Pancreatic Ductal Adenocarcinoma Metastasis Research Articles

CALB2 drives pancreatic cancer metastasis through inflammatory reprogramming of the tumor microenvironment

BackgroundEarly dissemination to distant organs accounts for the dismal prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). Chronic, dysregulated, persistent and unresolved inflammation provides a preferred tumor microenvironment (TME) for tumorigenesis, development, and metastasis. A better understanding of the key regulators that maintain inflammatory TME and the development of predictive biomarkers to identify patients who are most likely to benefit from specific inflammatory-targeted therapies is crucial for advancing personalized cancer treatment.MethodsThis study identified cell-specific expression of CALB2 in human PDAC through single-cell RNA sequencing analysis and assessed its clinicopathological correlations in tissue microarray using multi-color immunofluorescence. Co-culture systems containing cancer-associated fibroblasts (CAFs) and patient-derived organoids (PDOs) in vitro and in vivo were employed to elucidate the effects of CALB2-activated CAFs on PDAC malignancy. Furthermore, CUT&RUN assays, luciferase reporter assays, RNA sequencing, and gain- or loss-of-function assays were used to unravel the molecular mechanisms of CALB2-mediated inflammatory reprogramming and metastasis. Additionally, immunocompetent KPC organoid allograft models were constructed to evaluate CALB2-induced immunosuppression and PDAC metastasis, as well as the efficacy of inflammation-targeted therapy.ResultsCALB2 was highly expressed both in CAFs and cancer cells and correlated with an unfavorable prognosis and immunosuppressive TME in PDAC patients. CALB2 collaborated with hypoxia to activate an inflammatory fibroblast phenotype, which promoted PDAC cell migration and PDO growth in vitro and in vivo. In turn, CALB2-activated CAFs upregulated CALB2 expression in cancer cells through IL6-STAT3 signaling-mediated direct transcription. In cancer cells, CALB2 further activated Ca2+-CXCL14 inflammatory axis to facilitate PDAC metastatic outgrowth and immunosuppression. Genetic or pharmaceutical inhibition of CXCL14 significantly suppressed CALB2-mediated metastatic colonization of PDAC cells in vivo and extended mouse survival.ConclusionsThese findings identify CALB2 as a key regulator of inflammatory reprogramming to promote PDAC metastatic progression. Combination therapy with αCXCL14 monoclonal antibody and gemcitabine emerges as a promising strategy to suppress distant metastasis and improve survival outcomes in PDAC with CALB2 overexpression.

Targeted inhibition of CHKα and mTOR in models of pancreatic ductal adenocarcinoma: A novel regimen for metastasis

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic malignancy for which there are currently no effective anti-metastatic therapies. Herein, we employed single-cell RNA sequencing and metabolomics analysis to demonstrate that metastatic cells highly express focal adhesion kinase (FAK), which promotes metastasis by remodeling choline kinase α (CHKα)-dependent choline metabolism. We designed a novel CHKα inhibitor, CHKI-03, and verified its efficacy in inhibiting metastasis in multiple preclinical models. Classical and newly synthesized small-molecule inhibitors have previously been used to assess the therapeutic potential of targeting mTOR and CHKα in various animal models. Mechanistically, FAK activated mTOR and its downstream HIF-1α, thereby elevating CHKα expression and promoting the proliferation, migration, and invasion of PDAC cells, as well as tumor growth and metastasis. Consistently, high expression levels of both FAK and CHKα are correlated with poor prognosis in patients with PDAC. Notably, CHK1-03 inhibited CHKα expression and also suppressed mTORC1 phosphorylation, disrupting the mTORC1-CHKα positive feedback loop. In addition, the combination of CHKI-03 and the mTORC1 inhibitor rapamycin synergistically inhibited tumor growth and metastasis in PDX models. The combination of CHKI-03 and rapamycin demonstrates considerable therapeutic efficacy in PDO models resistant to gemcitabine. Our findings reveal a pivotal mechanism underlying PDAC metastasis regulated by mTORC1-CHKα loop-dependent choline metabolism reprogramming, highlighting the therapeutic potential of this novel regimen for treating PDAC metastasis.

