Mouse Model Of Pancreatic Cancer Research Articles

Inhibitory effects of the combination of rapamycin with gemcitabine plus paclitaxel on the growth of pancreatic cancer tumors

We previously examined the antitumor effects of short interfering RNA nanoparticles targeting mammalian target of rapamycin (mTOR) in an orthotopic pancreatic cancer mouse model. We herein report the inhibitory effects of the mTOR inhibitor rapamycin on tumor growth in a novel established mouse model of pancreatic cancer using human pancreatic cancer cell line-derived organoids. Gemcitabine, 5-fluorouracil, and gemcitabine plus nab-paclitaxel are clinically used to treat advanced pancreatic cancer. In vitro assays showed that rapamycin strongly inhibited cell invasion, while gemcitabine, 5-fluorouracil, and gemcitabine plus paclitaxel primarily inhibited cell proliferation with minimal effects on invasion. In vivo mouse experiments demonstrated that rapamycin exhibited superior antitumor activity to S-1 (a metabolically activated prodrug of 5-fluorouracil) and another mTOR inhibitor, everolimus, while its efficacy was similar to that of gemcitabine plus paclitaxel (which was used instead of nab-paclitaxel due to concerns about allergic reactions in mice to human albumin) in a mouse model of pancreatic cancer using human pancreatic cancer cell line-derived organoids. Furthermore, the combination of rapamycin with gemcitabine plus paclitaxel exerted synergistic inhibitory effects on the growth of pancreatic cancer tumors. Although the inhibition of tumor growth was significantly stronger in everolimus-treated mice than in control mice, there were no additive anti-growth effects when combined with gemcitabine plus paclitaxel. The present results suggest that the combination of rapamycin with gemcitabine plus paclitaxel achieved the greatest reduction in tumor volumes in the mouse xenograft model and, thus, has significant clinical promise.

Induction of macrophage efferocytosis in pancreatic cancer via PI3Kγ inhibition and radiotherapy promotes tumour control

BackgroundThe immune suppression mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unknown, but preclinical studies have implicated macrophage-mediated immune tolerance. Hence, pathways that regulate macrophage phenotype are of strategic interest, with...

Investigating the effects of stereotactic body radiation therapy on pancreatic tumor hypoxia and microvasculature in an orthotopic mouse model using intravital fluorescence microscopy

Despite decades of improvements in cytotoxic therapy, the current standard of care for locally advanced pancreatic cancer (LAPC) provides, on average, only a few months of survival benefit. Stereotactic Body Radiation Therapy (SBRT), a technique that accurately delivers high doses of radiation to tumors in fewer fractions, has emerged as a promising therapy to improve local control of LAPC; however, its effects on the tumor microenvironment and hypoxia remain poorly understood. To explore how SBRT affects pancreatic tumors, we combined an orthotopic mouse model of pancreatic cancer with an intravital microscopy platform to visualize changes to the in vivo tumor microenvironment in real-time. Mice received SBRT (5 × 8 Gy) or were left untreated, and were imaged before and 1, 4, 7, and 14 days after treatment (n = 7/group). A fluorescent human pancreatic cancer cell line (BxPC3-DsRed) engineered to express GFP under hypoxic conditions (driven by hypoxia-inducible factor, HIF) was used to monitor tumor hypoxia. Immunohistochemical staining was also performed on tissues to validate in vivo data. Our findings demonstrate a persistent decrease in pancreatic tumor hypoxia as early as one day after SBRT. This coincided with a decrease in both tumor cell proliferation and cell density in the SBRT group. Reduced demand for oxygen after SBRT (due to cell death and growth arrest from treatment) significantly contributed to reoxygenation of the pancreatic TME. Understanding how this reoxygenation phenomenon occurs in a dose-dependent manner will help improve dosing and fractionation schemes for clinical SBRT.

The RAGE Inhibitor TTP488 (Azeliragon) Demonstrates Anti-Tumor Activity and Enhances the Efficacy of Radiation Therapy in Pancreatic Cancer Cell Lines.

