A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

A Biomimetic Tumor Model of Heterogeneous Invasion in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a complex, heterogeneous, and genetically unstable disease. Its tumor microenvironment (TME) is complicated by heterogeneous cancer cell populations and strong desmoplastic stroma. This complex and heterogeneous environment makes it challenging to discover and validate unique therapeutic targets. Reliable and relevant in vitro PDAC tumor models can significantly advance the understanding of the PDAC TME and may enable the discovery and validation of novel drug targets. In this study, an engineered tumor model is developed to mimic the PDAC TME. This biomimetic model, named ductal tumor-microenvironment-on-chip (dT-MOC), permits analysis and experimentation on the epithelial-mesenchymal transition (EMT) and local invasion with intratumoral heterogeneity. This dT-MOC is a microfluidic platform where a duct of murine genetically engineered pancreatic cancer cells is embedded within a collagen matrix. The cancer cells used carry two of the three mutations of KRAS, CDKN2A, and TP53, which are key driver mutations of human PDAC. The intratumoral heterogeneity is mimicked by co-culturing these cancer cells. Using the dT-MOC model, heterogeneous invasion characteristics, and response to transforming growth factor-beta1 are studied. A mechanism of EMT and local invasion caused by the interaction between heterogeneous cancer cell populations is proposed.

BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice.

BATF functions in T cells and B cells to control the host response to antigen and promote the production of class switched immunoglobulins. In this study, we demonstrate that BATF expression increases rapidly, and transiently, following B cell stimulation and use an inducible murine model of BATF deletion to show that this induction is necessary, and sufficient, for immunoglobulin (Ig) class switch recombination (CSR). We examine two genes (Nfil3 and miR155gh) that are positively regulated, and one gene (Wnt10a) that is negatively regulated by BATF during CSR. These genes play essential roles in CSR and each impacts the expression and/or function of the others. Our observations allow these targets of BATF regulation to be positioned in a network upstream of the activation of germline transcripts (GLT) from the IgH locus and of transcriptional activation of Aicda - the gene encoding the enzyme directing Ig gene rearrangements. This work extends the knowledge of the molecular control of CSR and, importantly, positions the induction and function of BATF as an early event in this process.

Induced Mist1 expression promotes remodeling of mouse pancreatic acinar cells.

BACKGROUND & AIMS: Early embryogenesis involves cell fate decisions that define the body axes and establish pools of progenitor cells. Development does not stop once lineages are specified; cells continue to undergo specific maturation events, and changes in gene expression patterns lead to their unique physiological functions. Secretory pancreatic acinar cells mature postnatally to synthesize large amounts of protein, polarize, and communicate with other cells. The transcription factor MIST1 is expressed by only secretory cells and regulates maturation events. MIST1-deficient acinar cells in mice do not establish apical-basal polarity, properly position zymogen granules, or communicate with adjacent cells, disrupting pancreatic function. We investigated whether MIST1 directly induces and maintains the mature phenotype of acinar cells. METHODS: We analyzed the effects of Cre-mediated expression of Mist1 in adult Mist1-deficient (Mist1(KO)) mice. Pancreatic tissues were collected and analyzed by light and electron microscopy, immunohistochemistry, real-time polymerase chain reaction analysis, and chromatin immunoprecipitation. Primary acini were isolated from mice and analyzed in amylase secretion assays. RESULTS: Induced expression of Mist1 in adult Mist1(KO) mice restored wild-type gene expression patterns in acinar cells. The acinar cells changed phenotypes, establishing apical-basal polarity, increasing the size of zymogen granules, reorganizing the cytoskeletal network, communicating intercellularly (by synthesizing gap junctions), and undergoing exocytosis. CONCLUSIONS: The exocrine pancreas of adult mice can be remodeled by re-expression of the transcription factor MIST1. MIST1 regulates acinar cell maturation and might be used to repair damaged pancreata in patients with pancreatic disorders.

Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.