Abstract B073: Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) that is refractory to treatment frequently leads to extensive systemic and multi-organ metastases. PDAC is characterized as an immune “cold” cancer; however, the persistence of this “cold” tumor immune microenvironment (TIME) across distant metastases, and the mechanisms by which metastases influence their respective TIME, remain poorly understood. In this study, we conducted spatial protein expression profiling of the TIME using the Nanostring GeoMx Digital Spatial Profiling (DSP) platform. We employed a 43- plex panel on 54 tissue samples, including 14 primary tumors and 40 matched metastases from organs commonly affected by PDAC metastases (liver, lung, and peritoneum), obtained through a rapid (“warm”) autopsy program. We quantified protein expression in three cellular compartments: PanCK+ epithelial cells, α-SMA+ fibroblasts, and CD45+ immune cells, across 181 regions of interest (ROIs). Our analysis revealed a significant enrichment of CD45+ immune cells in lung metastases compared to other organ sites, with a notably lower presence of these cells in liver metastases. Focusing on the immune cell compartment, we analyzed the CD45 segments for further investigation. After normalization using housekeeping proteins, including Histone H3, GAPDH, and ribosomal protein S6, we found that the immune profiles were most distinct between liver and lung metastases. Specifically, liver metastases exhibited the greatest deviation in immune profile compared to primary PDAC, lung, and peritoneal metastases. We scored protein expression signatures for various immune cell subsets based on specific marker expressions. When comparing liver to lung metastases, scores for T cells, myeloid cells, checkpoint markers, macrophages, and interferon signaling were significantly lower in the liver, underscoring the immune “coldness” of the liver metastasis environment. Our cohort included six untreated patients and nine patients treated with chemotherapies. We compared protein expressions in each organ site between untreated and treated patients. Notably, treatment induced divergent expression patterns of multiple proteins between liver and lung metastases. While treatment significantly reduced the expression of several immune markers, including CD45 (total immune), CD3 (T cell), CD4 (T helper cell), CD45RO (memory T cell), CD163 (M2 macrophage), and B7-H3 (checkpoint marker), in lung metastases, these markers were significantly upregulated in liver metastases of treated patients. This finding indicates a pronounced divergence in TIME modulation in response to treatment between liver and lung metastases, potentially fostering a treatment-resistant environment in PDAC patients with multi- organ metastasis. Consequently, this urges the need to develop tailored therapeutic strategies for PDAC metastasis, taking into account the distinct immune landscapes of different metastasis sites. Citation Format: Jimin Min, Vittorio Branchi, Kimal I Rajapakshe, Nisha Narayanan, Guangsheng Pei, Linghua Wang, Jean L Grem, Thomas C Caffrey, Paul M Grandgenett, Michael A Hollingsworth, Paola A Guerrero, Anirban Maitra. Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B073.