Pancreatic cancer is the third leading cause of cancer-related mortality in the United States, with rising incidence and mortality. The receptor for advanced glycation end products (RAGE) and its ligands significantly contribute to pancreatic cancer progression by enhancing cell proliferation, fostering treatment resistance, and promoting a pro-tumor microenvironment via activation of the nuclear factor-kappa B (NF-κB) signaling pathways. This study validated pathway activation in human pancreatic cancer and evaluated the therapeutic efficacy of TTP488 (Azeliragon), a small-molecule RAGE inhibitor, alone and in combination with radiation therapy (RT) in preclinical models of pancreatic cancer. Human (Panc1) and murine (Pan02) pancreatic cancer cell lines exhibited elevated levels of RAGE and its ligands compared to normal pancreatic tissue. In vitro, Azeliragon inhibited RAGE-mediated NF-κB activation and ligand-mediated cell proliferation in pancreatic cancer cell lines. Target engagement of Azeliragon was confirmed in vivo, as determined by decreased NF-κB activation. Azeliragon demonstrated significant growth delay in mouse models of pancreatic cancer and additive effects when combined with RT. Additionally, Azeliragon modulated the immune suppressive tumor microenvironment in pancreatic cancer by reducing immunosuppressive cells, including M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells, while enhancing CD8+ T cell infiltration. These findings suggest that Azeliragon, by inhibiting RAGE-mediated signaling and modulating immune response, may serve as an effective anti-cancer agent in pancreatic cancer.

A Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth and Induces Antitumor Immunity in Gastrointestinal Cancer.

RAS is a common driver of cancer that was considered undruggable for decades. Recent advances have enabled the development of RAS inhibitors, but the efficacy of these inhibitors remains limited by resistance. Here, we developed a pan-RAS inhibitor, ADT-007, that binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme, and RASWT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases that were expressed in RASWT and normal cells but repressed in RAS mutant cancer cells. ADT-007 displayed unique advantages over KRAS mutant-specific, pan-KRAS, and pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms that lead to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth. Thus, ADT-007 has the potential to address the complex RAS mutational landscape of many human cancers and to improve treatment of RAS-driven tumors.

Exploring the clinical significance of TPX2 in pancreatic cancer: from biomarker to immunotherapy.

Pancreatic cancer (PC) is a highly aggressive malignancy characterized by a dismal prognosis. The present study is designed to elucidate the pivotal role of Xenopus kinesin-like protein 2 (TPX2) as a biomarker with substantial clinical prognostic significance in PC. By conducting a comprehensive analysis of RNA sequencing data and protein expression profiles obtained from multiple databases, we observed a pronounced upregulation of TPX2 expression in PC tissues compared to normal pancreatic tissues. Importantly, TPX2 emerged as an independent prognostic factor, demonstrating remarkable diagnostic accuracy. Notably, its expression levels were found to be significantly associated with the PC immune microenvironment and sensitivity to various therapeutic modalities. Functional assays revealed that the silencing of TPX2 markedly inhibited PC cell proliferation, metastasis, and the growth of subcutaneous tumors in PC mouse models. These effects were potentially mediated by the activation of CD8+ T cell immune responses and the inhibition of cell cycle progression and adhesion mechanisms. Taken together, our findings indicate that TPX2 may serve as a critical biomarker for the diagnosis and clinical management of patients with PC.

Prolonged survival by combination treatment with a standardized herbal extract from Japanese Kampo-medicine (Juzentaihoto) and gemcitabine in an orthotopic transplantation pancreatic cancer model.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its poor prognosis. Traditional Japanese herbal medicine (Kampo), such as Juzentaihoto (a standardized combination of 10 herbal extracts), has shown immune modulatory effects, modulation of microcirculation, and amelioration of fatigue. It is administered to patients to prevent deterioration of cachexia and counteract side effects of chemotherapy. The effect of Juzentaihoto with or without standard chemotherapy (Gemcitabine) on survival and tumor microenvironment was studied in an immunocompetent pancreatic cancer mouse model. Following tumor development ±12 days after orthotopic implantation of murine pancreatic cancer cells (KPC) into the pancreas of C57BL/6 mice, the mice were treated with Gemcitabine, Juzentaihoto, their combination (Gem/Juz) or NaCl (Ctr.). Combination treatment significantly prolonged survival (+38%) of tumor bearing mice, compared to controls as well as Gemcitabine or Juzentaihoto monotherapy. Macrophage (CD68+) infiltration in pancreatic tumors was significantly enhanced in Gem/Juz - treated animals, compared with controls (p < 0,001), with significant increases of both, macrophages (CD68+) and for lymphocytes (CD45+), especially at the tumor front. In vitro, Juz- or Gem/Juz-treated KPC tumor cells secreted significantly more macrophage-chemoattractant cytokines, e.g., CCL2, CCL20, and CXCL2, whilst Juz- and Gem/Juz-treated macrophages (MH-S) secreted cytokines of the M1 phenotype, e.g., IL6, TNF-α, and IL12. It has been shown that tumor cells recruit and polarize macrophages towards tumor-associated macrophages (TAM). Our results indicate a change in macrophage polarization which not only induced anti-tumor immune-cell activity and cytokine release, but also suggests amelioration of Gemcitabine efficacy as DNA-analogue and as partial antitumor antigen. We propose that the increased survival of tumor bearing mice after Gem/Juz combination treatment is due to the restored cytotoxicity of Gemcitabine and changes in the tumor-microenvironment - induced by Juzentaihoto - such as an increased number of M1 macrophages.

