Pancreatic Ductal Adenocarcinoma Liver Metastases Research Articles

A rat-based preclinical platform facilitating transcatheter hepatic arterial infusion in immunodeficient rats with liver xenografts of patient-derived pancreatic ductal adenocarcinoma

Liver metastases from pancreatic ductal adenocarcinoma (PDAC) are highly fatal. A rat-based patient-derived tumor xenograft (PDX) model is available for transcatheter therapy. This study aimed to create an immunodeficient rat model with liver xenografts of patient-derived primary PDAC and evaluate efficacy of hepatic arterial infusion chemotherapy with cisplatin in this model. Three patient-derived PDACs were transplanted into the livers of 21 rats each (totally, 63 rats), randomly assigned into hepatic arterial infusion, systemic venous infusion, and control groups (n = 7 each) four weeks post-implantation. Computed tomography evaluated tumor volumes before and four weeks after treatment. Post-euthanasia, resected tumor specimens underwent histopathological examination. A liver-implanted PDAC PDX rat model was established in all 63 rats, with first CT identifying all tumors. Four weeks post-treatment, arterial infusion groups exhibited significantly smaller tumor volumes than controls for all three tumors on second CT. Xenograft tumors histologically maintained adenocarcinoma features compared to original patient tumors. Ki67 expression was significantly lower in arterial infusion groups than in the other two for the three tumors, indicating reduced tumor growth in PDX rats. A liver-implanted PDAC PDX rat model was established as a rat-based preclinical platform. Arterial cisplatin infusion chemotherapy represents a potential therapy for PDAC liver metastasis.

Macrophage-fibroblast JAK/STAT dependent crosstalk promotes liver metastatic outgrowth in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease for which better therapies are urgently needed. Fibroblasts and macrophages are heterogeneous cell populations able to enhance metastasis, but the role of a macrophage-fibroblast crosstalk in regulating their pro-metastatic functions remains poorly understood. Here we deconvolve how macrophages regulate metastasis-associated fibroblast (MAF) heterogeneity in the liver. We identify three functionally distinct MAF populations, among which the generation of pro-metastatic and immunoregulatory myofibroblastic-MAFs (myMAFs) critically depends on macrophages. Mechanistically, myMAFs are induced through a STAT3-dependent mechanism driven by macrophage-derived progranulin and cancer cell-secreted leukaemia inhibitory factor (LIF). In a reciprocal manner, myMAF secreted osteopontin promotes an immunosuppressive macrophage phenotype resulting in the inhibition of cytotoxic T cell functions. Pharmacological blockade of STAT3 or myMAF-specific genetic depletion of STAT3 restores an anti-tumour immune response and reduces metastases. Our findings provide molecular insights into the complex macrophage–fibroblast interactions in tumours and reveal potential targets to inhibit PDAC liver metastasis.

Abstract 4414: Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting

Abstract Introduction: The metastasis of a pancreatic ductal adenocarcinoma (PAAD) is a diagnosis of exclusion and one of the most common causes of cancer of unknown primary (CUP). We have recently developed a genome-wide DNA methylation-based neural network classifier that can accurately differentiate between liver metastasis of a PAAD and intrahepatic cholangiocarcinoma (iCCA) (PAAD-iCCA-Classifier). Therefore, the aim of our study was to test whether our PAAD-iCCA-Classifier can be extended to be used to correctly diagnose PAAD metastases from other sites in CUP setting. Methods: For this purpose, we enhanced the anomaly detection layer of the classifier by incorporating ten mimicker carcinomas to be excluded by this layer. We used a validation set 1 (n=3786) including primary PAAD (n=242), PAAD liver metastases (n=20), iCCA (n=151) and 10 other mimicker carcinomas (n=3373) and a validation set 2 (n=26) including primary PAAD from a real-life clinical cohort from an independent institution to validate the classifier. Next, we tested the classifier on 16 PAAD initially considered CUP samples (test set) from different sites: peritoneum, lung, liver, and lymph node. The clinical history and diagnostic imaging of these samples were used to confirm PAAD as the most probable origin. We further performed differentially methylated probes (DMP) and copy number alterations (CNA) analysis of primary PAAD and metastatic PAAD from different locations. Results: The improved version of the PAAD-iCCA-Classifier achieved an accuracy of 98.43% on the validation set 1 and was able to exclude most of the mimicker carcinomas. On validation set 2, the classifier achieved an accuracy of 88.46%. Medical history, imaging and immunohistochemical analysis of the test set samples confirmed the diagnosis of PAAD. The DNA methylation classifier correctly labeled 15/16 PAAD metastatic samples as PAAD (93.75% accuracy). We observed that the classifier performance was negatively affected by a high CD3+ immune infiltrate and positively affected by high tumor purity and high proliferation rate. CNA revealed that PAAD liver metastases have a distinct CNA profile characterized by chromosome 6, 9 (CDKN2A/B) and 18q (SMAD4) deletions. DMP analysis showed that PAAD liver metastases have global hypomethylation of both promoter- and enhancer-associated CpGs compared to primary PAAD and PAAD peritoneal carcinomatosis. Finally, gene ontology analysis revealed that different epithelial-mesenchymal transition pathways are activated in PAAD liver metastases compared to PAAD peritoneal carcinomatosis. Conclusion: Our tool performs well in classifying metastatic PAAD samples and could be of great clinical use when a PAAD origin is suspected in the case of a CUP. DMP and CNA profiles show that PAAD liver metastases may have a distinct DNA methylation and copy number profile compared to primary and peritoneal carcinomatosis PAAD. Citation Format: Teodor G. Calina, Eilís Perez, Simon Schallenberg, Horst David, Erik Knutsen, David Capper, Mihnea P. Dragomir. Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4414.