Abstract A059: Understanding the events that promote pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy due to overwhelming metastatic burden. PDAC cells migrate and invade by two distinct epithelial-to-mesenchymal transition phenotypes: collective (partial-EMT) or single-cell (complete-EMT). The mechanism(s) by which these invasive phenotypes intravasate is unknown. The Tumor Microenvironment of Metastasis (TMEM) doorway, which is formed by a tumor cell, macrophage and an endothelial cell in direct contact, causes opening of the vasculature in which tumor cells intravasate. Intravasation via TMEM doorway is inhibited by Tie2 blockade. Whether this occurs in PDAC and whether the EMT phenotype affects intravasation is unknown. We hypothesize that PDAC cells intravasate through TMEM-dependent mechanisms regardless of EMT phenotype. Tumor samples from murine models were stained for TMEM doorways using a triple immunohistochemistry (IHC) stain (tumor cells expressing the actin regulatory protein Mena, macrophages expressing CD68, and endothelial cells expressing CD31). TMEM doorway opening was assessed in murine models of PDAC treated with or without Tie-2 inhibitor by analyzing TMEM-mediated extravasation of high-molecular weight dextran (155kD-TMR dextran) by Intravital Imaging, IHC and immunofluorescence (IF) of serial slides and circulating tumor cells. An intravasation trans-endothelial migration assay was also performed to assess the intravasation event by TMEM doorway in vitro. A student t-test was used for the analyses. TMEM doorways were observed in all PDAC murine models. For the first time in PDAC, focal extravasation of dextran events followed by a tumor cell intravasation was visualized by intravital imaging. PDAC tumor bearing mice treated with Tie2 inhibitor had a 2-fold decrease in the number of circulating tumor cells compared to control (958 ± 499.8 vs 534 ± 479.9 cells/mL blood, p=0.0257). The intravasation trans-endothelial migration assay showed that C-EMT cells have a higher migration rate than P-EMT cells (17.8 ± 5.53 vs 10.94 ± 3.84 migrating cells p=0.01), and this ratio increases with the presence of macrophages (23.75 ± 5 vs 15.54 ± 3.09 migrating cells p=0.005). The Tie-2 inhibition decreases the number of C-EMT and P-EMT migrating cells (7.69 ± 1.3 vs 3.67 ± 1.2 migrating cells p=0.01). Orthotopic tumors from C-EMT phenotype grow at a faster rate than the P-EMT (0.49mg ± 0.09 vs 0.12mg ± 0.05 p<0.05). Tumors from C-EMT and P-EMT cells will be analyzed for TMEM doorway score, TMEM activity and stemness signatures around TMEM doorway to determine if TMEM doorway is a niche for stemness in PDAC. TMEM doorways are present in PDAC mouse model. Inhibition of Tie2 leads to decreased TMEM doorway function and circulating tumor cells in PDAC. In addition, regarding the EMT phenotype, TMEM doorways are functional mechanisms of intravasation in vitro. This is a novel mechanism in PDAC and suggests a promising therapeutic target for decreasing intravasation and potential metastasis in PDAC. Citation Format: Erika Pereira Zambalde, Lina Ariyan, Francisco Puerta-Martinez, Yuqin Zhu, John Condeelis, Ben Stanger, John McAuliffe. Understanding the events that promote pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A059.

Abstract A075: Establishing the interaction mechanism of oncoprotein, Mucin1, and extracellular vesicle marker protein, Alix, in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive disease with devastating mortality rates largely due to a poor understanding of its progression mechanism. Small extracellular vesicles (sEVs) are used by cancer cells to aid in their progression, and tumor-derived EVs (TEVs) show a distinct protein cargo composition potentially contributing to this rapid progression and metastasis of PDAC. To investigate this phenomenon, we isolated sEVs from conditioned media of pancreatic cancer cell lines using ultracentrifugation and characterized them by transmission electron microscopy (TEM), nanoparticle tracking analysis, and immunoblotting. These sEVs were then used in several studies to investigate critical TEV cargo sorting machinery. For instance, PDAC-derived sEVs have selective enrichment of the oncogenic transmembrane protein Mucin1 (MUC1). Preliminary MUC1KO studies from our lab revealed that MUC1 potentially regulates sEV formation, where we saw a clear impact on sEV cargo in the absence of MUC1. Additional studies found that the cytoplasmic adaptor protein ALG-2-Interacting Protein X (Alix), a protein known to play a critical role during TEV formation, regulates MUC1’s presence in sEVs. From this discovery, we then identified a novel MUC1-Alix interaction, which was confirmed using several techniques, including co-immunoprecipitation (CoIP). To define the Alix-interacting domain of MUC1, we utilized in silico modeling and localized surface plasmon resonance (LSPR) of MUC1.CT and Alix to quantify the interaction. This revealed the cytoplasmic tail of MUC1 (MUC1.CT) as the Alix-interacting domain. Moving forward, our central hypothesis for this study is that this MUC1.CT-Alix interaction utilizes a conserved binding motif that targets Alix to the endosomal membrane and contributes to the formation of MUC1+ sEVs. While a distinct sEV biogenesis pathway employed by cancer cells has not yet been defined, our studies will provide insight into the potential sEV sorting mechanism of MUC1 and other cancer biomarkers and advance the understanding of PDAC progression. Citation Format: Kristine V Hoagstrom, Ying Huang, Gloria E Borgstahl, Michael A Hollingsworth. Establishing the interaction mechanism of oncoprotein, Mucin1, and extracellular vesicle marker protein, Alix, in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A075.