Leveraging CAR macrophages targeting c-Met for precision immunotherapy in pancreatic cancer: insights from single-cell multi-omics.

Pancreatic cancer is known for its poor prognosis and resistance to conventional therapies, largely due to the presence of cancer stem cells (CSCs) and aggressive angiogenesis. Effectively targeting these CSCs and associated angiogenic pathways is crucial for effective treatment. This study leverages single-cell multi-omics to explore a novel therapeutic approach involving Chimeric Antigen Receptor (CAR) macrophages engineered to target the c-Met protein on pancreatic CSCs. We employed single-cell RNA sequencing to analyze pancreatic cancer tissue, identifying c-Met as a key marker of CSCs. CAR macrophages were engineered using a lentiviral system to express a c-Met-specific receptor. The phagocytic efficiency of these CAR macrophages against pancreatic CSCs was assessed in vitro, along with their ability to inhibit angiogenesis. The in vivo efficacy of CAR macrophages was evaluated in a mouse model of pancreatic cancer. CAR macrophages demonstrated high specificity for c-Met + CSCs, significantly enhancing phagocytosis and reducing the secretion of angiogenic factors such as VEGFA, FGF2, and ANGPT. In vivo, these macrophages significantly suppressed tumor growth and angiogenesis, prolonging survival in pancreatic cancer-bearing mice. CAR macrophages targeting c-Met represent a promising therapeutic strategy for pancreatic cancer, offering targeted elimination of CSCs and disruption of tumor angiogenesis. This study highlights the potential of single-cell multi-omics in guiding the development of precision immunotherapies.

4-1BBL-Armed Oncolytic Herpes Simplex Virus Exerts Antitumor Effects in Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a notably poor response to therapy due to its immunosuppressive tumor microenvironment (TME) and intrinsic drug resistance. The oncolytic virus (OV) represents a promising therapeutic strategy capable of transforming the "cold" immunological profile of PDAC tumors to a "hot" one by reshaping the TME. 4-1BB (CD137), a crucial member of the tumor necrosis factor receptor superfamily, plays a significant role in T-cell activation and function. Methods: In this study, we constructed an oncolytic herpes simplex virus armed with 4-1BBL (oHSV-4-1BBL), the ligand for the 4-1BB receptor, and investigated its therapeutic effects in two mouse models of pancreatic cancer, Pan02_HVEM and KPC. Results: We found that oHSV-4-1BBL remarkably inhibited tumor growth and extended the median survival time in both models. To amplify the therapeutic effect, we further combined oHSV-4-1BBL with PD-1 antibody. This combination therapy not only further suppressed tumor growth but also extended the median survival time by an additional 11 days compared to oHSV (armed with GFP as a control) combined with PD-1 antibody treatment, with some mice achieving complete tumor regression. Conclusions: Our findings confirm the potential of combining oncolytic viral therapy with 4-1BB targeting in enhancing the treatment of pancreatic cancer.

Novel Amanitin-based Antibody Drug Conjugates (ATAC®) targeting TROP2 for the treatment of Pancreatic Cancer.

Trophoblast cell surface antigen 2 (TROP2) exhibits aberrant expression in pancreatic cancer, correlating with metastasis, advanced tumor stage and poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. TROP2 has been recognized as a promising therapeutic target for antibody drug conjugates (ADCs), as evidenced by the approval of the anti-TROP2 ADC Trodelvy® for the treatment of triple negative breast cancer. In this study we report the generation of novel second-generation amanitin based ADCs (ATAC®s) targeting TROP2, comprising the humanized RS7 antibody of Trodelvy® (hRS7) and the highly potent payload amanitin. The specific in vitro binding, efficient antigen internalization, and high cytotoxicity of hRS7 ATAC®s with half maximal effective concentration (EC50) values in the picomolar range in TROP2-expressing cells constituted the foundation for preclinical in vivo evaluation. The hRS7 ATAC®s demonstrated a significant reduction in tumor growth in vivo in subcutaneous xenograft mouse models of pancreatic cancer and triple negative breast cancer at well-tolerated doses. The antitumor efficacy correlated with the level of TROP2 expression on the tumors and the in vivo tumor uptake of the ATAC®s. The long half-life of 9.7-10.7 days of hRS7 ATAC®s without premature payload release in serum supported a high therapeutic index. Notably, the efficacy of the hRS7 ATAC®s was superior to that of Trodelvy® with complete tumor eradication in both, refractory pancreatic and triple negative breast cancer xenograft models. In summary, hRS7 ATAC®s represent a highly effective and well-tolerated targeted therapy, and our data support their development for pancreatic cancer and other TROP2-expressing tumors.

