Pancreatic Ductal Adenocarcinoma Cells Research Articles

Abstract A022: Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer

Abstract Although immune therapy has made impressive progress in cancer treatment, pancreatic ductal adenocarcinoma (PDAC) remains an exception, likely due to its immunosuppressive tumor microenvironment that prevents immune responses against the tumor. Our current work aims to reactivate the suppressive immune infiltrate using radiation therapy and oxidative stress to potentiate antitumor immune responses in PDAC. Our lab previously found that PDAC cells are dependent on the antioxidant peroxiredoxin-4 (PRDX4) expression. PRDX4 prevents excessive levels of reactive oxygen species in cells and while high PRDX4 expression is associated with a more aggressive disease, it is dispensable in normal tissue. As such, disrupting redox homeostasis by inhibiting PRDX4 represents a targetable vulnerability in cancer cells. To investigate the radio-sensitizing potential of PRDX4, we knocked-down (KD) PRDX4 in human pancreatic tumor cells as well as in mouse cancer cells obtained from the spontaneous KPC model (driven by the KRas and P53 mutations), in combination with radiation. Presence and quantification of DNA damage were performed by immunofluorescence, and impaired cell survival was analyzed by clonogenic assays. Changes in immune secretion were assessed by RT-qPCR and Elisa and in vitro macrophage phenotyping was performed upon exposure to tumor cell conditioned media. Our data so far show an accumulation of cytosolic DNA upon PRDX4 KD in PDAC cells, with the presence of micronuclei as well as less aggregated DNA. Interestingly, targeting PRDX4 before radiation amplifies the release of radiation-induced cytosolic DNA while significantly decreasing cell growth and survival, compared to radiation alone. Cytosolic DNA has been shown to trigger innate immune signaling in tumor cells. Consistently, PRDX4 loss induces the upregulation of many pro-inflammatory genes, including CCL5, CCL20, and CXCL10, in a STING-dependent manner. Combining PRDX4 KD with radiation leads to a higher gene upregulation of these immune stimulatory chemokines, as well as an increased protein secretion within tumor cell media. These findings support an amplified immunomodulatory effect of radiation when combined with PRDX4 loss in PDAC cells. To assess whether targeting PRDX4 can remodel the tumor microenvironment, we applied tumor conditioned media from cancer cells on isolated naive mouse CD45+/CD11c+/F4/80+ macrophages. Media from cells treated with PRDX4 KD + radiation strongly increased the migration ability of macrophages while modulating the expression of a subset of chemokine-encoding genes in immune cells. Using the spontaneous KPC model depleted for PRDX4, along with syngeneic orthotopic models, we are now aiming to confirm this radio-sensitizing and immune-modulatory potential of PRDX4 in vivo. The aggressiveness of PDAC requires research towards novel treatment strategies and our data strongly support evidence for targeting cancer metabolism, particularly antioxidant systems, in synergy with existing cancer therapies, like radiation and immune therapy. Citation Format: Lucie Malbeteau, Emily Poulton, Vishal Pandya, Marianne Koritzinsky. Disrupting redox homeostasis initiates anti-tumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A022.

Abstract PR015: Sepsis-induced inflammation alters natural killer cell-mediated surveillance of liver metastasis

Abstract A majority of pancreatic ductal adenocarcinoma (PDAC) patients who undergo surgery for primary tumor resection will develop lethal metastases, despite having no evidence of metastasis at the time of their initial diagnosis. Liver metastases constitute nearly half of recurrences detected within the first six months following PDAC resection. Several clinical and experimental studies provide evidence that perioperative inflammation, including infectious complications like sepsis, positively correlates with liver metastasis across several malignancies. Despite these observations, it is still unknown whether there is a causal link between perioperative inflammation and PDAC liver metastasis. To gain mechanistic insight into the regulation of PDAC liver metastasis in the context of perioperative inflammation, we established mouse models that combine experimental PDAC liver metastasis with either the cecal ligation and puncture (CLP) model of polymicrobial sepsis or the lipopolysaccharide (LPS) model of acute sterile endotoxemia. Mice challenged with CLP prior to seeding PDAC cells in the liver had increased metastatic burden at endpoint compared to sham laparotomy controls. In contrast, mice challenged with LPS prior to PDAC cell seeding had reduced metastatic burden at endpoint compared to PBS-injected controls. We hypothesized that these opposing pro- and anti-metastatic effects of CLP and LPS, respectively, were due to qualitatively distinct inflammatory responses elicited in the liver. Our profiling of the host response to either CLP or LPS revealed shared systemic changes, including activation of the acute-phase protein response. However, LPS-associated inflammation uniquely upregulated a distinct set of interferon-regulated genes in the liver important for modifying immune cell activity (i.e., Ccl2, Ccl5, Cxcl9, Cxcl10). Accordingly, immunostaining and flow cytometry of the liver revealed that LPS uniquely increased the abundance of activated natural killer (NK) cells and MPO+ myeloid cells. We utilized Tlr4 -/- mice, which lack the primary receptor for LPS, to determine that the anti-metastatic effect of LPS required a host-facilitated response downstream of TLR4-mediated signaling. Through a series of genetic models and short-term depletion experiments, we determined that NK cells restrict PDAC cell seeding and outgrowth in our experimental liver metastasis model, and that this protective effect is further enhanced by activation of type-I interferon signaling in the host. Our findings confirm prior work identifying NK cell-mediated surveillance of disseminated cancer cells as an important mechanism in protecting against liver metastasis and show sterile endotoxemia and its associated inflammation establish a protective ‘anti-metastatic niche’ marked by enhanced NK cell activity. Further insights into the establishment of this niche may reveal potential therapeutic strategies to reduce the incidence of liver metastasis among resected PDAC patients. Citation Format: Nicole Sivetz, Patrick J. Cunniff, Christopher R. Vakoc, Semir Beyaz, Mikala Egeblad. Sepsis-induced inflammation alters natural killer cell-mediated surveillance of liver metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR015.