Abstract 4130: Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dire prognosis, in large part due to the late stage at which it is diagnosed in most patients. High-risk individuals (HRIs) identified through family history or germline genetic testing are candidates for imaging-based screening, yet even with intensive surveillance, metastasis often occurs between scans. Our study aims to address this critical clinical challenge by investigating the epigenetic determinants of PDAC metastasis and developing novel strategies for metastasis prevention in HRIs. Methods: Innovative transplantation models of PDAC liver and peritoneal metastasis incorporating DNA barcodes for subclonal tracking were developed to facilitate the identification of subclones with high or low metastatic potential. An in-depth epigenomic characterization and comparison of these subclones was performed utilizing assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq. Results: Metastasis-high subclones demonstrate widespread chromatin accessibility differences relative to their metastasis-low peers. These differences are linked to expression changes for genes involved in motility, differentiation, survival, and particularly inflammation. Top epigenetically activated genes in metastasis-high subclones include the pro-inflammatory cytokines IL-1α and IL-23α and the pro-inflammatory cytokine receptor IL-18R1. Discussion: These findings highlight the role of epigenetic changes in PDAC metastasis, especially activation of inflammatory pathways resulting from altered chromatin accessibility. These insights provide a foundation for exploring anti-inflammatory agents for PDAC metastasis prevention. Targeting inflammation, alongside imaging-based screening, could significantly improve outcomes for HRIs. Citation Format: Jesse Handler, Reza Kalhor. Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4130.

Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis.

Pancreatic ductal adenocarcinoma is a highly metastatic disease and macrophages support liver metastases. Efferocytosis, or engulfment of apoptotic cells by macrophages, is an essential process in tissue homeostasis and wound healing, but its role in metastasis is less well understood. Here, we found that the colonization of the hepatic metastatic site is accompanied by low-grade tissue injury and that efferocytosis-mediated clearance of parenchymal dead cells promotes macrophage reprogramming and liver metastasis. Mechanistically, progranulin expression in macrophages is necessary for efficient efferocytosis by controlling lysosomal acidification via cystic fibrosis transmembrane conductance regulator and the degradation of lysosomal cargo, resulting in LXRα/RXRα-mediated macrophage conversion and upregulation of arginase 1. Pharmacological blockade of efferocytosis or macrophage-specific genetic depletion of progranulin impairs macrophage conversion, improves CD8+ T cell functions, and reduces liver metastasis. Our findings reveal how hard-wired functions of macrophages in tissue repair contribute to liver metastasis and identify potential targets for prevention of pancreatic ductal adenocarcinoma liver metastasis.

Exosome-derived tRNA fragments tRF-GluCTC-0005 promotes pancreatic cancer liver metastasis by activating hepatic stellate cells