Abstract B092: PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine and glucose for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that reguates a wide variety of cell signaling pathways, inlcuding KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demosntrate that suppression of PP2A accelerates the formation of KRAS-driven precursor lesions in an in vivo mouse model of PDAC, implicating PP2A as a critical regulator of mutant KRAS phenotypes. Here, we show that acute PP2A activation prevents the fusion of macropinosomes with the lysosome, robbing PDAC cells of critical metabolic nutrients and driving cell death. Furthermore, we find PP2A associates with the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, support a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M Pfeffer, Indiraa Doraivel, Sara N Filippelli, Brittany N Heil, Brittany L Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B092.

Abstract B075: Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant cancer- associated fibroblasts (CAFs) which contribute to a tumor-promoting, chemotherapy-resistant and immunosuppressive tumor microenvironment (TME). However, CAF-ablating therapeutic strategies have thus far failed in clinical trials. One potential reason is that PDAC CAFs are heterogeneous and plastic. It was recently discovered that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) can originate from pancreatic stellate cells (PSCs) via TGF-β and IL-1 signaling, respectively. However, their functional heterogeneity remains to be elucidated. We hypothesize that distinct CAF subtypes exhibit different tumor-promoting roles mediated by targetable signaling mechanisms. To this end, we aimed at characterizing the functions of TGF- β-dependent myCAFs in shaping the TME and influencing PDAC progression. Methods: We generated novel myCAF-depleted PDAC mouse models by orthotopically injecting PDAC organoids derived from KPC (Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre) mice into Fap-Tgfbr2 knockout (Fap-tTA; Rosa26 LSLtdTom/+ ; tet-O-Cre; Tgfbr2 fl/fl ) mice. The pancreatic tumors were analyzed by flow cytometry, histology and single-cell RNA-sequencing (scRNAseq). In a complementary approach, we generated myCAF-enriched PDAC mouse models by orthotopically co-injecting KPC PDAC organoids and novel TGF-β-driven ‘myCAF-locked’ murine PSCs. In addition, to further dissect myCAF-malignant cell signaling crosstalk, KPC PDAC organoids and myCAF-locked PSCs were co-cultured and then flow-sorted for bulk RNA- sequencing. Results: MyCAF-depleted PDAC tumors from Fap-Tgfbr2 knockout mice had a significant decrease in myCAFs, and a significant expansion of iCAFs, compared to control mice. Additionally, Masson’s trichrome staining indicated significant reduction in collagen content in the TME upon myCAF depletion. Moreover, scRNAseq analysis revealed that myCAF depletion shaped the composition of innate immune cells, including macrophages and neutrophils. Notably, although primary tumor growth was not altered, liver and lung metastases decreased, while diaphragm metastases and ascites increased, in myCAF-depleted PDAC mice. Conversely, liver and lung metastases increased, while diaphragm metastases and ascites decreased in myCAF-enriched PDAC mouse models, suggesting that TGF-β-dependent myCAFs play a role in promoting distant metastases. In line with this, transcriptomic analysis of PDAC organoids co-cultured with myCAF-locked PSCs showed enrichment of pathways in epithelial-mesenchymal transition, hypoxia, VEGF, WNT and cell-matrix interactions. Conclusion: Our study demonstrates that TGF-β-dependent CAFs are not only largely responsible for collagen deposition in PDAC, but also possess immune-modulating properties. Moreover, our study supports a role of TGF-β-dependent CAFs in promoting PDAC metastasis to the liver and the lungs, potentially through upregulating pro-metastatic pathways in the malignant cells. Citation Format: Priscilla S.W. Cheng, Muntadher Jihad, Judhell S. Manansala, Weike Luo, Gianluca Mucciolo, Sara Pinto Teles, Giulia Biffi. Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B075.

Platelet-Derived Growth Factor C Facilitates Malignant Behavior of Pancreatic Ductal Adenocarcinoma by Regulating SREBP1 Mediated Lipid Metabolism.