Abstract C004: Glucocorticoids establish a metastatic-promoting tumor microenvironment in a PDAC mouse model

Abstract Disseminated cancer cells (DCCs) arriving to a new organ may either proliferate and form metastasis, get eliminated by immune cells, or enter a quiescent stage. The factors that determine the DCCs’ fate, including getting quiescent DCCs to re-initiate proliferation and successfully evade the immune system, remain poorly understood, in part due to the lack of robust mouse models. We established a pancreatic ductal adenocarcinoma (PDAC) mouse model of dormant DCCs in the liver. An “immunization protocol” was used to mimic PDAC resection in patients: KPC1199 PDAC cells were injected to establish primary tumors and allow an adaptive immune response to develop against the cancer cells; the tumors were surgically resected; and new KPC1199 cells were intrasplenically injected to establish experimental liver metastases. The overwhelming majority of the KPC1199 cells were eliminated. No metastases spontaneously formed during 20 months of observation, but about 3-8 sporadic, single DCCs were found per liver cross section, and 94% of these DCCs were negative for proliferation markers. Although the model is based on entraining an adaptive immune response, T cell depletion resulted in only 25-30% of mice developing just 1-3 metastases each. This suggests that T cell depletion results in metastasis from the small percentage of DCCs that were proliferating. The DCC-hosting mice therefore represents of model to identify factors that can trigger further DCC proliferation and metastatic recurrence. Patients with PDAC often experience significant stress, leading to excess of endogenous glucocorticoids (GCs). Additionally, synthetic GCs are used during e.g., chemotherapy to manage side effects. To mimic elevated GC levels in patients, we treated DCC-hosting mice with synthetic GCs resulting in an increased percentage of proliferating DCCs (increasing from <6% of the examined cells in control mice to about 36%). Yet, no metastases formed. GC treatment also decreased T cell and increased neutrophil liver infiltration. Neutrophils can form protease-containing neutrophil extracellular traps (NETs), which we previously showed trigger DCC proliferation by proteolytic remodeling of the extracellular matrix protein laminin. We found that neutrophils from GC-treated mice spontaneously formed more NETs, and additionally, NET-generated laminin remodeling was observed in the liver metastasis. Together, the data suggest, that GC treatment drive quiescent DCCs to proliferate via neutrophils but alone this is insufficient to cause metastases. Consistent with this interpretation, T cell depletion combined with GC treatment resulted in multiple metastases in all mice examined. Of note, deleting the GC receptor in the DCCs did not reduce metastases, supporting that GCs act on the host and not the cancer cells. In conclusion, our data suggest that for quiescent DCCs to form metastases, they need signals that trigger proliferation and simultaneously, they must overcome immune surveillance. Elevated GC levels or GC treatment in immune suppressed individuals represents such a scenario. Citation Format: Xiao Han, Xueyan He, Yuan Gao, Sicheng Pan, Mikala Egeblad. Glucocorticoids establish a metastatic-promoting tumor microenvironment in a PDAC mouse model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C004.

