Mouse Model Of Pancreatic Cancer Research Articles

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGFβ depletion

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGFβ depletion

Abstract B007: Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis due to two key features. The first is metastasis at presentation, occurring in approximately 80% of PDAC patients, with 90% of these metastases occurring in the liver. The second is cachexia, which compromises treatment tolerance and reduces the quality of life for patients. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. In this study, we optimized an orthotopic mouse model of PDAC by altering implantation sites, subcloning parental murine PDAC cells, adjusting the number of transplanted cells, and varying the age of recipient mice to study liver metastasis and cancer cachexia. These modifications significantly increased the latency of tumor development and the time course of the disease, concurrently increasing the rate of liver metastasis to approximately 70%. Furthermore, reliable cachexia endpoints were also achieved in both PDAC mice with and without metastases, mirroring the condition in patients. We also found that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. Together, this model is likely to be advantageous in both advancing our understanding of the mechanisms of PDAC cachexia and in evaluating novel therapeutics. Citation Format: David J Wang, Victoria Spadafora, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Micheal C Ostrowski, Denis C Guttridge. Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia [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 B007.

Abstract C001: Transforming Growth Factor-β Blockade in Pancreatic Cancer Enhances Sensitivity to Combination Chemotherapy in Mice

Abstract Background: TGFβ plays a critical role in promoting pancreatic tumor progression, attenuating CD8 T cell proliferation and cytokine production, enhancing the differentiation of myofibroblasts and the deposition of extracellular matrix. However, single agent TGFβ blockade has shown limited efficacy in mouse models of pancreatic cancer. Methods: We evaluated the TGFβ- blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)- paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment. Results: Blockade of TGFβ with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFβ blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2-/-) mice. TGFβ blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFβ blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFβ blockade increased chemotherapy-induced cell death in vivo. Conclusions: TGFβ regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFβ blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens. This study provided the scientific rationale for the evaluation of TGFβ with both chemotherapy backbones in the clinical setting. Ongoing evaluation of samples from the stopped clinical trials will inform as to the role of TGFb in human PDAC. Citation Format: Li Qiang, Megan Hoffman, Lestat Ali, Jaime Ivan Castillo Silva, Lauren Kageler, Ayantu Temesgen, Patrick Lenehan, Jennifer Wang, Elisa Bello, Victoire Cardot-Ruffino, Giselle Uribe, Annan Yang, Michael Dougan, Andrew Aguirre, Srivatsan Raghavan5, Marc Pelletier, Viviana Cremasco, Stephanie Dougan. Transforming Growth Factor-β Blockade in Pancreatic Cancer Enhances Sensitivity to Combination Chemotherapy in Mice [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 C001.

Abstract A066: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression. Citation Format: Audrey M Hendley, Sudipta Ashe, Atsushi Urano, Martin Ng, Tuan A Phu, Xianlu Laura Peng, Changfei Luan, Anna-Marie Finger, Gun Ho Jang, Natanya R Kerper, David I Berrios, David Jin, Jonghyun Lee, Irene R Riahi, Oghenekevwe M Gbenedio, Christina Chung, Jeroen P Roose, Jen Jen Yeh, Steven Gallinger, Andrew V Biankin, Grainne M O’Kane, Vasilis Ntranos, David K Chang, David W Dawson, Grace E Kim, Valerie M Weaver, Robert L Raffai, Matthias Hebrok. nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support 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 A066.