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.

The Impact of a Ketogenic Diet on Late-Stage Pancreatic Carcinogenesis in Mice: Efficacy and Safety Studies.

High-fat diets (HFDs) have been associated with an increased risk of pancreatic cancer. In contrast, ketogenic diets (KDs) have been shown to display anti-tumor characteristics. The objective of this work was to evaluate the efficacy of a KD on late-stage pancreatic carcinogenesis in a genetically modified mouse model of pancreatic cancer [LSL-KrasG12D/+; Ptf1-Cre (KC) mice], as well as its liver safety, and to compare it to that of an HFD. Six-month-old female and male KC mice were randomly allocated to either a control diet (CD) (%kcal: 20% fat, 15% protein, 65% carbohydrates), an HFD (%kcal: 40% fat, 15% protein, 45% carbohydrate) or a KD (%kcal: 84% fat, 15% protein, 1% carbohydrate) and fed these diets for 6 months. HFD-fed, but not KD-fed, mice showed a 15% increase in body weight, plus elevated serum insulin (2.4-fold increase) and leptin (2.9-fold increase) levels, compared to CD-fed mice. At the pancreas level, no differences in pancreatic cancer incidence rates were observed among the diet groups. Regarding the liver safety profile, the HFD-fed mice had higher serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), when compared to the CD and KD groups. In addition, upon histologic examination, an HFD, but not a KD, showed a ~2-fold increase in both macro- and microsteatosis, as well as 35% and 32% higher levels of TLR4 and NF-κB activation, respectively, compared to CD-fed mice. In summary, although a KD intervention alone did not prevent pancreatic carcinogenesis, our data suggests that a KD modulates insulin signaling and hepatic lipid metabolism, highlighting its beneficial effects on healthspan and liver function when compared to an HFD.

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGF-β depletion.

The dual tumor-suppressive and -promoting functions of TGF-β signaling has made its targeting challenging. We examined the effects of TGF-β depletion by AVID200/BMS-986416 (TGF-β-TRAP), a TGF-β ligand trap, on the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) murine models with different organ-specific metastasis. Our study demonstrated that TGF-β-TRAP potentiates the efficacy of anti-programmed cell death 1 (anti-PD-1) in a PDAC orthotopic murine model with liver metastasis tropism, significantly reducing liver metastases. We further demonstrated the heterogeneous response of cytotoxic effector T cells to combination TGF-β-TRAP and anti-PD-1 treatment across several tumor models. Single-nuclear RNA sequencing suggested that TGF-β-TRAP modulates cancer-associated fibroblast (CAF) heterogeneity and suppresses neutrophil degranulation and CD4+ T cell response to neutrophil degranulation. Ligand-receptor analysis indicated that TGF-β-TRAP may modulate the CCL5/CCR5 axis as well as costimulatory and checkpoint signaling from CAFs and myeloid cells. Notably, the most highly expressed ligands of CCR5 shifted from the immunosuppressive CCL5 to CCL7 and CCL8, which may mediate the immune agonist activity of CCR5 following TGF-β-TRAP and anti-PD-1 combination treatment. This study suggested that TGF-β depletion modulates CAF heterogeneity and potentially reprograms CAFs and myeloid cells into antitumor immune agonists in PDAC, supporting the validation of such effects in human specimens.

Non‐Natural MUC1 Glycopeptide Homogeneous Cancer Vaccine with Enhanced Immunogenicity and Therapeutic Activity

AbstractGlycopeptides 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 observed when the vaccine was combined with an anti‐programmed cell death protein 1 (anti‐PD‐1) checkpoint inhibitor. Our strategy reduces batch‐to‐batch variability and enhances both immunogenicity and therapeutic potential. This site‐specific approach disputes a prevailing dogma where glycoconjugate vaccines require multivalent display of antigens.