Abstract PR007: Pancreatic cancer cachexia is mediated by tumor-derived PTHrP

Abstract Purpose: Our prior work has established that metastasis is initiated by PTHrP-driven mechanisms in pancreatic ductal adenocarcinoma (PDAC). PTHLH (the gene encoding the PTHrP protein) is directly adjacent to and co-amplified along with KRAS, the major oncogenic driver in PDAC patients. The KRAS-PTHLH amplicon is a marker of the highly aggressive squamous/quasi-mesenchymal/basal-like PDAC patient subtypes, is associated with increases in metastasis and cancer cachexia, and correlates with decreased overall patient survival. We have deleted Pthlh in the autochthonous Kras- and Tp53-driven pancreatic cancer mouse model (i.e. KPC mice) and found that the resulting KPC-Pthlh LoxP mice (herein KPC-PthrpcKO) live nearly twice as long as KPC controls. Intriguingly, recent evidence has emerged for PTHrP’s role in cachexia-associated adipose tissue wasting and we posit that the dramatic survival extension in KPC-PthrpcKO mice may be due to reduced cachexia. Results: In PDAC patients, PTHrP is co-amplified along with KRAS and correlates with significantly decreased overall survival. We generated KPC-PthrpcKO mice and showed that they have reduced tumor burden and dramatically increased overall survival relative to KPC controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly extended survival. Upon further analysis, we observed that the overall body condition of KPC-PthrpcKO mice (and anti- PTHrP treated KPC mice) was greatly improved, with less loss of adipose and muscle tissue. Mechanistic studies revealed that tumor cell-derived PTHrP signaling to adipocytes in white adipose tissue depots mediates cachexia. Specifically, we found that adipose tissue wasting and lipolysis were greatly reduced upon deletion or pharmacological inhibition of PTHrP. In the same vein, RNA-seq of cachectic adipose tissue revealed that PTHrP mediates adipose wasting by turning off de novo lipogenesis (DNL), a process critical for the generation of fatty acids in adipocytes. PTHrP binds to its cognate receptor, PTH1R, on adipocytes and blocks fatty acid synthesis by turning off the essential DNL transcription factors CHREBP and SREBP, likely through a PTH1R-PKA-CREB1 signaling axis. Thus, the genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia observed upon orthotopic implantation, leading to a reduction in overall survival. Therefore, PTHrP is both necessary and sufficient to induce cachexia in pancreatic cancer. Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving pancreatic cancer cachexia and adipose tissue remodeling, and future studies will look to translate anti-PTHrP therapy into clinical trials. Citation Format: Jason Pitarresi. Pancreatic cancer cachexia is mediated by tumor-derived PTHrP [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR007.

Abstract B009: Characterization and CRISPR perturbation of cancer-associated fibroblast heterogeneity in vitro models

Abstract Cancer-associated fibroblasts (CAFs) are attractive therapeutic targets for cancer due to their abundance in the tumor microenvironment (TME) and extensive pro- and antitumorigenic functions. However, formulating CAF-targeting strategies remains challenging due to the complexity of CAF biology. CAFs specialize into phenotypically and functionally distinct subtypes that may respond differently to specific treatments. Thus, designing CAF-targeting therapies necessitates a thorough understanding of CAF heterogeneity and preclinical model systems that can reliably prototype CAF biology observed in tumors. However, CAF heterogeneity is still not well-understood for many solid tumors, including the stroma-rich pancreatic ductal adenocarcinoma (PDAC), and the translatability of stroma models has not been comprehensively evaluated. In this work, we developed in vitro cell lines of tumor-CAF cocultures using primary CAFs derived from PDAC and used single-cell transcriptomics and CRISPR perturbation to investigate CAF heterogeneity and subtype-specific responses. We found that coculturing primary CAFs with the BxPC3 PDAC cell line could capture the tumor-CAF crosstalks underlying CAF activation. We characterized eight subtypes, including myofibroblastic, inflammatory, extracellular matrix (ECM)-like, and interferon CAFs, which could be preserved with immortalization. We also delineated the trajectories of CAF subtype interconvertibility, highlighting inflammatory CAFs as the likely initial diseased state and ECM CAFs as a “reservoir” that seeded the later states. By correlating the in vitro subtypes with clinical data of immunotherapy responses, we established that the in vitro cell lines could model the immuno-modulatory and immunoresistant phenotypes of CAFs. Finally, using an immortalized tumor-CAF coculture line, we performed Perturb-seq single-cell perturbation to perturb several stroma genes of interest, including TGFBR1, SPATS2L, AEBP1, and PTGS1, and evaluated the subtype-specific effects of the perturbations. We found that perturbing TGFBR1 resulted in the expected eradication of myofibroblastic CAFs, while the other perturbations resulted in subtype-specific modulations of metabolic and extracellular matrix related pathways. Overall, our findings underscored that in vitro tumor-CAF coculture models could serve as a translatable system that effectively captured CAF biology and heterogeneity observed in tumors and highlighted some similarities between PDAC CAFs and previously characterized breast cancer CAFs. We also demonstrated that single-cell perturbation could be a useful approach for interrogating the distinct responses of different CAF subtypes to perturbations. Citation Format: Elysia Saputra, Shamsudheen Karuthedath Vellarikkal, Lixia Li, Hong Sun, Suchitra Natarajan, Federica Piccioni, Alex M. Tamburino, Xin Yu, Aleksandra K. Olow. Characterization and CRISPR perturbation of cancer-associated fibroblast heterogeneity in vitro models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B009.