Lipid metabolism reprogramming stands as a fundamental hallmark of cancer cells. Unraveling the core regulators of lipid biosynthesis holds the potential to find promising therapeutic targets in pancreatic ductal adenocarcinoma (PDAC). Here, it is demonstrated that platelet-derived growth factor C (PDGFC) orchestrated lipid metabolism, thereby facilitated the malignant progression of PDAC. Expression of PDGFC is upregulated in PDAC cohorts and is corelated with a poor prognosis. Aberrantly high expression of PDGFC promoted proliferation and metastasis of PDAC both in vitro and in vivo. Mechanistically, PDGFC accelerated the malignant progression of PDAC by upregulating fatty acid accumulation through sterol regulatory element-binding protein 1 (SREBP1), a key transcription factor in lipid metabolism. Remarkably, Betulin, an inhibitor of SREBP1, demonstrated the capability to inhibit proliferation and metastasis of PDAC cell lines, along with attenuating the process of liver metastasis in vivo. Overall, the study underscores the pivotal role of PDGFC-mediated lipid metabolism in PDAC progression, suggesting PDGFC as a potential biomarker for PDAC metastasis. Targeting PDGFC-induced lipid metabolism emerges as a promising therapeutic strategy for metastatic PDAC, with the potential to improve clinical outcomes.

Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate.

Identifying the key molecular pathways that enable metastasis by analyzing the eventual metastatic tumor is challenging because the state of the founder subclone likely changes following metastatic colonization. To address this challenge, we labeled primary mouse pancreatic ductal adenocarcinoma (PDAC) subclones with DNA barcodes to characterize their pre-metastatic state using ATAC-seq and RNA-seq and determine their relative in vivo metastatic potential prospectively. We identified a gene signature separating metastasis-high and metastasis-low subclones orthogonal to the normal-to-PDAC and classical-to-basal axes. The metastasis-high subclones feature activation of IL-1 pathway genes and high NF-κB and Zeb/Snail family activity and the metastasis-low subclones feature activation of neuroendocrine, motility, and Wnt pathway genes and high CDX2 and HOXA13 activity. In a functional screen, we validated novel mediators of PDAC metastasis in the IL-1 pathway, including the NF-κB targets Fos and Il23a , and beyond the IL-1 pathway including Myo1b and Tmem40 . We scored human PDAC tumors for our signature of metastatic potential from mouse and found that metastases have higher scores than primary tumors. Moreover, primary tumors with higher scores are associated with worse prognosis. We also found that our metastatic potential signature is enriched in other human carcinomas, suggesting that it is conserved across epithelial malignancies. This work establishes a strategy for linking cancer cell state to future behavior, reveals novel functional regulators of PDAC metastasis, and establishes a method for scoring human carcinomas based on metastatic potential.

KHSRP Stabilizes m6A-Modified Transcripts to Activate FAK Signaling and Promote Pancreatic Ductal Adenocarcinoma Progression.

N6-methyladenosine (m6A) is the most prevalent RNA modification and is associated with various biological processes. Proteins that function as readers and writers of m6A modifications have been shown to play critical roles in human malignancies. Here, we identified KH-type splicing regulatory protein (KHSRP) as an m6A binding protein that contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). High KHSRP levels were detected in PDAC and predicted poor patient survival. KHSRP deficiency suppressed PDAC growth and metastasis in vivo. Mechanistically, KHSRP recognized and stabilized FAK pathway mRNAs, including MET, ITGAV and ITGB1, in an m6A-dependent manner, which led to activation of downstream FAK signaling that promoted PDAC progression. Targeting KHSRP with a PROTAC showed promising tumor suppressive effects in mouse models, leading to prolonged survival. Together, these findings indicate that KHSRP mediates FAK pathway activation in an m6A-dependent manner to support PDAC growth and metastasis, highlighting the potential of KHSRP as a therapeutic target in pancreatic cancer.