Early metastasis is the primary factor in the very poor prognosis of pancreatic ductal adenocarcinoma (PDAC), with liver metastasis being the most common form of distant metastasis in PDAC. To investigate the mechanism of PDAC liver metastasis, we found that PDAC cells can promote the formation of pre-metastatic niches (PMNs) through exosomes to facilitate liver metastasis in the early stage. In our study, hepatic stellate cells (HSCs) were treated with PDAC-derived exosomes (PDAC-exo), and the activation of HSCs was detected. A novel transfer RNA-derived fragment, the tRF-GluCTC-0005 was obtained by small RNA sequencing from serum exosomes of PDAC patients. Bioinformatics analysis and RNA pull-down assays revealed the interaction between WDR1 and tRF-GluCTC-0005. A KPC transgenic mouse model and an AAV-mediated sh-WDR1 mouse model were used to detect the mechanism of liver metastasis in vivo. Finally, the dual luciferase reporter assay, protein mutation truncation assay, Co-IP assay, and flow cytometry assay were used to explore the molecular mechanism in HSCs activation and PMNs formation. We found that the tRF-GluCTC-0005 in exosomes binds to the 3’ untranslated region of the mRNA of the WDRl in HSCs and increases mRNA stability. The N-terminals of WDR1 bind to the YAP protein directly, inhibit YAP phosphorylation, and promote the expression of YAP transcription factors. The tRF-GluCTC-0005 in PDAC-exo significantly recruits myeloid-derived suppressor cells (MDSCs) in the liver, creating a PMNs immunosuppressive microenvironment and further advancing liver metastasis from PDAC. Our results suggest that the key of PDAC liver metastasis is the activation of HSCs through upregulation of WDR1 by tRF-GluCTC-0005 in exosomes, which mediates the infiltration of MDSCs to form PMNs.

Abstract A018: Exercise stimulates anti-tumoral immunity in metastatic PDAC

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients are metastatic at presentation with limited treatment options and a poor overall 5-year survival of 3%. The liver is the predominant site of distant metastasis in PDAC. Our group has previously shown that aerobic exercise can inhibit tumorigenesis in a primary PDAC mouse model by promoting anti-tumor immunity, however, the effects of exercise in the metastatic setting have not been explored. As such, we developed a model of PDAC liver metastasis by isolating primary pancreatic and liver metastatic cell lines from 18–20-week-old LSL-KRasG12D/+; LSL-Trp53R172H/+; p48Cre; Rosa26LSL-tdTomato/+ (KPCTpancreas or KPCTliver, respectively) mice. KPCTpancreas cells were surgically implanted into the pancreata of syngeneic wild-type mice. One week after pancreatic implantation, KPCTliver cells were implanted into the liver either by direct injection into the parenchyma or via the portal vein. The portal vein method enables us to account for the extravasation step of the metastatic cascade, while the direct parenchymal injection offers a less invasive surgery. Both models preserve the spleen, which is the largest secondary lymphoid organ and essential to the immune response. Treadmill running was initiated in the aerobic exercise cohort 5 days per week for 2 weeks or mice were allowed to rest. Endpoint analysis revealed that aerobic exercise treatment decreased tumor burden in both primary tumors and liver metastasis as measured by tumor weights and tdTomato fluorescence imaging when compared to the rested controls. Immune profiling of the liver metastases revealed elevated IL-15Rɑ+ CD8+ T cells as well as CD19+ B cells in the exercise cohort. While identification of IL-15Rɑ+ CD8+ T cells is consistent with the immune changes we have previously reported in the primary pancreatic tumor of exercised mice, the latter changes reveal organ specific immune cell recruitment not observed in the primary pancreatic tumor of exercised mice. The combined immune profiling and phenotypic changes suggest that aerobic exercise reduces PDAC metastatic growth by modulating systemic and intra-tumoral immunity. The induction of an anti-tumorigenic immune response by aerobic exercise could open avenues for potential pharmacological intervention in downstream pathways to aid in the treatment of metastatic PDAC. Citation Format: Mansour E. Riachi, Carolina G. Alcantara Hirsch, Erica Ma, Beny Shapiro, Ammar Javed, Christopher L. Wolfgang, Dafna Bar-Sagi. Exercise stimulates anti-tumoral immunity in metastatic PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A018.

Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.

Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis invitro and invivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma livermetastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ Tcells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ Tcells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory Tcell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive Tcells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Single cell transcriptomic analyses implicate an immunosuppressive tumor microenvironment in pancreatic cancer liver metastasis

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease refractory to all targeted and immune therapies. However, our understanding of PDAC microenvironment especially the metastatic microenvironment is very limited partly due to the inaccessibility to metastatic tumor tissues. Here, we present the single-cell transcriptomic landscape of synchronously resected PDAC primary tumors and matched liver metastases. We perform comparative analysis on both cellular composition and functional phenotype between primary and metastatic tumors. Tumor cells exhibit distinct transcriptomic profile in liver metastasis with clearly defined evolutionary routes from cancer cells in primary tumor. We also identify specific subtypes of stromal and immune cells critical to the formation of the pro-tumor microenvironment in metastatic lesions, including RGS5+ cancer-associated fibroblasts, CCL18+ lipid-associated macrophages, S100A8+ neutrophils and FOXP3+ regulatory T cells. Cellular interactome analysis further reveals that the lack of tumor-immune cell interaction in metastatic tissues contributes to the formation of the immunosuppressive microenvironment. Our study provides a comprehensive characterization of the transcriptional landscape of PDAC liver metastasis.