HPB SO33 - Transcriptomic characterisation of the diabetogenic pancreatic ductal adenocarcinoma phenotype

Abstract Background New-onset diabetes mellitus (NOD) can manifest as a consequence of pancreatic ductal adenocarcinoma (PDAC), although not all PDAC patients develop NOD. For many patients, hyperglycaemia begins 3 years prior to PDAC diagnosis, with 30% of PDAC patients experiencing NOD by the time their cancer is diagnosed. The mechanisms underlying the onset of diabetes mellitus (DM) in PDAC and its consequences for disease development, detection and treatment are not well understood. The aim of this study was to characterise the transcriptomic differences between PDAC tumours that present with NOD compared to those that do not. Method Eight tumour samples from patients undergoing surgery for PDAC (4 without DM (HbA1c <35), 4 with NOD (HbA1c >65, within 3 years of cancer diagnosis) underwent single cell-RNA sequencing (scRNAseq) after taking intra-operative biopsies using Chromium 10X Genomics technology. Unique cell clusters were identified using CellRanger and cell types annotated using scATOMIC and singleR datasets. Manual annotation was undertaken when results were not agreeable after consulting the literature. Differential expression between groups was performed using DESeq2. Significant genes were identified as having a log2 fold-change >1.5 and p<0.05. Gene set enrichment was performed using Reactome. Analysis was conducted in RStudio. Results Single cell suspensions were created with a mean cell count of 1.2 million cells and a mean viability of 90%. PDAC cells and the full complement of tumour microenvironmental cells were recovered. Significant differential expression was seen in the PDAC cells themselves, CD4+ cells, macrophages, mast cells and fibroblasts. Cancer associated fibroblasts (CAFs) seemed not to differ. PDAC cells with NOD seemed to be enriched in MYC oncogenes whereas those without DM seemed to be enriched in KRAS mutations. There was a mixture of “basal” and “classical” transcriptional subtypes between the two groups. Conclusion Differences between diabetogenic PDAC and non-diabetogenic PDAC have been observed at the transcriptional level. Diabetogenic tumours seem to be promoted by MYC oncogenes and be more inflammatory than non-diabetogenic tumours. These findings require validation which will be done on independent samples using spatial transcriptomics to allow endocrine-exocrine crosstalk to be determined. Understanding the molecular pathways in diabetogenic pancreatic tumours could enhance future strategies to detect PDAC earlier and to target diabetes-associated vulnerabilities for therapeutic purposes.

A collagenase-decorated Cu-based nanotheranostics: remodeling extracellular matrix for optimizing cuproptosis and MRI in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), characterized by a dense extracellular matrix (ECM), presents significant therapeutic challenges due to its poor prognosis and high resistance to chemotherapy. Current chemodrugs and diagnostic agents largely fail to cross the barrier posed by the ECM, which severely limits the PDAC theranostics. This study introduces a novel theranostic strategy using thioether-hybridized hollow mesoporous organosilica nanoparticles (dsMNs) for the co-delivery of copper (Cu) and disulfiram (DSF), aiming to induce cuproptosis in PDAC cells. Our approach leverages the ECM-degrading enzyme collagenase, integrated with dsMNs, to enhance drug penetration by reducing matrix stiffness. Furthermore, the innovative use of a pancreatic cancer cell membrane coating on the nanoparticles enhances tumor targeting and stability (dsMCu-D@M-Co). The multifunctional platform not only facilitates deep drug penetration and triggers cuproptosis effectively but also utilizes the inherent properties of Cu to serve as a T1-weighted magnetic resonance imaging (MRI) contrast agent. In vitro and in vivo assessments demonstrate significant tumor size reduction in PDAC-bearing mice, highlighting the dual functionality of our platform in improving therapeutic efficacy and diagnostic precision. This integrated strategy represents a significant advancement in the management of PDAC, offering a promising new direction for overcoming one of the most lethal cancers.Graphical