Abstract B041: Fibroblast STAT3 Signaling in Pancreatic Cancer: Implications for Immunotherapy and Tumor Microenvironment Modulation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer distinguished by a dense stromal microenvironment. The current investigation delves into the fibroblast-specific STAT3 signaling in the PDAC tumor microenvironment (TME) and its effects on immune cell infiltration and extracellular matrix (ECM) modulation. An association was identified between heightened levels of phosphorylated STAT3 (pSTAT3) in tumor-associated fibroblasts (CAFs) and a more unfavorable prognosis, particularly among male patients. Comparative analysis of gene expression patterns in normal fibroblasts and those deficient in STAT3, extracted from mouse models of pancreatic cancer, unveiled distinct gene expression profiles related to the inflammatory signaling pathway (IL6, ADM, CXCL11, CD80, VEGFA, TGFB1, STC1, STC2). Utilizing ChIP-Seq analysis with an anti-pSTAT3 antibody, it was confirmed that STAT3 binds to the regulatory regions of these genes, supporting its regulatory role in the inflammatory signaling pathway. Moreover, the absence of STAT3 in CAFs resulted in a decline in M2 macrophages and an upsurge in M1 macrophages within the TME, indicating a potential function of STAT3 in influencing macrophage polarization. Noteworthy observations also revealed that STAT3 signaling in CAFs impacts ECM remodeling, as illustrated by reduced collagen deposition. Furthermore, the deficiency of STAT3 in CAFs led to a notable reduction in the quantity of neutrophils within the TME in our mouse model. Additionally, a plausible involvement of STAT3 in neutrophil recruitment and activation through the CXCL1/CXCR2 axis was indicated. Alterations in the infiltration of neutrophils and macrophages due to the absence of STAT3 could profoundly influence the immune response against PDAC. The modified composition of ECM following STAT3 deletion might also impact the infiltration and functioning of immune cells. These outcomes underscore the significance of STAT3 signaling in CAFs as a promising therapeutic target in PDAC, offering potential for modulating the tumor microenvironment, augmenting anti-tumor immunity, and advancing patient outcomes. Citation Format: Samaneh Saberi, Cameron Bumbleburg, Caroline Everett, Julia E Lefler, Victoria Jordan, Lu Han, Sudarshana Sharma, Michael C. Ostrowski. Fibroblast STAT3 Signaling in Pancreatic Cancer: Implications for Immunotherapy and Tumor Microenvironment Modulation [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 B041.

Abstract B031: Alternate pain management strategies: targeting pancreatic cancer-related pain with Flavonoid

Abstract Purpose For up to 90% of cancer patients, pain is a substantial symptom, contributing to morbidity and, possibly, mortality. In this study we sought to test the hypothesis that a flavonoid, derived from Cannabis, and previously shown to suppress cancer cell growth in vitro and tumor progression in mice in vivo, could also reduce the pain caused by pancreatic tumors in vivo. Methods A subcutaneous tumor in the dorsum of C57BL/6 mouse was generated by sub- dermal injection of KPC mouse pancreatic cancer cell line (ATCC). All the mice were video recorded to observe and analyze their spontaneous pain-related behaviors, including facial washing, dorsal hunching, dorsal skin twitch, licking of dorsal skin and overall locomotion around the cage. Two five minute recording sessions were analyzed by a blinded observer during times before the cancer cell injection (Baseline), after tumor growth, to about 8mm diameter (30d and 37d post-injection) and just before and then 24h after intra-tumor injection of the flavonoid ( Group A: FBL-03G, 1 nmole in 100µL) or Vehicle(100µL; Group B). Pain-related behavioral changes were averaged from the two recording sessions and analyzed from Baseline, just before FBL-03G and 24h after and compared between test compound-and vehicle injected groups (n=7 in each), using non-parametric statistics. Results Facial washing (events/5min) was: reduced to 87% of Baseline in tumor-bearing mice, then increased by 150% at 24h post-injection in Group A (FBL-03G); reduced to 65% of Baseline in tumor-bearing mice of Group B, then reduced a further 9% by Vehicle. Dorsal hunching, which events occur only after tumor growth (Baseline =0), was reduced at 24h by 62% in Group A but increased by 75% in Group B. Dorsal twitch, also absent before tumor growth, was reduced by 64% in Group A, but increased by 19% in Group B. Dorsal skin licking was reduced from Baseline by 33% in tumor-bearing mice of Group A, then increased by 76% after FBL-03g injection whereas licking, reduced by 22% by tumors in Group B and then by an additional 4% by vehicle (Group B). Tumors reduced locomotion by 58% in Group A, which was reversed by 32% after FBL-03g (Group A); vehicle injection resulted in a 38% reduction in locomotion (Group B). Thus, in 4 of the 5 pain-related behaviors from tumors quantitated symptoms were reversed by FBL-03G and oppositely affected, or not at all, by Vehicle. Conclusions These results indicate a substantial relief of spontaneous pain-related pain in this mouse model of pancreatic cancer. Such an acute effect occurs much more rapidly than the previously published in vitro suppression of KPC cell growth (assayed after 9-12d of treatment) or the reduction of tumor size (1-7 weeks) in vivo and suggest that the anti-hyperalgesic affect is due to mechanisms that do not involve reduction in tumor mass. The findings, using a single dose and a small number of mice, appear promising for further studies that examine a range of doses or FBL-03G and its actions in intra-pancreatic tumors. Citation Format: Sayeda Yasmin-Karim, Suheera Haq, Mike Makrigiorgos, Gary Strichartz, Geraud Richards, Natasha Rayman. Alternate pain management strategies: targeting pancreatic cancer-related pain with Flavonoid [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 B031.