OBJECTIVE: Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated. DESIGN: Expression of HNF6 and Sox9 was measured by immunostaining in normal and diseased human pancreas. The function of the factors was tested in cultured cells and in mouse models of ADM by a combination of gain and loss of function experiments. RESULTS: Expression of HNF6 and Sox9 was ectopically induced in acinar cells in human ADM as well as in mouse models of ADM. HNF6 and, to a lesser extent, Sox9 were required for repression of acinar genes, for modulation of ADM-associated changes in cell polarity and for activation of ductal genes in metaplastic acinar cells. CONCLUSIONS: HNF6 and Sox9 are new biomarkers of ADM and constitute candidate targets for preventive treatment in cases when ADM may lead to cancer. This work also shows that ectopic activation of transcription factors may underlie metaplastic processes occurring in other organs.

Polo-like kinase 1 facilitates loss of Pten tumor suppressor-induced prostate cancer formation.

Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the molecular mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress and that nuclear function of Pten, but not its phosphatase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a critical regulator of the cell cycle. We show that overexpression of Plk1 correlates with genetic inactivation of Pten during prostate neoplasia formation. Significantly, we find that elevated Plk1 is critical for Pten-depleted cells to adapt to mitotic stress for survival and that reintroduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.

Nonproteolytic properties of murine alternatively spliced tissue factor: implications for integrin-mediated signaling in murine models.

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a ß3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via ß3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

Extensive pancreas regeneration following acinar-specific disruption of Xbp1 in mice.

BACKGROUND & AIMS: Progression of diseases of the exocrine pancreas, which include pancreatitis and cancer, is associated with increased levels of cell stress. Pancreatic acinar cells are involved in development of these diseases and, because of their high level of protein output, they require an efficient, unfolded protein response (UPR) that mediates recovery from endoplasmic reticulum (ER) stress following the accumulation of misfolded proteins. METHODS: To study recovery from ER stress in the exocrine organ, we generated mice with conditional disruption of Xbp1 (a principal component of the UPR) in most adult pancreatic acinar cells (Xbp1fl/fl). We monitored the effects of constitutive ER stress in the exocrine pancreas of these mice. RESULTS: Xbp1-null acinar cells underwent extensive apoptosis, followed by a rapid phase of recovery in the pancreas that included expansion of the centroacinar cell compartment, formation of tubular complexes that contained Hes1- and Sox9-expressing cells, and regeneration of acinar cells that expressed Mist1 from the residual, surviving Xbp1+ cell population. CONCLUSIONS: XBP1 is required for homeostasis of acinar cells in mice; ER stress induces a regenerative response in the pancreas that involves acinar and centroacinar cells, providing the needed capacity for organ recovery from exocrine pancreas disease.

The Xbp1-regulated transcription factor Mist1 restricts antibody secretion by restraining Blimp1 expression in plasma cells.

Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23-Cre Bhlha15 fl/fl mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared with Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 (Prdm1) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1 Gfp/+ Cd23-Cre Bhlha15 fl/fl mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.

Targeting Plk1 Sensitizes Pancreatic Cancer to Immune Checkpoint Therapy.

Polo-like kinase 1 (Plk1) plays an important role in cell-cycle regulation. Recent work has suggested that Plk1 could be a biomarker of gemcitabine response in pancreatic ductal adenocarcinoma (PDAC). Although targeting Plk1 to treat PDAC has been attempted in clinical trials, the results were not promising, and the mechanisms of resistance to Plk1 inhibition is poorly understood. In addition, the role of Plk1 in PDAC progression requires further elucidation. Here, we showed that Plk1 was associated with poor outcomes in patients with PDAC. In an inducible transgenic mouse line with specific expression of Plk1 in the pancreas, Plk1 overexpression significantly inhibited caerulein-induced acute pancreatitis and delayed development of acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Bioinformatics analyses identified the regulatory networks in which Plk1 is involved in PDAC disease progression, including multiple inflammation-related pathways. Unexpectedly, inhibition or depletion of Plk1 resulted in upregulation of PD-L1 via activation of the NF-κB pathway. Mechanistically, Plk1-mediated phosphorylation of RB at S758 inhibited the translocation of NF-κB to nucleus, inactivating the pathway. Inhibition of Plk1 sensitized PDAC to immune checkpoint blockade therapy through activation of an antitumor immune response. Together, Plk1 suppresses PDAC progression and inhibits NF-κB activity, and targeting Plk1 can potentiate the efficacy of immunotherapy in PDAC. SIGNIFICANCE: Inhibition of Plk1 induces upregulation of PD-L1 expression in pancreatic ductal adenocarcinoma, stimulating antitumor immunity and sensitizing tumors to immunotherapy.

