Orthotopic Pancreatic Cancer Model Research Articles

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.

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.

Harnessing the Power of Sugar-Based Nanoparticles: A Drug-Free Approach to Enhance Immune Checkpoint Inhibition against Glioblastoma and Pancreatic Cancer.

Cancer cells have a high demand for sugars and express diverse carbohydrate receptors, offering opportunities to improve delivery with multivalent glycopolymer materials. However, effectively delivering glycopolymers to tumors while inhibiting cancer cell activity, altering cellular metabolism, and reversing tumor-associated macrophage (TAM) polarization to overcome immunosuppression remains a challenging area of research due to the lack of reagents capable of simultaneously achieving these objectives. Here, the glycopolymer-like condensed nanoparticle (∼60 nm) was developed by a one-pot carbonization reaction with a single precursor, promoting multivalent interactions for the galactose-related receptors of the M2 macrophage (TAM) and thereby regulating the STAT3/NF-κB pathways. The subsequently induced M2-to-M1 transition was increased with the condensed level of glycopolymer-like nanoparticles. We found that the activation of the glycopolymer-like condensed galactose (CG) nanoparticles influenced monocarboxylate transporter 4 (MCT-4) function, which caused inhibited lactate efflux (similar to inhibitor effects) from cancer cells. Upon internalization via galactose-related endocytosis, CG NPs induced cellular reactive oxygen species (ROS), leading to dual functionalities of cancer cell death and M2-to-M1 macrophage polarization, thereby reducing the tumor's acidic microenvironment and immunosuppression. Blocking the nanoparticle-MCT-4 interaction with antibodies reduced their toxicity in glioblastoma (GBM) and affected macrophage polarization. In orthotopic GBM and pancreatic cancer models, the nanoparticles remodeled the tumor microenvironment from "cold" to "hot", enhancing the efficacy of anti-PD-L1/anti-PD-1 therapy by promoting macrophage polarization and activating cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs). These findings suggest that glycopolymer-like nanoparticles hold promise as a galactose-elicited adjuvant for precise immunotherapy, particularly in targeting hard-to-treat cancers.

Mesenchymal stem cell-delivered paclitaxel nanoparticles exhibit enhanced efficacy against a syngeneic orthotopic mouse model of pancreatic cancer

Pancreatic cancer is considered the deadliest among various solid tumors, with a five-year survival rate of 13 %. One of the major challenges in the management of advanced pancreatic cancer is the inefficient delivery of chemotherapeutics to the tumor site. Even though nanocarriers have been developed to improve tumoral delivery of chemotherapeutics, less than 1 % of the drugs reach tumors, rendering inadequate concentration for effective inhibition of tumors. As a potential alternative, mesenchymal stem cells (MSCs) can effectively deliver their cargo to tumor sites because of their resistance to chemotherapeutics and inherent tumor tropism. In this study, we used MSCs for the delivery of dibenzocyclooctyne (DBCO)-functionalized paclitaxel (PTX)-loaded poly(lactide-co-glycolide)-b-poly (ethylene glycol) (PLGA) nanoparticles. MSCs were modified to generate artificial azide groups on their surface, allowing nanoparticle loading via endocytosis and surface conjugation via click chemistry. This dual drug loading strategy significantly improves the PTX-loading capacity of azide-expressed MSCs (MSC-Az, 55.4 pg/cell) compared to unmodified MSCs (28.1 pg/cell). The in vitro studies revealed that PTX-loaded MSC-Az, nano-MSCs, exhibited cytotoxic effects against pancreatic cancer without altering their inherent phenotype, differentiation abilities, and tumor tropism. In an orthotopic pancreatic tumor model, nano-MSCs demonstrated significant inhibition of tumor growth (p < 0.05) and improved survival (p < 0.0001) compared to PTX solution, PTX nanocarriers, and Abraxane. Thus, nano-MSCs could be an effective delivery system for targeted pancreatic cancer chemotherapy and other solid tumors.