Identifying a baicalein-related prognostic signature contributes to prognosis prediction and tumor microenvironment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant and lethal human cancers in the world due to its high metastatic potential, and patients with PDAC have a poor prognosis, yet quite little is understood regarding the underlying biological mechanisms of its high metastatic capacity. Baicalein has a dramatic anti-tumor function in the treatment of different types of cancer. However, the therapeutic effects of baicalein on human PDAC and its mechanisms of action have not been extensively understood. In order to explore the biological characteristic, molecular mechanisms, and potential clinical value of baicalein in inhibiting the metastatic capacity of PDAC. We performed several in vitro, in vivo, and in silico studies. We first examined the potential regulation of baicalein in the metastatic capacity of PDAC cells. We showed that baicalein could dramatically suppress liver metastasis of PDAC cells with highly metastatic potential in mice model. The high-throughput sequencing analysis was employed to explore the biological roles of baicalein in PDAC cells. We found that baicalein might be involved in the infiltration of Cancer-Associated Fibroblasts (CAF) in PDAC. Moreover, a baicalein-related risk model and a lncRNA-related model were built by Cox analysis according to the data set of PDAC from TCGA database which suggested a clinical value of baicalein. Finally, we revealed a potential downstream target of baicalein in PDAC, we proposed that baicalein might contribute to the infiltration of CAF via FGFBP1. Thus, we uncovered a novel role for baicalein in regulation of PDAC liver metastasis that may contribute to its anti-cancer effect. We proposed that baicalein might suppress PDAC liver metastasis via regulation of FGFBP1-mediated CAF infiltration. Our results provide a new perspective on clinical utility of baicalein and open new avenues for the inhibition of liver-metastasis of PDAC.

Pancreatic cancer-derived small extracellular vesical ezrin activates fibroblasts to exacerbate cancer metastasis through STAT3 and YAP-1 signaling pathways.

Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC), play an important role in tumorigenesis, metastasis, and chemoresistance. Tumor-derived small extracellular vesicles (sEVs), which mediate cell-to-cell communication between cancer cells and fibroblasts, are also critical for cancer progression and metastasis. However, it remains unclear how PDAC cell-derived sEVs activate fibroblasts, which contributes to tumor progression. Here, we report that ezrin (EZR) expression in PDAC cell-derived sEVs (sEV-EZR) can activate fibroblasts, resulting in increased migration ability and high expression of α-SMA, PDGFRB, and high production of extracellular matrix in fibroblasts. Reciprocally, sEV-EZR-activated fibroblasts enhanced PDAC cell proliferation, invasion, and metastasis to the liver in animal models. Conversely, fibroblasts treated with PDAC cell-derived sEVs with EZR knockdown resulted in the reduced metastatic ability of PDAC. Mechanistically, we demonstrated that PDAC cell-derived sEV-EZR increases the STAT3 and YAP-1 signaling pathways to induce fibroblast activation, and the activated fibroblasts promote PDAC cell proliferation, invasion, and liver metastasis. Inhibition of the STAT3 and YAP-1 signaling pathways by gene knockdown can abrogate sEV-EZR-induced effects. These findings suggest that targeting the interaction between PDAC cell-derived sEV-EZR and fibroblasts is a potential therapeutic strategy for PDAC.

Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

To explore the mechanism of coadaptation and the potential drivers of pancreatic ductal adenocarcinoma (PDAC) metastasis to the liver, we study key molecules involved in this process and their translational value. Premetastatic niche (PMN) and macrometastatic niche (MMN) formation in a mouse model is observed via CT combined with 3D organ reconstruction bioluminescence imaging, and then we screen slit guidance ligand 2 (SLIT2) and its receptor roundabout guidance receptor 1 (ROBO1) as important factors. After we confirm the expression and distribution of SLIT2 and ROBO1 in samples from PDAC patients and several mouse models, we discover that SLIT2-ROBO1-mediated coadaptation facilitated the implantation and outgrowth of PDAC disseminated tumour cells (DTCs) in the liver. We also demonstrate the dependence receptor (DR) characteristics of ROBO1 in a follow-up mechanistic study. A neutralizing antibody targeting ROBO1 significantly attenuate liver metastasis of PDAC by preventing the coadaptation effect. Thus, we demonstrate that coadaptation is supported by the DR characteristics in the PMN and MMN.