XBP1 controls maturation of gastric zymogenic cells by induction of MIST1 and expansion of the rough endoplasmic reticulum.

BACKGROUND & AIMS: The transition of gastric epithelial mucous neck cells (NCs) to digestive enzyme-secreting zymogenic cells (ZCs) involves an increase in rough endoplasmic reticulum (ER) and formation of many large secretory vesicles. The transcription factor MIST1 is required for granulogenesis of ZCs. The transcription factor XBP1 binds the Mist1 promoter and induces its expression in vitro and expands the ER in other cell types. We investigated whether XBP1 activates Mist1 to regulate ZC differentiation. METHODS: Xbp1 was inducibly deleted in mice using a tamoxifen/Cre-loxP system; effects on ZC size and structure (ER and granule formation) and gastric differentiation were studied and quantified for up to 13 months after deletion using morphologic, immunofluorescence, quantitative reverse-transcriptase polymerase chain reaction, and immunoblot analyses. Interactions between XBP1 and the Mist1 promoter were studied by chromatin immunoprecipitation from mouse stomach and in XBP1-transfected gastric cell lines. RESULTS: Tamoxifen-induced deletion of Xbp1 (Xbp1Δ) did not affect survival of ZCs but prevented formation of their structure. Xbp1Δ ZCs shrank 4-fold, compared with those of wild-type mice, with granulogenesis and cell shape abnormalities and disrupted rough ER. XBP1 was required and sufficient for transcriptional activation of MIST1. ZCs that developed in the absence of XBP1 induced ZC markers (intrinsic factor, pepsinogen C) but showed abnormal retention of progenitor NC markers. CONCLUSIONS: XBP1 controls the transcriptional regulation of ZC structural development; it expands the lamellar rough ER and induces MIST1 expression to regulate formation of large granules. XBP1 is also required for loss of mucous NC markers as ZCs form.

Mature chief cells are cryptic progenitors for metaplasia in the stomach.

BACKGROUND & AIMS: Gastric cancer evolves in the setting of a pathologic mucosal milieu characterized by both loss of acid-secreting parietal cells and mucous cell metaplasias. Indeed, mucous cell metaplasia is considered the critical preneoplastic lesion for gastric cancer. Previous investigations have shown that infection of mice with Helicobacter felis or induction of acute parietal cell loss with the drug DMP-777 leads to the emergence of a type of metaplasia designated spasmolytic polypeptide-expressing metaplasia (SPEM). We have hypothesized that SPEM arises from proliferating cells in gland bases, either from a cryptic progenitor cell or by transdifferentiation of mature chief cells. METHODS: Taking advantage of the chief cell-restricted expression of Mist1-Cre-ER(T2), we used lineage mapping to examine whether SPEM lineages were derived from chief cells in 3 independent models of induction by DMP-777 treatment, L-635 treatment, or H felis infection. RESULTS: Treatment of mice with L-635 for 3 days led to rapid parietal cell loss, induction of a prominent inflammatory infiltrate, and emergence of SPEM. In all 3 models, SPEM developed, at least in part, from transdifferentiation of chief cells. We further found that acute parietal cell loss in the setting of inflammation (L-635 treatment) led to more rapid induction and expansion of SPEM derived from transdifferentiation of chief cells. CONCLUSIONS: These studies provide direct evidence by lineage tracing that SPEM evolves from differentiated chief cells. Thus, mature gastric chief cells have the ability to act as cryptic progenitors and reacquire proliferative capacity within the context of mucosal injury and inflammation.

MIST1 regulates the pancreatic acinar cell expression of Atp2c2, the gene encoding secretory pathway calcium ATPase 2.