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 PR-01: Systemic targeting of therapeutic RNA to pancreatic ductal adenocarcinoma via a novel, cell-penetrating, and nucleic acid-binding monoclonal antibody

Abstract There is intense interest in developing nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors (PRRs), particularly for treating “cold” tumors like pancreatic ductal adenocarcinoma (PDAC). However, selective delivery of intact and functional RNAs after intravenous administration in a tumor-specific manner to avoid systemic toxicity has been challenging. Many current efforts rely on the direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for pancreatic tumors and metastases. Here, we present a groundbreaking and unique cancer treatment approach using a lupus-derived, cell-penetrating antibody to deliver nucleic acids to PDAC tumors in vivo. This antibody, a modified version of the initially reported 3E10-D31N, designated TMAB3, forms non-covalent complexes with RNAs. We tested our delivery platform in genetically engineered mouse models (Pdx-1 Cre KrasG12D) and flank and orthoptic allographs of pancreatic cancer (Pdx-1 Cre KrasG12D, p53R172H/- KPC cells) in immunocompetent models. Using 3p-hpRNA, an agonist of the pattern recognition receptor retinoic acid-inducible gene-I (RIG-I), we observed robust anti-tumor efficacy of systemically administered TMAB3/3p-hpRNA complexes in an orthotopic pancreatic cancer model. Treatment tripled animal survival and decreased tumor growth, specifically targeting the malignant cells, with a 1500-fold difference in RNA delivery into tumor cells versus non-malignant cells within the tumor mass. While an immunosuppressive microenvironment typically characterizes PDAC, we found that RIG-I stimulation via TMAB3/3p-hpRNA treatment elicited a potent anti-tumoral immune response characterized by induction of apoptosis in tumor cells and infiltration of CD8+ T cells using single-cell RNASeq and confirming using FACS and immunocompromised mice. In vitro and in vivo studies demonstrated that cancer-cell delivery was mediated by nucleoside transporter ENT2, which is highly expressed in PDAC compared to healthy and benign tissues. These results were recapitulated in orthogonal models of immunologically cold tumors, including melanoma and medulloblastoma. Together, these studies demonstrate that the TMAB3 antibody can 1) precisely localize and enter pancreatic tumor cells, 2) promote CD8+ T cell infiltrates, and 3) rapidly deliver RNA payloads specifically to tumors. Given the known stromal barrier in pancreatic ductal adenocarcinoma, TMAB3 provides a novel targeted delivery platform for a broad range of therapeutics for these difficult-to-treat tumors. Citation Format: Diana Martinez-Saucedo, Elias Quijano, Zaira Ianniello, Madison Rackear, Yanfeng Liu, Denise Hegan, Haoting Chen, Xinning Shan, Robert Tseng, Deanne Yugawa, Natasha Pinto-Medici, Sumedha Chowdhury, Ranjit S Bindra, Marie E Robert, W. Mark Saltzman, Luisa F Escobar-Hoyos, Peter M Glazer. Systemic targeting of therapeutic RNA to pancreatic ductal adenocarcinoma via a novel, cell-penetrating, and nucleic acid-binding monoclonal antibody [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 PR-01.

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.

Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

Low migratory dendritic cell (DC) levels pose a challenge in cancer immune surveillance, yet their impact on tumor immune status and immunotherapy responses remains unclear. We present clinical evidence linking reduced migratory DC levels to immune-cold tumor status, resulting in poor patient outcomes. To address this, we develop an autologous DC-based nanovaccination strategy using patient-derived organoid or cancer cell lysate-pulsed cationic nanoparticles (cNPs) to load immunogenic DC-derived microvesicles (cNPcancer cell@MVDC). This approach transforms immune-cold tumors, increases migratory DCs, activates Tcells and natural killer cells, reduces tumor growth, and enhances survival in orthotopic pancreatic and lung cancer models, surpassing conventional methods. Invivo imaging reveals superior cNPcancer cell@MVDC accumulation in tumors and lymph nodes, promoting immune cell infiltration. Mechanistically, cNPs enrich mitochondrial DNA, enhancing cGAS-STING-mediated DC activation and migration. Our strategy shifts cold tumors to a hot state, enhancing antitumor immunity for potential personalized cancer treatments.

Identifying the Morphological and Molecular Features of a Cell-Based Orthotopic Pancreatic Cancer Mouse Model during Growth over Time.