The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease soon to become the second leading cause of cancer deaths in the US. Beside surgery, current therapies have narrow clinical benefits with systemic toxicities. FOLFIRINOX is the current standard of care, one component of which is 5- Fluorouracil (5-FU), which causes serious gastrointestinal and hematopoietic toxicities and is vulnerable to resistance mechanisms. Recently, we have developed polymeric fluoropyrimidines (F10, CF10) which unlike 5-FU, are, in principle, completely converted to the thymidylate synthase inhibitory metabolite FdUMP, without generating appreciable levels of ribonucleotides that cause systemic toxicities while displaying much stronger anti-cancer activity. Here, we confirm the potency of CF10 and investigate enhancement of its efficacy through combination with inhibitors in vitro targeting replication stress, a hallmark of PDAC cells. CF10 is 308-times more potent as a single agent than 5-FU and was effective in the nM range in primary patient derived models. Further, we find that activity of CF10, but not 5-FU, is enhanced through combination with inhibitors of ATR and Wee1 that regulate the S and G2 DNA damage checkpoints and can be reversed by addition of dNTPs indicative of CF10 acting, at least in part, through inducing replication stress. Our results indicate CF10 has the potential to supersede the established benefit of 5-FU in PDAC treatment and indicate novel combination approaches that should be validated in vivo and may be beneficial in established regimens that include 5-FU.

MGST1 facilitates novel KRASG12D inhibitor resistance in KRASG12D-mutated pancreatic ductal adenocarcinoma by inhibiting ferroptosis

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a low 5-year survival rate. Treatment options for PDAC patients are limited. Recent studies have shown promising results with MRTX1133, a KRASG12D inhibitor that demonstrated potent antitumor activity in various types of tumors with KRASG12D mutation. Resistance to KRAS inhibitors is frequently occurred and one of the main reasons for treatment failure. Understanding resistance mechanisms to novel KRAS inhibitors is crucial to ensure sustained and durable remissions.MethodsTwo KRASG12D inhibitor MRTX1133-resistant PDAC cell lines were established in vitro. The resistance mechanisms to KRASG12D inhibitor MRTX1133 against PDAC in vitro and in vivo were characterized by RNA sequencing, reverse transcript polymerase chain reaction, cytotoxicity test, plasmid transfection, lentivirus transfection, lipid peroxidation detection, malondialdehyde levels detection, glutathione levels detection, western blot, immunofluorescence, nude mice tumorigenesis experiment and immunohistochemistry.ResultsThe bioinformatics analysis and transcriptome sequencing showed that ferroptosis was involved in the resistant effect of the KRASG12D inhibitor treatment, and MGST1 was the key molecule against MRTX1133-induced ferroptosis. Increased expression of MGST1 weakened the cytotoxicity of MRTX1133 by inhibiting lipid peroxidation-induced ferroptosis in KRASG12D inhibitor-resistant PDAC cells. Knockdown or overexpression of MGST1 conferred sensitivity or resistance to KRASG12D inhibitor MRTX1133, respectively. Mechanismly, increased nuclear localization and higher levels of active β-catenin were observed in MRTX1133-resistant PDAC cells, which contributed to higher MGST1 expression. Knockdown of CTNNB1 or TCF4 can decreased MGST1 expression. Additionally, we found that PKF-118-310, an antagonist of β-catenin/Tcf4 complex, repressed MGST1 expression. In both in vitro and in vivo models, a synergistic effect was observed when combining MRTX1133 and PKF-118-310 in KRASG12D inhibitor MRTX1133-resistant PDAC cells and tumors.ConclusionOur data showed that KRASG12D inhibitor MRTX1133 combined with PKF-118-310 could enhance the effectiveness of MRTX1133 treatment response through induction of ferroptosis via inhibiting MGST1 expression in MRTX1133-resistant PDAC cells and tumors. This evidence may provide a promising strategy to overcome KRASG12D inhibitor MRTX1133 resistance in PDAC patients with KRASG12D mutations.Graphical

Endosomal Trafficking Bypassed by the RAB5B-CD109 Interplay Promotes Axonogenesis in KRAS-Mutant Pancreatic Cancer.

Perineural invasion (PNI) represents a unique biological feature associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC), especially in the presence of KRAS mutations. Extracellular vesicle (EV)-packaged circular RNAs (circRNAs) function as essential mediators of tumor microenvironment communication, triggering PDAC cell invasion and distant metastasis. However, the regulatory mechanisms of EV-packaged circRNAs in the PNI of KRAS-mutant PDAC have not yet been elucidated. Herein, a KRASG12D mutation-responsive EV-packaged circRNA, circPNIT, which positively correlated with PNI in PDAC patients is identified. Functionally, KRASG12D PDAC-derived EV-packaged circPNIT promoted axonogenesis and PNI both in vitro and in vivo. Mechanistically, the circPNIT-mediated Rab5B-CD109 interplay bypassed traditional endosomal trafficking to anchor Rab5B to the lipid rafts of multivesicular bodies and packaged circPNIT into CD109+ EVs. Subsequently, CD109+ EVs delivered circPNIT to neurons by binding to TRPV1 and facilitating DSCAML1 transcription-induced axonogenesis, which in turn enhanced the PNI by activating the GFRα1/RET pathway. Importantly, circPNIT-loaded CD109+ EVs are established to dramatically promote PNI in a KRASG12D/+ Trp53R172H/+ Pdx-1-Cre mouse model. Collectively, the findings highlight the mechanism underlying how EV-packaged circRNAs mediate the PNI of KRAS-mutant PDAC cells through the Rab5B endosomal bypass, identifying circPNIT as an effective target for the treatment of neuro-metastatic PDAC.

