impact local and systemic inflammation and, thus, are involved in the disease development and progression. Notably, multiple miRs act on pancreatic acinar cells and regulate the transduction of signals between pancreatic acinar cells, pancreatic stellate cells, and immune cells, and provide a complex interaction network between these cells. Importantly, recent studies from various animal models and patients' data combined with advanced detection techniques support their importance in diagnosing and treating pancreatitis. In this review, we plan to provide an up-to-date summary of the role of miRs in the development and progression of pancreatitis.

PDAC-on-chip for in vitro modeling of stromal and pancreatic cancer cell crosstalk.

Pancreatic ductal adenocarcinoma (PDAC) mainly develops in the head of the pancreas, within the acino-ductal unit composed of acinar and ductal cells surrounded by pancreatic stellate cells (PSCs). PSCs strongly influence the tumor microenvironment by triggering an intense stromal deposition, which plays a key role in tumor progression and limits drug perfusion. We have developed a microfluidic in vitro model recreating the in vivo tumor-stroma crosstalk to replicate the steps of PDAC evolution towards the establishment of an efficient in vitro platform for innovative therapy validation. The multilayer PDAC-on-chip was designed to culture the PDAC cells and the PSCs embedded in a type I collagen gel in the top and bottom layers, respectively. The presence of a biomimetic nanofibrous membrane in the middle of the chip permits the control of interactions between the two cell lines and the easy analysis of the effects of the crosstalk on cell behavior. First, the PDAC-stromal cell relationship was evaluated under co-culture conditions on 24-well inserts including the PCL/Gel electrospun membrane. This simplified model shows that human fibroblasts change their morphology and secrete larger amounts of IL-6 cytokines in the presence of tumor cells, confirming the activation of stromal cells under co-culture. Then, the PDAC-on-chip system was validated by demonstrating that human fibroblasts seeded in a 3D collagen matrix in the bottom microchannel also change to a myofibroblast-like shape with increased expression of α-SMA and secrete larger amounts of IL-6 cytokines. This microfluidic system is suitable for the evaluation of drug efficacy and serves as a powerful tool for understanding the early evolution steps of PDAC.