Pancreatic ductal adenocarcinoma (PDAC), characterized by hypovascularity, hypoxia, and desmoplastic stroma is one of the deadliest malignancies in humans, with a 5-year survival rate of only 7%. The anatomical location of the pancreas and lack of symptoms in patients with early onset of disease accounts for late diagnosis. Consequently, 85% of patients present with non-resectable, locally advanced, or advanced metastatic disease at diagnosis and rely on alternative therapies such as chemotherapy, immunotherapy, and others. The response to these therapies highly depends on the stage of disease at the start of therapy. It is, therefore, vital to consider the stages of PDAC models in preclinical studies when testing new therapeutics and treatment modalities. We report a standardized induction of cell-based orthotopic pancreatic cancer models in mice and the identification of vital features of their progression by ultrasound imaging and histological analysis of the level of pancreatic stellate cells, mature fibroblasts, and collagen. The results highlight that early-stage primary tumors are secluded in the pancreas and advance towards infiltrating the omentum at week 5-7 post implantation of the BxPC-3 and Panc-1 models investigated. Late stages show extensive growth, the infiltration of the omentum and/or stomach wall, metastases, augmented fibroblasts, and collagen levels. The findings can serve as suggestions for defining growth parameter-based stages of orthotopic pancreatic cancer models for the preclinical testing of drug efficacy in the future.

Abstract 3323: Novel Pan-RAS inhibitor ADT-007: A unique mechanism of antitumor selectivity in pancreatic and colorectal carcinoma models

Abstract Multiple types of RAS mutations activate RAS isoforms, HRAS, NRAS, or KRAS, that drive oncogenic signaling leading to uncontrolled cell proliferation and tumor development. RAS mutations occur de novo in approximately one-third of all human cancers, and are especially prevalent in pancreatic, colorectal, and lung tumors. Where RAS mutations are less frequent in other cancers, such as breast cancer, RAS can be pathologically activated by growth factor receptors that engage numerous downstream effectors. The currently FDA-approved KRAS-targeted drugs are designed to covalently inactivate only G12C KRAS mutants, however, this mutation is only prevalent in lung cancer. The most common mutations in pancreatic ductal adenocarcinoma (PDAC) are KRAS-G12D, KRAS-G12V, and KRAS-G12R. Our novel class of pan-RAS inhibitors, exemplified by ADT-007, addresses the urgent need for pan-RAS inhibitors with antitumor activity in cancers harboring these common mutations. These pan-RAS inhibitors have unique pharmacological properties that allow for selective killing of cancer cells harboring RAS mutations. ADT-007 inhibited growth of an array of carcinoma tumor cell lines in vitro with single digit nM IC50 values regardless of RAS mutant allele, and inhibited RAS activation and MAPK/AKT signaling. Because it is a pan-RAS inhibitor, ADT-007 may not be susceptible to commonly reported mechanisms of resistance to covalent KRAS inhibitors, such as activation of wild type (WT) alleles or secondary RAS mutations. Differentiated normal epithelial or hepatic cells, and tumor cells with WT RAS were insensitive to ADT-007 due to enzymatic detoxification of ADT-007 via glucuronidation, representing a novel mechanism of tumor selectivity. An orally bioavailable prodrug of ADT-007 (ADT-1004) is well tolerated in vivo, and produced sustained concentrations of ADT-007 well above IC50 values in plasma and even higher levels in tumors. Daily oral administration of ADT-1004 significantly inhibited RAS signaling and tumor growth in orthotopic and PDX mouse models of pancreatic cancer. Citation Format: Junwei Wang, Adam B. Keeton, Sindhu Ramesh, Peyton Johnson, Yulia Y. Maxuitenko, Jeremy B. Foote, Chung-Hui Huang, Kristy L. Berry, Khalda Fadlalla, Bandi D. Reddy, Ganji P. Nagaraju, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Xi Chen, Bassel F. El-Rayes, Donald J. Buchsbaum, Gary A. Piazza. Novel Pan-RAS inhibitor ADT-007: A unique mechanism of antitumor selectivity in pancreatic and colorectal carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3323.