MIST1 is a transcription factor expressed in pancreatic acinar cells and other serous exocrine cells. Mice harboring a targeted deletion of the Mist1 gene (Mist1(-/-)) exhibit alterations in acinar regulated exocytosis and aberrant Ca(2+) signaling that are normally controlled by acinar cell Ca(2+)-ATPases. Previous studies indicated that total sarcoendoplasmic reticulum Ca(2+)-ATPases (SERCA) and plasma membrane Ca(2+)-ATPases (PMCA) remained unaffected in Mist1(-/-) acinar cultures. Therefore, we have assessed the expression of Atp2c2, the gene that encodes the secretory pathway Ca(2+)-ATPase 2 (SPCA2). We revealed a dramatic decrease in pancreatic expression of Atp2a2 mRNA and SPCA2 protein in Mist1(-/-) mice. Surprisingly, this analysis indicated that the acinar-specific Atp2c2 mRNA is a novel transcript, consisting of only the 3' end of the gene and the protein and localizes to the endoplasmic reticulum. Expression of SPCA2 was also lost in Mist1(-/-) secretory cells of the salivary glands and seminal vesicles, suggesting that Atp2c2 transcription is regulated by MIST1. Indeed, inducible MIST1 expression in Mist1(-/-) pancreatic acinar cells restored normal Atp2c2 expression, supporting a role for MIST1 in regulating the Atp2c2 gene. Based on these results, we have identified a new Atp2c2 transcript, the loss of which may be linked to the Mist1(-/-) phenotype.

Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise through a multistep model comprised of putative precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). Recent genetically engineered mouse models of PDAC demonstrate a comparable morphologic spectrum of murine PanIN (mPanIN) lesions. The histogenesis of PanIN and PDAC in both mice and men remains controversial. The most faithful genetic models activate an oncogenic Kras(G12D) knockin allele within the pdx1- or ptf1a/p48-expression domain of the entire pancreatic anlage during development, thus obscuring the putative cell(s)-of-origin from which subsequent mPanIN lesions arise. In our study, activation of this knockin Kras(G12D) allele in the Elastase- and Mist1-expressing mature acinar compartment of adult mice resulted in the spontaneous induction of mPanIN lesions of all histological grades, although invasive carcinomas per se were not seen. We observed no requirement for concomitant chronic exocrine injury in the induction of mPanIN lesions from the mature acinar cell compartment. The acinar cell derivation of the mPanINs was established through lineage tracing in reporter mice, and by microdissection of lesional tissue demonstrating Cre-mediated recombination events. In contrast to the uniformly penetrant mPanIN phenotype observed following developmental activation of Kras(G12D) in the Pdx1-expressing progenitor cells, the Pdx1-expressing population in the mature pancreas (predominantly islet beta cells) appears to be relatively resistant to the effects of oncogenic Kras. We conclude that in the appropriate genetic context, the differentiated acinar cell compartment in adult mice retains its susceptibility for spontaneous transformation into mPanIN lesions, a finding with potential relevance vis-à-vis the origins of PDAC.

Engineering of a functional pancreatic acinus with reprogrammed cancer cells by induced PTF1a expression.

A pancreatic acinus is a functional unit of the exocrine pancreas producing digest enzymes. Its pathobiology is crucial to pancreatic diseases including pancreatitis and pancreatic cancer, which can initiate from pancreatic acini. However, research on pancreatic acini has been significantly hampered due to the difficulty of culturing normal acinar cells in vitro. In this study, an in vitro model of the normal acinus, named pancreatic acinus-on-chip (PAC), is developed using reprogrammed pancreatic cancer cells. The developed model is a microfluidic platform with an epithelial duct and acinar sac geometry microfabricated by a newly developed two-step controlled "viscous-fingering" technique. In this model, human pancreatic cancer cells, Panc-1, reprogrammed to revert to the normal state upon induction of PTF1a gene expression, are cultured. Bioinformatic analyses suggest that, upon induced PTF1a expression, Panc-1 cells transition into a more normal and differentiated acinar phenotype. The microanatomy and exocrine functions of the model are characterized to confirm the normal acinus phenotypes. The developed model provides a new and reliable testbed to study the initiation and progression of pancreatic cancers.

CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.

Thrombin-PAR1 signaling in pancreatic cancer promotes an immunosuppressive microenvironment.