Establishment of a nomogram model for predicting distant metastasis in pancreatic ductal adenocarcinoma: a comparative analysis of different lymph node staging systems based on the SEER database

The purpose of this study was to compare the predictive value of different lymph node staging systems and to develop an optimal prognostic nomogram for predicting distant metastasis in pancreatic ductal adenocarcinoma (PDAC). Our study involved 6364 patients selected from the Surveillance, Epidemiology, and End Results (SEER) database and 126 patients from China. Independent risk factors for distant metastasis were screened by univariate and multivariate logistic regression analyses, and a model-based comparison of different lymph node staging systems was conducted. Furthermore, we developed a nomogram for predicting distant metastasis using the optimal performance lymph node staging system. The lymph node ratio (LNR), log odds of positive lymph nodes (LODDS), age, primary site, grade, tumor size, American Joint Committee on Cancer (AJCC) 7th Edition T stage, and radiotherapy recipient status were significant predictors of distant metastasis in PDAC patients. The model with the LODDS was a better fit than the model with the LNR. We developed a nomogram model based on LODDS and six clinical parameters. The area under the curve (AUC) and concordance index (C-index) of 0.753 indicated that this model satisfied the discrimination criteria. Kaplan–Meier curves indicate a significant difference in OS among patients with different metastasis risks. LODDS seems to have a superior ability to predict distant metastasis in PDAC patients compared with the AJCC 8th Edition N stage, PLN and LNR staging systems. Moreover, we developed a nomogram model for predicting distant metastasis. Clinicians can use the model to detect patients at high risk of distant metastasis and to make further clinical decisions.

Multiphoton excited polymerized biomimetic models of collagen fiber morphology to study single cell and collective migration dynamics in pancreatic cancer

The respective roles of aligned collagen fiber morphology found in the extracellular matrix (ECM) of pancreatic cancer patients and cellular migration dynamics have been gaining attention because of their connection with increased aggressive phenotypes and poor prognosis. To better understand how collagen fiber morphology influences cell-matrix interactions associated with metastasis, we used Second Harmonic Generation (SHG) images from patient biopsies with Pancreatic ductal adenocarcinoma (PDAC) as models to fabricate collagen scaffolds to investigate processes associated with motility. Using the PDAC BxPC-3 metastatic cell line, we investigated single and collective cell dynamics on scaffolds of varying collagen alignment. Collective or clustered cells grown on the scaffolds with the highest collagen fiber alignment had increased E-cadherin expression and larger focal adhesion sites compared to single cells, consistent with metastatic behavior. Analysis of single cell motility revealed that the dynamics were characterized by random walk on all substrates. However, examining collective motility over different time points showed that the migration was super-diffusive and enhanced on highly aligned fibers, whereas it was hindered and sub-diffusive on un-patterned substrates. This was further supported by the more elongated morphology observed in collectively migrating cells on aligned collagen fibers. Overall, this approach allows the decoupling of single and collective cell behavior as a function of collagen alignment and shows the relative importance of collective cell behavior as well as fiber morphology in PDAC metastasis. We suggest these scaffolds can be used for further investigations of PDAC cell biology. Statement of significancePancreatic ductal adenocarcinoma (PDAC) has a high mortality rate, where aligned collagen has been associated with poor prognosis. Biomimetic models representing this architecture are needed to understand complex cellular interactions. The SHG image-based models based on stromal collagen from human biopsies afford the measurements of cell morphology, cadherin and focal adhesion expression as well as detailed motility dynamics. Using a metastatic cell line, we decoupled the roles of single cell and collective cell behavior as well as that arising from aligned collagen. Our data suggests that metastatic characteristics are enhanced by increased collagen alignment and that collective cell behavior is more relevant to metastatic processes. These scaffolds provide new insight in this disease and can be a platform for further experiments such as testing drug efficacy.

HnRNPAB promotes pancreatic ductal adenocarcinoma extravasation and liver metastasis by stablizing MYC mRNA.

Heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) is considered a cancer-promoting heterogeneous nuclear ribonucleoprotein in many cancers, but its function in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. HnRNPAB was highly expressed in PDAC tissues compared to normal pancreatic tissues, and high expression of hnRNPAB was associated with poor overall survival and recurrence-free survival in PDAC patients. HnRNPAB promotes migration and invasion of PDAC cells in vitro. In xenograft tumor mouse models, hnRNPAB deprivation significantly attenuated liver metastasis. HnRNPAB mRNA and protein levels are positively associated with MYC in PDAC cells. Mechanistically, hnRNPAB bound to MYC mRNA and prolonged its half-life of MYC mRNA. HnRNPAB induced PDAC cells to secret CXCL8 via MYC, which promoted neutrophils recruitment and facilitated tumor cells entrancing into the hepatic parenchyma. These findings point to a novel regulatory mechanism via which hnRNPAB promotes PDAC metastasis. Implications: Hnrnpab participates in the post-transcriptional regulation of the oncogene MYC by binding and stabilizing MYC mRNA, thereby promoting liver metastasis in PDAC.