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 observed when the vaccine was combined with an anti-programmed cell death protein 1 (anti-PD-1) checkpoint inhibitor. Our strategy reduces batch-to-batch variability and enhances both immunogenicity and therapeutic potential. This site-specific approach disputes a prevailing dogma where glycoconjugate vaccines require multivalent display of antigens.

Engineering Radiocatalytic Nanoliposomes with Hydrophobic Gold Nanoclusters for Radiotherapy Enhancement.

Chemoradiation therapy is on the forefront of pancreatic cancer care, and there is a continued effort to improve its safety and efficacy. Liposomes are widely used to improve chemotherapy safety, and may accurately deliver high-Z element- radiocatalytic nanomaterials to cancer tissues. In this study, the interaction between X-rays and long-circulating nanoliposome formulations loaded with gold nanoclusters is explored in the context of oxaliplatin chemotherapy for desmoplastic pancreatic cancer. Hydrophobic gold nanoclusters stabilized with dodecanethiol (AuDDT) are efficiently incorporated in nanoliposomal bilayers. AuDDT-nanoliposomes significantly augmented radiation-induced •OH production, which is most effective with monochromatic X-rays at energies that exceed the K-shell electron binding energy of Au (81.7keV). Cargo release assays reveal that AuDDT-nanoliposomes can permeabilize lipid bilayers in an X-ray dose- and formulation-dependent manner. The radiocatalytic effect of AuDDT-nanoliposomes significantly augments radiotherapy and oxaliplatin-chemoradiotherapy outcomes in 3D pancreatic microtumors. The PEGylated AuDDT-nanoliposomes display high tumor accumulation in an orthotopic mouse model of pancreatic cancer, showing promise for nanoliposomes as carriers for radiocatalytic nanomaterials. Altogether, compelling proof for chemo-radiation dose-enhancement using AuDDT-nanoliposomes is presented. Further improving the nanoliposomal loading of high-Z elements will advance the safety, efficacy, and translatability of such chemoradiation dose-enhancement approaches.

Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases.

Background: Oncogenic mutations in the KRAS gene are detected in >90% of pancreatic cancers (PC). In genetically engineered mouse models of PC, oncogenic KRAS drives the formation of precursor lesions and their progression to invasive PC. The Yes-associated Protein (YAP) is a transcriptional coactivator required for transformation by the RAS oncogenes and the development of PC. In Ras-driven tumors, YAP can also substitute for oncogenic KRAS to drive tumor survival after the repression of the oncogene. Ras oncoproteins exert their transforming properties through their downstream effectors, including the PI3K kinase, Rac1 GTPase, and MAPK pathways. Methods: To identify Ras effectors that regulate YAP, YAP levels were measured in PC cells exposed to inhibitors of oncogenic K-Ras and its effectors. Results: In PC cells, the inhibition of Rac1 leads to a time-dependent decline in YAP protein, which could be blocked by proteosome inhibitor MG132. This YAP degradation after Rac1 inhibition was observed in a range of cell lines using different Rac1 inhibitors, Rac1 siRNA, or expression of dominant negative Rac1T17N mutant. Several E3 ubiquitin ligases, including SCFβTrCP, regulate YAP protein stability. To be recognized by this ligase, the βTrCP degron of YAP (amino acid 383-388) requires its phosphorylation by casein kinase 1 at Ser384 and Ser387, but these events must first be primed by the phosphorylation of Ser381 by LATS1/2. Using Flag-tagged mutants of YAP, we show that YAP degradation after Rac1 inhibition requires the integrity of this degron and is blocked by the silencing of βTrCP1/2 and by the inhibition of casein kinase 1. Unexpectedly, YAP degradation after Rac1 inhibition was still observed after the silencing of LATS1/2 or in cells carrying a LATS1/2 double knockout. Conclusions: These results reveal Rac1 as an oncogenic KRAS effector that contributes to YAP stabilization in PC cells. They also show that this regulation of YAP by Rac1 requires the SCFβTrCP ligase but occurs independently of the LATS1/2 kinases.

All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer.