Abstract B004: Targeting Metabolic Vulnerabilities in Pancreatic Cancer: The Synergistic Anti- Tumor Effects of a Ketogenic Diet and IDH1 Inhibition

Abstract Background: A ketogenic diet, characterized by high fat (90% kCal) and low carbohydrates (5% kCal), induces ketosis. Previous research suggests that a ketogenic diet shifts cancer cell metabolism from glycolysis to the tricarboxylic acid (TCA) cycle in the mitochondria. While the overall impact on cancer growth remains uncertain, most studies suggest an anti-cancer effect. We hypothesize that understanding the molecular and biochemical adaptations of pancreatic cancer cells to a ketogenic diet will reveal metabolic vulnerabilities and lead to rational therapeutic strategies that complement the diet. Methods: Mice were fed a ketogenic diet, with control mice on a normal diet. Pancreatic cancer xenografts were monitored for growth and subjected to biochemical analyses. Tumor metabolites were measured using LC/GC-MS, and oxidative stress was assessed by NADP+/NADPH, NAD+/NADH levels, and lipid peroxidation assays. Enzyme expression was determined by Western blot. In vitro, mitochondrial function was analyzed using the Seahorse FX Analyzer, and cell growth was measured by PicoGreen DNA quantitation. Results: A ketogenic diet slowed pancreatic cancer growth in multiple in vivo models, including MIA-PaCa2, KPC, and patient-derived xenografts. TCA metabolites increased in xenografts exposed to the ketogenic diet, with a significant upregulation of TCA cycle-associated enzymes. Specifically, the expression of BDH1 (a ketolytic enzyme), IDH1 (supports antioxidant defense and mitochondrial function), and IDH2 (mitochondrial homolog of IDH1) were elevated. Conversely, the glycolytic enzyme G3P was reduced. Additionally, a ketogenic diet induced oxidative stress in pancreatic cancer in vivo. Combining a ketogenic diet with an IDH1 inhibitor impaired antioxidant defense and mitochondrial function, resulting in significantly reduced tumor proliferation compared to either treatment alone. In vitro studies indicated that low glucose and fatty acids primarily drive the reduced proliferation of pancreatic cancer cells on a ketogenic diet, while exogenous ketone bodies are utilized for energy under glucose deprivation, generally promoting tumor growth. Conclusion: A ketogenic diet exerts a strong anti-tumor effect in multiple pancreatic cancer mouse models, likely due to low glucose and increased fatty acid load. The metabolic shift towards an oxidative phosphorylation (OXPHOS) phenotype makes pancreatic cancer cells particularly vulnerable to antioxidant and mitochondrial inhibitors. Citation Format: Omid Hajihassani, Mehrdad Zarei, Soubhi Tahan, Peter Gallagher, William Beegan, John Speers, James Choi, Aya Murray, Alexander Loftus, Helen Cheng, Anusha Mudigonda, Karen Ji, Jonathan Hue, Hallie Graor, Jordan Winter. Targeting Metabolic Vulnerabilities in Pancreatic Cancer: The Synergistic Anti- Tumor Effects of a Ketogenic Diet and IDH1 Inhibition [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 B004.