Essentials Elimination of PDAC tumor cell PAR1 increased cytotoxic T cells and reduced tumor macrophages. PAR1KO PDAC cells are preferentially eliminated from growing tumors. Thrombin-PAR1 signaling in PDAC tumor cells drives an immunosuppressive gene signature. Csf2 and Ptgs2 are thrombin-PAR1 downstream immune suppressor genes in PDAC tumor cells. ABSTRACT: Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prothrombotic state and a lack of host antitumor immune responsiveness. Linking these two key features, we previously demonstrated that tumor-derived coagulation activity promotes immune evasion. Specifically, thrombin-protease-activated receptor-1 (PAR1) signaling in mouse PDAC cells drives tumor growth by evading cytotoxic CD8a+ cells. Methods Syngeneic mixed cell tumor growth, transcriptional analyses, and functional tests of immunosuppressive response genes were used to identify cellular and molecular immune evasion mechanisms mediated by thrombin-PAR-1 signaling in mouse PDAC tumor cells. Results Elimination of tumor cell PAR1 in syngeneic graft studies increased cytotoxic T lymphocyte (CTL) infiltration and decreased tumor-associated macrophages in the tumor microenvironment. Co-injection of PAR1-expressing and PAR1-knockout (PAR-1KO ) tumor cells into immunocompetent mice resulted in preferential elimination of PAR-1KO cells from developing tumors, suggesting that PAR1-dependent immune evasion is not reliant on CTL exclusion. Transcriptomics analyses revealed no PAR1-dependent changes in the expression of immune checkpoint proteins and no difference in major histocompatibility complex-I cell surface expression. Importantly, thrombin-PAR1 signaling in PDAC cells upregulated genes linked to immunosuppression, including Csf2 and Ptgs2. Functional analyses confirmed that both Csf2 and Ptgs2 are critical for PDAC syngeneic graft tumor growth and overexpression of each factor partially restored tumor growth of PAR1KO cells in immunocompetent mice. Conclusions Our results provide novel insight into the mechanisms of a previously unrecognized pathway coupling coagulation to PDAC immune evasion by identifying PAR1-dependent changes in the tumor microenvironment, a PAR1-driven immunosuppressive gene signature, and Csf2 and Ptgs2 as critical PAR1 downstream targets.

Loss of the acinar-restricted transcription factor Mist1 accelerates Kras-induced pancreatic intraepithelial neoplasia.

BACKGROUND & AIMS: Invasive pancreatic ductal adenocarcinoma is thought to originate from duct-like lesions called pancreatic intraepithelial neoplasia (PanIN). PanINs progress from low grade (PanIN-1) to high grade (PanIN-3) as the cells attain molecular alterations to key regulatory genes, including activating mutations in the KRAS protooncogene. Despite a well-documented progression model, our knowledge of the initiator cells of PanINs and the transcriptional networks and signaling pathways that impact PanIN formation remains incomplete. METHODS: In this study, we examined the importance of the acinar-restricted transcription factor Mist1 to KrasG12D-induced mouse PanIN (mPanIN) formation in 3 different mouse models of pancreatic cancer. RESULTS: In the absence of Mist1 (Mist1KO), KrasG12D-expressing mice exhibited severe exocrine pancreatic defects that were rescued by ectopic expression of Mist1 in acinar cells. mPanIN development was greatly accelerated in Mist1KO/KrasG12D/+ pancreata, and in vitro assays revealed that Mist1KO acinar cells were predisposed to convert to a ductal phenotype and activate epidermal growth factor receptor (EGFR) and Notch-signaling pathways. CONCLUSIONS: We propose that convergence of EGFR, Notch, and Kras pathways in acinar cells lacking Mist1 leads to enhanced mPanIN formation.

An engineered pancreatic cancer model with intra-tumoral heterogeneity of driver mutations.

Pancreatic ductal adenocarcinoma (PDAC) is a complex disease with significant intra-tumoral heterogeneity (ITH). Currently, no reliable PDAC tumor model is available that can present ITH profiles in a controlled manner. We develop an in vitro microfluidic tumor model mimicking the heterogeneous accumulation of key driver mutations of human PDAC using cancer cells derived from genetically engineered mouse models. These murine pancreatic cancer cell lines have KPC (Kras and Trp53 mutations) and KIC genotypes (Kras mutation and Cdkn2a deletion). Also, the KIC genotypes have two distinct phenotypes - mesenchymal or epithelial. The tumor model mimics the ITH of human PDAC to study the effects of ITH on the gemcitabine response. The results show gemcitabine resistance induced by ITH. Remarkably, it shows that cancer cell-cell interactions induce the gemcitabine resistance potentially through epithelial-mesenchymal-transition. The tumor model can provide a useful testbed to study interaction mechanisms between heterogeneous cancer cell subpopulations.