Abstract 1598: Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models

Abstract Background &amp; Aim: Progression and therapeutic resistance in pancreatic cancer depends on the ability of cancer cells to adopt specific cell states. These phenotypes largely depend on environmental tissue niches. Here, we investigated therapy-induced tumor dynamics in vivo at single-cell spatial and temporal resolution in different subtypes of PDAC using several genetically engineered mouse models, which reflect distinct human PDAC ecosystems. Methods &amp; Results: We established orthotopic tumor models of pancreatic cancer with distinct tumor microenvironment (TME) formation (reactive vs deserted stroma), which were monitored for tumor growth to define tumor response patterns under MAPK inhibition. Serial ultrasound-guided biopsy sampling was performed to collect tissues from the same tumors at pre-treatment, responsive and recurrence periods.Transcriptomic analysis of biopsies using the Mfuzz algorithm delineated highly dynamic gene and pathway activities during treatment. Reactive, but not deserted, TME showed an upregulation of immune-related pathways (antigen presentation, T cell receptor, NK cytotoxicity) immediately after MAPK inhibition in regressed tumors that dropped dramatically during recurrence of tumors, in which upregulation of TGF-b signaling was observed. In the deserted stroma model, on the other hand, an enrichment in drug metabolic pathways during recurrence was observed.CAF cluster deconvolution analysis revealed an increase in a LRRC15+ CAF cluster, which was TGF-b-driven and immunosuppressive, during recurrence period only in the reactive model. The dynamic changes of immune cells and CAFs were validated at protein level by multiplex immunofluorescent imaging. Cmputational spatial analysis revealed a tumor-encapsulating and CD8+ cytotoxic T cell-repelling pattern exhibited by LRRC15+FAP+ CAFs, implying a potential role of this CAF subset in T cell suppression during recurrence in reactive subtype. Using spectral cell sorting with CellView image technology, specific CAF subsets, tumor cells as well as immune cell subsets based on 30+ markers and cell morphology were isolated, characterized and assessed by ex vivo functional assays to confirm the immunosuppressive and tumor protective mechanism mediated by CAFs. Conclusion: Longitudinal in vivo assessment of distinct PDAC subtypes provides deep mechanistic insights to accurately unlock specific response patterns of tumors with heterogeneous TME dynamics upon therapeutic perturbation, leveraging identification of key nodes for intervention. Citation Format: Jens T. Siveke, Rui Fang, Phyllis Fung-Yi Cheung, Jiajin Yang, Juanfei Peng, Kristina Althoff, Konstantinos Savvatakis. Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1598.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2367.

Efficient Assessment of Tumor Vascular Shutdown by Photodynamic Therapy on Orthotopic Pancreatic Cancer Using High-Speed Wide-Field Waterproof Galvanometer Scanner Photoacoustic Microscopy.

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities.

Biodistribution and therapeutic efficacy of a gold nanoparticle-based targeted drug delivery system against pancreatic cancer

Pancreatic cancer is characterized by desmoplasia; crosstalk between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) leads to the deposition of extracellular matrix proteins in the tumor environment resulting in poor vascularity. Targeting either PCCs or PSCs individually has produced mixed results, and there is currently no effective strategy to target both cell types simultaneously. Previously, we demonstrated, through in vitro cell culture experiments, that a specific gold nanoparticle-based nanoformulation containing the anti-EGFR antibody cetuximab (C225) as a targeting agent and gemcitabine as a chemotherapeutic agent effectively targets both PCCs and PSCs simultaneously. Herein, we extend our studies to test the ability of these in vitro tested nano formulations to inhibit tumor growth in an orthotopic co-implantation model of pancreatic cancer in vivo. Orthotopic tumors were established by co-implantation of equal numbers of PCCs and PSCs in the mouse pancreas. Among the various formulations tested, 5 nm gold nanoparticles coated with gemcitabine, cetuximab and poly-ethylene glycol (PEG) of molecular weight 1000 Da, which we named ACGP441000, demonstrated optimal efficacy in inhibiting tumor growth. The current study reveals an opportunity to target PCCs and PSCs simultaneously, by exploiting their overexpression of EGFR as a target, in order to inhibit pancreatic cancer growth.

Roflumilast inhibits tumor growth and migration in STK11/LKB1 deficient pancreatic cancer

Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.