The lncRNA LINC01605 promotes the progression of pancreatic ductal adenocarcinoma by activating the mTOR signaling pathway

BackgroundThis study investigated the molecular mechanism of long intergenic non-protein coding RNA 1605 (LINC01605) in the process of tumor growth and liver metastasis of pancreatic ductal adenocarcinoma (PDAC).MethodsLINC01605 was filtered out with specificity through TCGA datasets (related to DFS) and our RNA-sequencing data of PDAC tissue samples from Renji Hospital. The expression level and clinical relevance of LINC01605 were then verified in clinical cohorts and samples by immunohistochemical staining assay and survival analysis. Loss- and gain-of-function experiments were performed to estimate the regulatory effects of LINC01605 in vitro. RNA-seq of LINC01605-knockdown PDAC cells and subsequent inhibitor-based cellular function, western blotting, immunofluorescence and rescue experiments were conducted to explore the mechanisms by which LINC01605 regulates the behaviors of PDAC tumor cells. Subcutaneous xenograft models and intrasplenic liver metastasis models were employed to study its role in PDAC tumor growth and liver metastasis in vivo.ResultsLINC01605 expression is upregulated in both PDAC primary tumor and liver metastasis tissues and correlates with poor clinical prognosis. Loss and gain of function experiments in cells demonstrated that LINC01605 promotes the proliferation and migration of PDAC cells in vitro. In subsequent verification experiments, we found that LINC01605 contributes to PDAC progression through cholesterol metabolism regulation in a LIN28B-interacting manner by activating the mTOR signaling pathway. Furthermore, the animal models showed that LINC01605 facilitates the proliferation and metastatic invasion of PDAC cells in vivo.ConclusionsOur results indicate that the upregulated lncRNA LINC01605 promotes PDAC tumor cell proliferation and migration by regulating cholesterol metabolism via activation of the mTOR signaling pathway in a LIN28B-interacting manner. These findings provide new insight into the role of LINC01605 in PDAC tumor growth and liver metastasis as well as its value for clinical approaches as a metabolic therapeutic target in PDAC.

A targetable secreted neural protein drives pancreatic cancer metastatic colonization and HIF1a nuclear retention.

Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer secreted protein that becomes over-expressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver-the primary site of PDAC metastasis. NPTX1-AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia induced factor-1a (HIF1a) nuclear retention and function. NPTX1 is over-expressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1-AMIGO2 as druggable critical upstream regulators of the HIF1a hypoxic response in PDAC.

Necroptosis enhances ‘don’t eat me’ signal and induces macrophage extracellular traps to promote pancreatic cancer liver metastasis

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with dismal prognosis due to distant metastasis, even in the early stage. Using RNA sequencing and multiplex immunofluorescence, here we find elevated expression of mixed lineage kinase domain-like pseudo-kinase (MLKL) and enhanced necroptosis pathway in PDAC from early liver metastasis T-stage (T1M1) patients comparing with non-metastatic (T1M0) patients. Mechanistically, MLKL-driven necroptosis recruits macrophages, enhances the tumor CD47 ‘don’t eat me’ signal, and induces macrophage extracellular traps (MET) formation for CXCL8 activation. CXCL8 further initiates epithelial–mesenchymal transition (EMT) and upregulates ICAM-1 expression to promote endothelial adhesion. METs also degrades extracellular matrix, that eventually supports PDAC liver metastasis. Meanwhile, targeting necroptosis and CD47 reduces liver metastasis in vivo. Our study thus reveals that necroptosis facilitates PDAC metastasis by evading immune surveillance, and also suggest that CD47 blockade, combined with MLKL inhibitor GW806742X, may be a promising neoadjuvant immunotherapy for overcoming the T1M1 dilemma and reviving the opportunity for radical surgery.