Abstract A006: Generation of a flexible pancreatic cancer mouse model via somatic tissue engineering

Abstract Pancreatic cancer, recognized as one of the most lethal cancer types, exhibits a dismal five-year survival rate below 12%. The genetic and epigenetic heterogeneity in human cancer poses a tremendous challenge in understanding disease mechanisms and developing targeted therapeutics. Current mouse models in the field can recapitulate some features of human pathophysiology but have limitations. While tumor formation in patients is focal, most genetically engineered mouse models (GEMMs) express oncogenes throughout the pancreas. GEMMs also have limited flexibility to manipulate gene expression and model the heterogeneity of human cancer. Additionally, GEMMs require significant time for crossing and tumor formation. Tumor transplantation models offer greater flexibility, however, they often require immunodeficient mice, which limits their applications in modeling the tumor microenvironment. To overcome the limits of current mouse models, we combined the piggyBac transposon system, CRISPR/Cas9, and in vivo electroporation to engineer somatic pancreatic tissue to induce focal tumor formation in immune-competent mice. With this approach, we engineered wildtype pancreata to express oncogenic KrasG12D while knocking out multiple tumor suppressors. We optimized this protocol by generating multiplexed gRNA constructs and cell type-specific promoters to improve reproducibility. Low-grade tumors formed within 10 days after electroporation, progressed to higher grade within a month, and metastasized to peritoneal organs during disease progression. Mouse tumors also recapitulated histopathological features in human patients. Lastly, we used our novel mouse model to study how mutant Kras disrupts post-transcriptional regulation by microRNA to drive pancreatic tumorigenesis. This model offers a powerful platform for manipulating gene expression and functionality to model cancer heterogeneity, ultimately advancing precision oncology's prospects for treating human patients. Citation Format: Ziyue (Zoey) Yang, Mara Cardenas, Akihiko Miyauchi, Caroline Snider, Ronald Parchem. Generation of a flexible pancreatic cancer mouse model via somatic tissue engineering [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 A006.

Non-natural MUC1 Glycopeptide Homogeneous Cancer Vaccine with Enhanced Immunogenicity and Therapeutic Activity.

Glycopeptides derived from the glycoprotein mucin-1 (MUC1) have shown potential as tumor-associated antigens for cancer vaccine development. However, their low immunogenicity and non-selective conjugation to carriers present significant challenges for the clinical efficacy of MUC1-based vaccines. Here, we introduce a novel vaccine candidate based on a structure-guided design of an artificial antigen derived from MUC1 glycopeptide. This engineered antigen contains two non-natural amino acids and has an α-S-glycosidic bond, where sulfur replaces the conventional oxygen atom linking the peptide backbone to the sugar N-acetylgalactosamine. The glycopeptide is then specifically conjugated to the immunogenic protein carrier CRM197 (Cross-Reactive Material 197), a protein approved for human use. Conjugation involves selective reduction and re-bridging of a disulfide in CRM197, allowing the attachment of a single copy of MUC1. This strategy results in a chemically defined vaccine while maintaining both the structural integrity and immunogenicity of the protein carrier. The vaccine elicits a robust Th1-like immune response in mice and generates antibodies capable of recognizing human cancer cells expressing tumor-associated MUC1. When tested in mouse models of colon adenocarcinoma and pancreatic cancer, the vaccine is effective both as a prophylactic and therapeutic use, significantly delaying tumor growth. In therapeutic applications, improved outcomes were….

ZG16 enhances the maturation of dendritic cells via induction of CD40 and contributes to the antitumor immunity in pancreatic cancer.