GJB3 promotes pancreatic cancer liver metastasis by enhancing the polarization and survival of neutrophil.

Connexins are membrane expressed proteins, which could assemble into hexamers to transfer metabolites and secondary messengers. However, its roles in pancreatic cancer metastasis remains unknown. In this study, by comparing the gene expression patterns in primary pancreatic cancer patients primary and liver metastasis specimens, we found that Gap Junction Protein Beta 3 (GJB3) significantly increased in Pancreatic ductal adenocarcinoma (PDAC) liver metastasis. Animal experiments verified that GJB3 depletion suppressed the hepatic metastasis of PDAC cancer cells. Further, GJB3 over expression increased the neutrophil infiltration. Mechanistic study revealed that GJB3 form channels between PDAC tumor cells and accumulated neutrophil, which transfer cyclic adenosine monophosphate (cAMP) from cancer to neutrophil cells, which supports the survival and polarization. Taken together, our data suggesting that GJB3 could act as a potential therapeutic target of PDAC liver metastasis.

A self-amplifying USP14-TAZ loop drives the progression and liver metastasis of pancreatic ductal adenocarcinoma

With a 5-year survival rate of approximately 10%, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies in humans. A poor understanding of the underlying biology has resulted in a lack of effective targeted therapeutic strategies. Tissue microarray and bioinformatics analyses have revealed that the downstream transcriptional coactivator of the Hippo pathway, transcriptional coactivator with PDZ-binding motif (TAZ), might be a therapeutic target in PDAC. Since pharmacological inhibition of TAZ is challenging, we performed unbiased deubiquitinase (DUB) library screening to explore the pivotal regulators of TAZ ubiquitination as potential targets in PDAC models. We found that USP14 contributed to Yes-associated protein (YAP)/TAZ transcriptional activity and stabilized TAZ but not YAP. Mechanistically, USP14 catalyzed the K48-linked deubiquitination of TAZ to promote TAZ stabilization. Moreover, TAZ facilitated the transcription of USP14 by binding to the TEA domain transcription factor (TEAD) 1/4 response element in the promoter of USP14. USP14 was found to modulate the expression of TAZ downstream target genes through a feedback mechanism and ultimately promoted cancer progression and liver metastasis in PDAC models in vitro and in vivo. In addition, depletion of USP14 led to proteasome-dependent degradation of TAZ and ultimately arrested PDAC tumour growth and liver metastasis. A strong positive correlation between USP14 and TAZ expression was also detected in PDAC patients. The small molecule inhibitor of USP14 catalytic activity, IU1, inhibited the development of PDAC in subcutaneous xenograft and liver metastasis models. Overall, our data strongly suggested that the self-amplifying USP14-TAZ loop was a previously unrecognized mechanism causing upregulated TAZ expression, and identified USP14 as a viable therapeutic target in PDAC.

Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy

The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

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.

CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

BackgroundCircular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC).MethodsCircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1.ResultsCircRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC.ConclusionThe upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT.Graphical abstract

Pirfenidone-loaded exosomes derived from pancreatic ductal adenocarcinoma cells alleviate fibrosis of premetastatic niches to inhibit liver metastasis.

Given the metastasis-promoting effect of pancreatic ductal adenocarcinoma (PDAC)-derived exosomes through activation of fibrotic premetastatic niches, targeting and intervening in premetastatic organs to inhibit distant metastasis have challenged researchers and clinicians. Herein, a self-biomimetic drug delivery system based on exosomes derived from PDAC (PF@PCCEs) was constructed to precisely deliver an antifibrotic drug (pirfenidone, PF) to fibrotic premetastatic organs. First, PDAC-derived exosomes were confirmed to remarkably promote liver fibrosis. Then the prepared PF@PCCEs were actively internalized by HSCs (hepatic stellate cells) and subsequently alleviated the activation of HSCs. Delivery of PF to the premetastatic liver affected the niche suitable for the colonization of circulating tumour cells, further suppressing liver metastasis of PDAC. Thus, the strategy for intervening in the formation of fibrotic premetastatic niches to inhibit liver metastasis of PDAC using PF@PCCEs might offer inspiration for the treatment of tumour metastasis.