Epigenetic Activation of the CMTM6-IGF2BP1-EP300 Positive Feedback Loop Drives Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a dismal prognosis. Gemcitabine-based chemotherapy has emerged as a first-line treatment for PDAC. However, the development of gemcitabine resistance often results in therapeutic failure. In order to uncover the underlying mechanisms of gemcitabine resistance, gemcitabine-resistant PDAC cell lines and patient-derived xenograft (PDX) models are established and subjected to RNA sequencing. It is found that CMTM6 is closely related to gemcitabine resistance in PDAC. Multi-omics analysis revealed that EP300-mediated H3K27ac modification is involved in the transcriptional activation of CMTM6, which maintains IGF2BP1 expression by preventing its ubiquitination. The m6A reader IGF2BP1 stabilizes the EP300 and MYC mRNAs by recognizing m6A modifications, forming a positive feedback loop that enhances tumor stemness and ultimately contributes to PDAC resistance. The combined application of the EP300 inhibitor inobrodib and gemcitabine exerts a synergistic effect on PDAC. Overall, these findings reveal that the EP300-CMTM6-IGF2BP1 positive feedback loop facilitates gemcitabine resistance via epigenetic reprogramming and the combined use of inobrodib and gemcitabine represents a promising strategy for overcoming chemoresistance in PDAC, warranting further investigation in clinical trials.

The Role of Dicer Phosphorylation in Gemcitabine Resistance of Pancreatic Cancer.

Dicer, a cytoplasmic type III RNase, is essential for the maturation of microRNAs (miRNAs) and is implicated in cancer progression and chemoresistance. Our previous research demonstrated that phosphorylation of Dicer at S1016 alters miRNA maturation and glutamine metabolism, contributing to gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on the role of Dicer phosphorylation at S1728/S1852 in GEM-resistant PDAC cells. Using shRNA to knock down Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells, we examined cell viability through MTT and clonogenic assays. We also expressed phosphomimetic Dicer 2E (S1728E/S1852E) and phosphomutant Dicer 2A (S1728A/S1852A) to evaluate their effects on GEM resistance and metabolism. Our results show that phosphorylation at S1728/S1852 promotes GEM resistance by reprogramming glutamine metabolism. Specifically, phosphomimetic Dicer 2E increased intracellular glutamine, driving pyrimidine synthesis and raising dCTP levels, which compete with gemcitabine's metabolites. This metabolic shift enhanced drug resistance. In contrast, phosphomutant Dicer 2A reduced GEM resistance. These findings highlight the importance of Dicer phosphorylation in regulating metabolism and drug sensitivity, offering insights into potential therapeutic strategies for overcoming GEM resistance in pancreatic cancer.

TGF-β Signaling Loop in Pancreatic Ductal Adenocarcinoma Activates Fibroblasts and Increases Tumor Cell Aggressiveness.

The interaction between cancer cells and cancer-associated fibroblasts (CAFs) is a key determinant of the rapid progression, high invasiveness, and chemoresistance of aggressive desmoplastic cancers such as pancreatic ductal adenocarcinoma (PDAC). Tumor cells are known to reprogram fibroblasts into CAFs by secreting transforming growth factor beta (TGF-β), amongst other cytokines. In turn, CAFs produce soluble factors that promote tumor-cell invasiveness and chemoresistance, including TGF-β itself, which has a major role in myofibroblastic CAFs. Such a high level of complexity has hampered progress toward a clear view of the TGFβ signaling loop between stromal fibroblasts and PDAC cells. Here, we tackled this issue by using co-culture settings that allow paracrine signaling alone (transwell systems) or paracrine and contact-mediated signaling (3D spheroids). We found that TGF-β is critically involved in the activation of normal human fibroblasts into alpha-smooth muscle actin (α-SMA)-positive CAFs. The TGF-β released by CAFs accounted for the enhanced proliferation and resistance to gemcitabine of PDAC cells. This was accompanied by a partial epithelial-to-mesenchymal transition in PDAC cells, with no increase in their migratory abilities. Nevertheless, 3D heterospheroids comprising PDAC cells and fibroblasts allowed monitoring the pro-invasive effects of CAFs on cancer cells, possibly due to combined paracrine and physical contact-mediated signals. We conclude that TGF-β is only one of the players that mediates the communication between PDAC cells and fibroblasts and controls the acquisition of aggressive phenotypes. Hence, these advanced in vitro models may be exploited to further investigate these events and to design innovative anti-PDAC therapies.