Dendritic cells (DCs) are critical mediators of antigen priming and T-cell activation. Zymogen granule protein 16 (ZG16) is demonstrated as an anti-oncogene in T-cell mediated antitumor immunity, but its effect on DCs is largely unknown. Herein, we wonder whether ZG16 affects the activation of DCs in pancreatic cancer. Firstly, the increased ZG16 expression was observed during the maturation of DCs derived from mouse bone marrow or human peripheral blood. Then, overexpression of ZG16 or exogenous introduction of recombinant ZG16 protein induced the expression of MHC II, CD86, CD84, and CCR7 on the surface of DCs, thereby facilitating the secretion of proinflammatory mediators IL-1β, IL-6, TNF-α, and IL-12/p70, supporting the promoting effect of ZG16 on DC maturation. By establishing the subcutaneous and orthotopic mouse models of pancreatic cancer, we confirmed that intraperitoneal injection of recombinant ZG16 protein (Re-mZG16) could induce tumor regression by stimulating DC maturation and enhancing antitumor responses of CD4 + , CD8 + , PD-1 + , and Ctla4+ cells. Besides, Re-mZG16 in combination with gemcitabine showed a synergistic effect in the treatment of pancreatic cancer. Mechanistically, we demonstrated that ZG16 inhibited the ubiquitination and degradation of CD40, which depended on the lectin domain of ZG16. In conclusion, this study provided a novel insight into the role of ZG16-CD40 axis in DC-based immunotherapy for pancreatic cancer.

Discovery of orally bioavailable phosphonate prodrugs of potent ENPP1 inhibitors for cancer treatment

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is the dominant hydrolase of 2′,3′-cyclic GMP-AMP (cGAMP). Inhibition of ENPP1 contributes to increased cGAMP concentration and stimulator of interferon gene (STING) activation, with the potential to boost immune response against cancer. ENPP1 is a promising therapeutic target in tumor immunotherapy. To date, orally bioavailable ENPP1 inhibitors with highly potent activity under physiological conditions have been rarely reported. Herein, we report our effort in the design and synthesis of two different series of ENPP1 inhibitors, and in the identification of a highly potent ENPP1 inhibitor 27 (IC50 = 1.2 nM at pH 7.5), which significantly enhanced the cGAMP-mediated STING activity in THP-1 cells. Phosphonate compound 27 has good preclinical pharmacokinetic profiles with low plasma clearance rate in mouse, rat, and dog. It has been developed as bis-POM prodrug 36 which successfully improves the oral bioavailability of 27. In the Pan02 syngeneic mouse model of pancreatic cancer, orally administered 36 showed synergistic effect in combination with radiotherapy.

Cascade-responsive size/charge bidirectional-tunable nanodelivery penetrates pancreatic tumor barriers.

The pancreatic tumor microenvironment presents multiple obstacles for polymer-based drug delivery systems, limiting tumor penetration and treatment efficacy. Here, we engineer a hyaluronidase/reactive oxygen species cascade-responsive size/charge bidirectional-tunable nanodelivery (btND, G/R@TKP/HA) for co-delivery of gemcitabine and KRAS siRNA, capable of navigating through tumor barriers and augmenting anticancer efficiency. When penetrating the tumor stroma barrier, the hyaluronic acid shell of the nanodelivery undergoes degradation by hyaluronidase in an extracellular matrix, triggering size tuning from large to small and charge tuning from negative to positive, thereby facilitating deeper penetration and cellular internalization. After endocytosis, the nanodelivery protonizes in the endo/lysosome, prompting rapid endo/lysosomal escape, effectively overcoming the lysosome barrier. Intracellular ROS further disrupt the nanodelivery, inducing its size tuning again from small to large and a positive charge decrease for high tumor retention and controlled drug release. The btND shows remarkable antitumor activity in pancreatic cancer mouse models, highlighting the efficacy of this approach in penetrating tumor barriers and enhancing anticancer outcomes.

Delivery of a STING Agonist Using Lipid Nanoparticles Inhibits Pancreatic Cancer Growth.