Earliest Metabolic Changes Associated with the Initiation of Pancreatic Cancer.

Pancreatic cancer is usually detected at a late stage, when tumors have already metastasized; therefore, it has a poor prognosis with a 5-year survival rate of 11% to 12%. A key to targeting this high mortality is to develop methods for detecting the disease at a stage in which it is still local to the pancreas. However, this needs a better understanding of the events that govern pancreatic cancer oncogenesis. In this issue of Cancer Research, Neuß and colleagues report metabolic changes associated with acinar-to-ductal metaplasia (ADM), an initiating event that leads to the formation of precursor lesions for pancreatic ductal adenocarcinoma (PDAC). Their findings reveal a switch to aerobic glycolysis, increased c-MYC signaling, and increased serine metabolism as driving factors for the ADM process. These findings are important as they demonstrate that metabolic changes that drive the proliferation and metastasis of full-blown PDAC begin in the earliest lesions. The data not only provide insights into how PDAC develops but also a potential explanation for previously described findings, such as circulating lesion cells can be detected even when no carcinoma in situ is present. In summary, this article is highly relevant for furthering our understanding of how metabolic reprogramming drives the earliest events leading to PDAC development and could lay the groundwork for developing methods for early detection or intervention. See related article by Neuß et al., p. 2297.

LINC01133 promotes pancreatic ductal adenocarcinoma epithelial–mesenchymal transition mediated by SPP1 through binding to Arp3

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited treatment methods. Long non-coding RNAs (lncRNAs) have been found involved in tumorigenic and progression. The present study revealed that LINC01133, a fewly reported lncRNA, was one of 16 hub genes that could predict PDAC patients’ prognosis. LINC01133 was over-expressed in PDAC tumors compared to adjacent pancreas and could promote PDAC proliferation and metastasis in vitro and in vivo, as well as inhibit PDAC apoptosis. LINC01133 expression positively correlated to secreted phosphoprotein 1 (SPP1) expression, leading to an enhanced epithelial-mesenchymal transition (EMT) process. LINC01133 bound with actin-related protein 3 (Arp3), the complex reduced SPP1 mRNA degradation which increased SPP1 mRNA level, ultimately leading to PDAC proliferation. This research revealed a novel mechanism of PDAC development and provided a potential prognosis indicator that may benefit PDAC patients.

MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray–based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein–coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.

Isoleucyl-tRNA synthetase 2 promotes pancreatic ductal adenocarcinoma proliferation and metastasis by stabilizing β-catenin

Isoleucyl-tRNA synthetase 2 (IARS2), originally regarded as an enzyme ligating isoleucine to the corresponding tRNA, has been identified as an oncogene recently. However, its function in pancreatic ductal adenocarcinoma (PDAC) remains to be discovered. Here we explored the biological role of IARS2 in PDAC. Up-regulated IARS2 was found in PDAC tissues and cell lines. Kaplan–Meier survival analysis indicated a worse prognosis in patients with high IARS2 expression. CCK-8, EdU, and colony formation assays showed IARS2 overexpression enhanced PDAC proliferation, which was reduced by IARS2 knockdown. Meanwhile, IARS2 down-regulation inhibited PDAC metastasis by impeding epithelial–mesenchymal transition. These results were also supported by subcutaneous xenograft and metastasis assays in vivo. To figure out underlying mechanisms, differential and enrichment analyses were conducted and the WNT signaling pathway was discovered. Our results demonstrated that there was no significant relationship between the WNT signaling pathway key factor CTNNB1 and IARS2 at the transcription level. However, cycloheximide assays showed that IARS2 reduced the β-catenin degradation rate. IARS2 inhibited the phosphorylation of β-catenin at the Ser33/37 site and regulated downstream targets of WNT signaling including c-MYC, c-JUN, and MMP7. The enhancement of proliferation and metastasis caused by IARS2 could be reversed by MSAB, an agent that promotes β-catenin degradation. In summary, IARS2 facilitates PDAC proliferation and metastasis by stabilizing β-catenin, which leads to WNT/β-catenin activation. IARS2 serves as an underlying prognosis marker and a potential therapeutic target for PDAC.