Discovery of novel TANK-Binding Kinase 1 (TBK1) inhibitor against pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options, underscoring the urgent need for developing new therapies. The upregulation of TBK1 activity plays a crucial role in multiple pancreatic cancer-related signaling pathways, suggesting that inhibiting the kinase activity of TBK1 could be a promising strategy. Herein, we discovered a novel TBK1 inhibitor, LIB3S0280, using a structure-based virtual screening (SBVS) strategy. In the anti-proliferative and viability assays, LIB3S0280 showed significant inhibition against pancreatic cancer cell lines that highly express TBK1 with the GI50 values of 2.24 and 4.71 μM and IC50 values of 6.64 and 10.98 μM at 96 h. For the downstream targets, LIB3S0280 can inhibit TBK1 downstream signaling by decreasing the phosphorylation of IκBα and AKT better than a known TBK1 inhibitor, BX-795. Furthermore, PDAC cells were arrested in G2/M and underwent apoptosis or senescence with the treatment of LIB3S0280. These findings suggest that TBK1 inhibitor LIB3S0280 has great potential as a lead compound in the further development of a novel treatment for PDAC.

Engineering of a Low Intrinsic Fluorescence and Chemical-Stable Fluorescent Probe Enables Highly Sensitive Detection of Biothiols and High-Fidelity Imaging of Dihydroartemisinin-Induced Ferroptosis

Ferroptosis modulation represents a novel therapeutic strategy for pancreatic ductal adenocarcinoma (PDAC), where precise control of ferroptotic levels is imperative for optimizing treatment efficacy. Dihydroartemisinin (DHA) has emerged as a promising candidate in PDAC therapy, potentially through its ability to induce ferroptosis and thereby enhance the synergistic effects of PDAC treatments. However, the mechanism study of DHA-induced ferroptosis is limited by the lack of accredited biomarkers and the absence of powerful fluorescent probe with good stability towards reactive oxygen species (ROS) and low intrinsic fluorescence, which is necessary for high-fidelity and sensitive monitoring ferroptosis in PDAC. In this innovative study, we engineered a novel fluorescent probe (BOD-NBD), characterized by minimal intrinsic fluorescence and exceptional stability against ROS, enabling highly sensitive and accurate detection and imaging of biothiols in PDAC. The probe achieved high signal-to-back ratio, high sensitivity and high specificity response to biothiols. Using this probe, significant depletion of biothiols in PDAC cells during ferroptosis was observed. The probe was further successfully applied for monitoring DHA-induced ferroptosis in Panc02 cells with reliable imaging results. Moreover, for the first time, we observed an interesting experimental finding that joint use of erastin and DHA or cisplatin and DHA could exacerbate the consumption of biothiols, which may imply an aggravation of ferroptosis in PDAC, demonstrating great significance in the development of novel therapeutic schedule for the treatment of PDAC.

The Far Side of Resistance to RAS Inhibitors.

In this issue, four articles highlight the critical role of nongenetic mechanisms and cell plasticity in mediating resistance to different classes of RAS inhibitors in pancreatic ductal adenocarcinoma and non-small cell lung cancer. See related article by Benitz et al., p. 2162 See related article by Dilly et al., p. 2135 See related article by Araujo et al., p. 2183 See related article by Singhal et al., p. 2122.

Genome-wide microRNA Analysis Identified miR-210-3p Over-expression in Pancreatic Cancer Tissues as a Predictor of their Local Invasiveness.

The severe malignancy of pancreatic ductal adenocarcinoma (PDAC) is mainly due to frequent local invasiveness and distant metastasis. As for local invasiveness, we previously reported that cancer-specific molecular alterations detected on resected PDAC specimen surfaces, so-called molecular surgical margin (MSM) positiveness, were significantly associated with postoperative locoregional recurrence and distant metastasis. However, due to anatomical limitations, achieving adequate surgical margins during pancreatic cancer resection is often challenging. Therefore, predicting local invasiveness based on the primary tumor's gene profile is crucial to avoid positive MSM. Genome-wide miRNA expression profiles were examined and compared between MSM-positive and negative cases. Candidate miRNAs were evaluated in another validation cohort, and their clinicopathological characteristics were examined. Mimic or inhibitor constructs of the candidate miRNA were transfected to PDAC cell lines to evaluate the miRNA function in the pancreatic cancer cell lines and detect the downstream targets. Among some candidates with highly expressed miRNAs in MSM-positive cases by miRNA expression array, recurrence-free survival (RFS) was significantly shorter in the miR-210-3p high expression group (p=0.015). High miR-210-3p was significantly associated with large tumor diameter (p=0.001), anterior invasion positive (p=0.010), and positive lymph node metastasis (p<0.001). miR-210-3p inhibition in PDAC cell lines resulted in decreased proliferation and invasiveness. The iron-sulfur cluster assembly enzyme (ISCU) gene was identified as a target of miR-210-3p. ISCU reduction was significantly observed in PDAC primary tumors with high levels of miR-210-3p, leading to mitochondrial dysfunction in miR-210-3p-overexpressing PDAC cell lines, as demonstrated by glycolysis stress tests. Highly expressed hypoxia-inducible miR-210-3p in primary PDAC tissues induces locally invasive characteristics through mitochondrial dysfunction by suppressing ISCU expression, which may result in poor postoperative RFS outcomes.