The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive and characterized by a large number of cancer-associated fibroblasts, myeloid-derived suppressor cells, and regulatory T cells. Stimulator of interferon genes (STING) is an endoplasmic reticulum receptor that plays a critical role in immunity. STING agonists have demonstrated the ability to inflame the TME, reduce tumor burden, and confer anti-tumor activity in mouse models. 2'3' cyclic guanosine monophosphate adenosine monophosphate (2'3'-cGAMP) is a high-affinity endogenous ligand of STING. However, delivering cGAMP to antigen-presenting cells and tumor cells within the cytosol remains challenging due to membrane impermeability and poor stability. In this study, we encapsulated 2'3'-cGAMP in a lipid nanoparticle (cGAMP-LNP) designed for efficient cellular delivery. We assessed the properties of the nanoparticles using a series of in-vitro studies designed to evaluate their cellular uptake, cytosolic release, and minimal cytotoxicity. Furthermore, we examined the nanoparticle's anti-tumor effect in a syngeneic mouse model of pancreatic cancer. The lipid platform significantly increased the cellular uptake of 2'3'-cGAMP. cGAMP-LNP exhibited promising antitumor activity in the syngeneic mouse model of pancreatic cancer. The LNP platform shows promise for delivering exogenous 2'3'-cGAMP or its derivatives in cancer therapy.

Near-infrared photoimmunotherapy as a complementary modality to in situ vaccine in a preclinical pancreatic cancer model

Pancreatic cancer is one of the most refractory malignancies. In situ vaccines (ISV), in which intratumorally injected immunostimulatory adjuvants activate innate immunity at the tumor site, utilize tumor-derived patient-specific antigens, thereby allowing for the development of vaccines in patients themselves. Near-infrared photoimmunotherapy (NIR-PIT) is a novel therapy that selectively kills cancer cells exclusively in the NIR-irradiated region. Extending our previous research showing that ISV using the unique nanoparticulate Toll-like receptor 9 (TLR9) ligand K3-SPG induced effective antitumor immunity, here we incorporated NIR-PIT into K3-SPG-ISV so that local tumor destruction by NIR-PIT augments the antitumor effect of ISV. In the mouse model of pancreatic cancer, the combination of K3-SPG-ISV and CD44-targeting NIR-PIT showed synergistic systemic antitumor effects and enhanced anti-programmed cell death-1 (PD-1) blockade. Mechanistically, strong intratumoral upregulation of interferon-related genes and dependency on CD8+ T cells were observed, suggesting the possible role of interferon and cytotoxic T cell responses in the induction of antitumor immunity. Importantly, this combination induced immunological memory in therapeutic and neoadjuvant settings. This study represents the first attempt to integrate NIR-PIT with ISV, offering a promising new direction for cancer immunotherapy, particularly for pancreatic cancer.

In vivo interaction screening reveals liver-derived constraints to metastasis

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2–semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.

Extracellular vesicle-packaged PIAT from cancer-associated fibroblasts drives neural remodeling by mediating m5C modification in pancreatic cancer mouse models.

Perineural invasion (PNI) is a biological characteristic commonly observed in pancreatic cancer. Although PNI plays a key role in pancreatic cancer metastasis, recurrence, and poor postoperative survival, its mechanism is largely unclarified. Clinical sample analysis and endoscopic ultrasonographic elasticity scoring indicated that cancer-associated fibroblasts (CAFs) were closely related to the occurrence of PNI. Furthermore, CAF-derived extracellular vesicles (EVs) were involved in PNI in dorsal root ganglion coculture and mouse sciatic nerve models. Next, we demonstrated that CAFs promoted PNI through extracellular vesicle transmission of PNI-associated transcript (PIAT). Mechanistically, PIAT specifically bound to YBX1 and blocked the YBX1-Nedd4l interaction to inhibit YBX1 ubiquitination and degradation. Furthermore, PIAT enhanced the binding of YBX1 and PNI-associated mRNAs in a 5-methylcytosine (m5C)-dependent manner. Mutation of m5C recognition motifs in YBX1 or m5C sites in downstream target genes reversed PIAT-mediated PNI. Consistent with these findings, analyses using a KPC mouse model demonstrated that the PIAT/YBX1 axis enhanced PNI through m5C modification. Clinical data suggested that the PIAT expression in the serum EVs of patients with pancreatic cancer was associated with the degree of neural invasion and prognosis. Our study revealed the important role of the PIAT/YBX1 signaling axis in the tumor microenvironment (TME) in promoting tumor cell PNI and provided a new target for precise interference with CAFs and RNA methylation in the TME to suppress PNI in pancreatic cancer.