As a double-stranded RNA-editing enzyme and an interferon-stimulated gene, double-stranded RNA-specific adenosine deaminase (ADAR1) suppresses interferon signaling and contributes to immunotherapy resistance. Suppression of ADAR1 overcomes immunotherapy resistance in preclinical models, but has not yet been translated to clinical settings. By conducting a screening of a subset of the FDA-approved drugs, we found that all-trans retinoic acid (ATRA, also known as tretinoin) caused ADAR1 protein degradation through ubiquitin-proteasome pathways and concomitantly increased PD-L1 expression in pancreatic and breast cancers. In addition, the combination of ATRA and PD-1 blockade reprogrammed the tumor microenvironment and unleashed antitumor immunity and thereby impeded tumor growth in pancreatic cancer mouse models. In a pilot clinical trial, a higher dose of ATRA plus the anti-PD-1 antibody nivolumab prolonged median overall survival in patients with chemotherapy-resistant pancreatic cancer compared to a lower dose of the same regimen. In this study, ATRA was the first drug to be found to cause ADAR1 degradation. We propose translation of a promising 2-pronged antitumor strategy using ATRA and nivolumab to convert immunologically "cold" into "hot" tumors susceptible to immune checkpoint blockade.

Contemporaneous Inflammatory, Angiogenic, Fibrogenic, and Angiostatic Cytokine Profiles of the Time-to-Tumor Development by Cancer Cells to Orchestrate Tumor Neovascularization, Progression, and Metastasis.

Cytokines can promote various cancer processes, such as angiogenesis, epithelial to mesenchymal transition (EMT), invasion, and tumor progression, and maintain cancer stem-cell-like (CSCs) cells. The mechanism(s) that continuously promote(s) tumors to progress in the TME still need(s) to be investigated. The data in the present study analyzed the inflammatory, angiogenic, fibrogenic, and angiostatic cytokine profiles in the host serum during tumor development in a mouse model of human pancreatic cancer. Pancreatic MiaPaCa-2-eGFP cancer cells were subcutaneously implanted in RAG2xCγ double mutant mice. Blood samples were collected before cancer cell implantation and every week until the end point of the study. The extracted serum from the blood of each mouse at different time points during tumor development was analyzed using a Bio-Plex microarray analysis and a Bio-Plex 200 system for proinflammatory (IL-1β, IL-10, IFN-γ, and TNF-α) and angiogenic and fibrogenic (IL-15, IL-18, basic FGF, LIF, M-CSF, MIG, MIP-2, PDGF-BB, and VEGF) cytokines. Here, we find that during cancer cell colonization for tumor development, host angiogenic, fibrogenic, and proinflammatory cytokine profiling in the tumor-bearing mice has been shown to significantly reduce host angiostatic and proinflammatory cytokines that restrain tumor development and increase those for tumor growth. The proinflammatory cytokines IL-15, IL-18, and IL-1β profiles reveal a significant host serum increase after day 35 when the tumor began to progress in growth. In contrast, the angiostatic cytokine profiles of TNFα, MIG, M-CSF, IL-10, and IFNγ in the host serum revealed a dramatic and significant decrease after day 5 post-implantation of cancer cells. OP treatment of tumor-bearing mice on day 35 maintained high levels of angiostatic and fibrogenic cytokines. The data suggest an entirely new regulation by cancer cells for tumor development. The findings identify for the first time how pancreatic cancer cells use host cytokine profiling to orchestrate the initiation of tumor development.

Targeting Human Pancreatic Cancer with a Fluorophore-Conjugated Mucin 4 (MUC4) Antibody: Initial Characterization in Orthotopic Cell Line Mouse Models.

Background/Objectives: Pancreatic cancer is the third leading cause of death related to cancer. The only possible cure presently is complete surgical resection; however, this is limited by difficulty in clearly defining tumor margins. Enhancement of the visualization of pancreatic ductal adenocarcinoma (PDAC) tumor margins using near-infrared dye-conjugated tumor-specific antibodies was pioneered by using anti-CEA, anti-CA19.9, and anti-MUC5AC in orthotopic mouse models of pancreatic cancer. Recently, an antibody to Mucin 4 (MUC4) conjugated to a fluorescent probe has shown promise in targeting colon tumors in orthotopic mouse models. Methods: In the present study, we targeted pancreatic cancer using an anti-MUC4 antibody conjugated to IRDye800 (anti-MUC4-IR800) in orthotopic mouse models. Two pancreatic cancer human cell lines were used, SW1990 and CD18/HPAF. Results: Anti-MUC4-IR800 targeted the two pancreatic cancer cell line tumors in orthotopic mouse models with high tumor-to-pancreas ratios and high tumor-to-liver ratios, with greater targeting seen in SW1990. Conclusions: The present results suggest anti-MUC4-IR800's potential to be used in fluorescence-guided surgical resection of pancreatic cancer.