Lymphocyte Antigen 6 Family Member D (LY6D) Affects Stem Cell Phenotype and Progression of Pancreatic Adenocarcinoma.

The membrane-bound protein lymphocyte antigen 6 family member D (LY6D), a marker of early B cell lineage is reportedly expressed in several human malignancies and has been implicated in cancer stemness. However, its expression and role in cancer stemness remain largely unexplored in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to clarify the role of LY6D in PDAC. We conducted functional analysis of LY6D to evaluate its impact on the malignant features of PDAC cells in vitro. Using our in-house developed stem cell separation technique, which isolates cells with low proteasome activity and CD44 v9 cell surface marker for cancer stem cells, we performed sphere formation and chemosensitivity tests and tumor formation assay in mice, through knockdown of LY6D expression. Immuno-histopathological analysis was also conducted to reveal the clinical significance of LY6D in PDAC. In vitro functional assays demonstrated that LY6D was critically involved in promoting the cancer malignant phenotype, including increased invasive ability, drug resistance, migration capacity, and cancer stemness. Immunohistopathological analysis revealed that high LY6D expression levels were associated with high recurrence rates and poorer prognosis in PDAC. Our study showed that LY6D is a novel prognostic indicator and plays a key role in regulation of cancer stemness in PDAC.

The Role of Heat Shock Factor 1 in Preserving Proteomic Integrity During Copper-Induced Cellular Toxicity.

Copper is crucial for many physiological processes across mammalian cells, including energy metabolism, neurotransmitter synthesis, and antioxidant defense mechanisms. However, excessive copper levels can lead to cellular toxicity and "cuproptosis", a form of programmed cell death characterized by the accumulation of copper within mitochondria. Tumor cells are less sensitive to this toxicity than normal cells, the mechanism for which remains unclear. We address this important issue by exploring the role of heat shock factor 1 (HSF1), a transcription factor that is highly expressed across several types of cancer and has a crucial role in tumor survival, in protecting against copper-mediated cytotoxicity. Using pancreatic ductal adenocarcinoma cells, we show that excessive copper triggers a proteotoxic stress response (PSR), activating HSF1 and that overexpressing HSF1 diminishes intracellular copper accumulation and prevents excessive copper-induced cell death and amyloid fibrils formation, highlighting HSF1's role in preserving proteasomal integrity. Copper treatment decreases the lipoylation of dihydrolipoamide S-acetyltransferase (DLAT), an enzyme necessary for cuproptosis, induces DLAT oligomerization, and induces insoluble DLAT formation, which is suppressed by overexpressing HSF1, in addition to enhancing the interaction between HSF1 and DLAT. Our findings uncover how HSF1 protects against copper-induced damage in cancer cells and thus represents a novel therapeutic target for enhancing copper-mediated cancer cell death.

CCL3 predicts exceptional response to TGFβ inhibition in basal-like pancreatic cancer enriched in LIF-producing macrophages

The TGFβ receptor inhibitor galunisertib showed promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the phase 2 H9H-MC-JBAJ study. Identifying biomarkers for this treatment remains essential. Baseline plasma levels of chemokine CCL3 were integrated with clinical outcomes in PDAC patients treated with galunisertib plus gemcitabine (n = 104) or placebo plus gemcitabine (n = 52). High CCL3 was a poor prognostic factor in the placebo group (mOS 3.6 vs. 10.1 months; p < 0.01) but a positive predictor for galunisertib (mOS 9.2 vs. 3.6 months; p < 0.01). Mechanistically, tumor-derived CCL3 activates Tgfβ signaling in macrophages, inducing their M2 phenotype and Lif secretion, sustaining a mesenchymal/basal-like ecotype. TGFβ inhibition redirects macrophage polarization to M1, reducing Lif and shifting PDAC cells to a more epithelial/classical phenotype, improving gemcitabine sensitivity. This study supports exploring TGFβ-targeting agents in PDAC with a mesenchymal/basal-like ecotype driven by high CCL3 levels.