An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8+ T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.

Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Coordination engineering of FeCo dual single-atom nanozymes with photothermal-enhanced cascaded catalysis for efficient pancreatic cancer immunotherapy

Cancer immunotherapy holds great promise in improving therapeutic outcomes. However, its effectiveness is significantly hindered by the inadequate immunogenicity and potent immuno-suppressive nature of the tumor microenvironment (TME). Herein, we elaborately design an advanced iron-cobalt dual-single-atom nanozyme (FeCo-DA) with adjacent Fe-N/O-C and Co-N/O-C pair sites. This design aims to induce potent immunogenic cell death (ICD), ultimately enhancing cancer immunotherapy by activating the immune microenvironment. Compared to Fe and Co single-atom nanozyme, FeCo-DA demonstrated superior photothermal effects and cascaded catalytic performance by simultaneously mimicking peroxidase (POD), catalase (CAT), and glutathione oxidase (GSH-OXD). The cascaded catalysis not only augmented oxidative stress but also exacerbated the redox imbalance through sustainable generation of hydroxyl radicals (∙OH) and depletion of glutathione (GSH). The comprehensive in vitro and vivo experiments demonstrated that FeCo-DA effectively induced immunogenic cell death (ICD) by releasing damage-associated molecular patterns (DAMPs). The photothermal-enhanced cascaded catalytic therapy exhibited remarkable therapeutic effects on a mouse model of pancreatic cancer. This work highlights the potential of structure engineering in enhancing the efficacy of dual single-atom nanozyme for ICD-based cancer immunotherapy.

Blocking M2-like macrophage polarization using decoy oligodeoxynucleotide-based gene therapy prevents immune evasion for pancreatic cancer treatment.

M2-like macrophages exhibit immunosuppressive activity and promote pancreatic cancer progression. Reactive oxygen species (ROS) affect macrophage polarization; however, the mechanism remains unclear. This study aimed to elucidate the underlying molecular basis and design a gene therapy to inhibit M2-like polarization. Microarray analysis and IF staining were performed in M1-like and M2-like macrophages to ascertain the expression of CYBB, a major intracellular ROS source. Co-culture assay and syngeneic orthotopic pancreatic cancer mouse models were used to study the mechanism of M2-like skewing. Decoy oligodeoxynucleotides (ODNs) were designed to manipulate CYBB transcription to inhibit M2-like polarization and control tumor growth. Lipopolysaccharide (LPS) treatment polarized U937 cells to M1-like macrophages in which CYBB expression was increased. In contrast, co-culture with PANC-1 cells induced M2-like polarization in U937 cells with CYBB downregulation. High CD204 M2-like expression in combination with low CYBB expression was associated with the worst prognosis in pancreatic cancer patients. STAT6 and HDAC2 in U937 cells were activated by cancer cell-derived IL-4 after coculture and then bound to the CYBB promoter to repress CYBB expression, resulting in M2-like polarization. DPI that inhibits ROS production could block this action. Knockdown of STAT6 and HDAC2 also inhibited M2-like polarization and maintained the M1-like phenotype of U937 cells after coculture. Decoy ODNs interrupting the binding of STAT6 to the CYBB promoter counteracted M2-like polarization and tumor growth and triggered antitumor immunity in vivo. Gene therapy using STAT6-CYBB decoy ODNs can inhibit M2-like polarization, representing a potential therapeutic tool for pancreatic cancer.