Abstract C062: Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting

Abstract Background: Patients with pancreatic cancer (PC) often experience cachexia, a multifactorial syndrome that includes weight loss, malnutrition, appetite suppression, and tissue wasting. Unfortunately, the factors secreted from the pancreas or other tissues that drive cachexia are still poorly defined. Lipocalin 2 (LCN2) is a cytokine involved in innate immunity that we have previously shown to be increased in the pancreas and circulation of patients with PC and mouse models of PC. Recent studies suggest that LCN2 contributes to appetite suppression in cancer cachexia in orthotopic models of PC. However, a reduction in muscle regeneration is also commonly seen in cachexia that is not repaired by increased nutrient intake. In a non-cancer setting, LCN2 has been implicated as an important factor in mediating skeletal muscle repair. Therefore, we hypothesized that elevated LCN2 in PC contributes to cachexia by inhibiting muscle progenitor cell proliferation and differentiation into new muscle fibers. Methods: Muscles from a syngeneic orthotopic mouse model of PC were assessed for muscle RING-finger protein-1 (MuRF1, an E3 ubiquitin ligase highly expressed in muscle during wasting) and LCN2 by qPCR. C2C12 mouse muscle myoblasts were cultured in proliferation or differentiation media with recombinant mouse LCN2. Proliferation across 3 days was assessed by MTS assay. Differentiation was assessed by imaging myotubes and western blotting for myosin heavy chain (MyHC) expression which increases during differentiation. PC genetically engineered mice ± a whole body LCN2 were assessed for pancreatic tumor and gastroc muscle size. One-way and Two-way ANOVAs were used to determine statistical significance. Results: LCN2 and MuRF1 expression are elevated in muscles of mice with PC. C2C12 cells treated with LCN2 decreased proliferation in a dose-dependent manner up to concentrations of LCN2 commonly observed in PC. LCN2 treatments at a physiologic concentration observed in PC delayed differentiation of C2C12 cells into myotubes by reducing myotube diameter and delaying MyHC expression. Deleting LCN2 in a PC genetic mouse model prevented body weight and gastroc weight loss but did not affect pancreas weight. Conclusions: Here, we show a relationship between muscle wasting and LCN2 expression during PC. Elevated circulating LCN2 may be contributing to decreased muscle regeneration observed in PC-associated cachexia by directly inhibiting muscle progenitor cell proliferation and differentiation. Targeting LCN2 in PC may be a viable method of slowing or preventing muscle wasting in PC to improve patient outcomes. Citation Format: Kristyn Gumpper-Fedus, Valentina Pita Grisanti, Ericka Velez Bonet, Martha A. Belury, Christopher Coss, Zobeida Cruz-Monserrate. Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C062.

Abstract C034: Genome-wide CRISPR screen identifies novel immunotherapeutic targets in pancreatic cancer

Abstract Background: Cancer-cell-intrinsic properties dictate the immune contexture of tumors, impacting the outcome of immunotherapy. Despite the increasing value of CRISPR/Cas9-based loss-of-function (LOF) genetic screens for systematically assessing gene knockout phenotype in tumor cells, it still present challenges, particularly when it comes to genomic editing in the context of large scale experiments and the identification of immunomodulatory genes in in vivo application. To address this, we have established an AsCpf1 (Cas12a)-based screen platform to enable comprehensive whole genome in vivo LOF screening and uncover novel immunotherapeutic targets for pancreatic cancer, which is notoriously resistant to current immunotherapies. Methods: We designed and constructed the optimized minimal CRISPR/AsCpf1 library that targets the entire mouse genome and is only one-fourth the size of the existing genome-wide Cas9-based library. To identify novel immune-related gene vulnerabilities in pancreatic cancer, we infected cell cultures derived from the classic p48-Cre KrasLSL-G12D/+ p53+/- genetically engineered pancreatic cancer mouse model with our AsCpf1 library for in vivo LOF screen in immune-competent and -incompetent mice. Next generation sequencing analysis was performed with the resulting tumors. The potential immunomodulatory hits were called out through BAGEL analysis, using a false discovery rate (FDR) &amp;lt; 0.05, and by comparing between the two mouse groups. Results: Our AsCpf1-based screen identified highly confident hits with potential immunomodulation properties. Notably, we observed depletions of various components of the MHC complex, including H13, specifically in the immune-competent background, implicating their requirement for the regulation of anti-tumor immunity. Depletion of H13 with CRISPR/Cas9 system significantly suppressed pancreatic tumor growth in T-cell dependent manner. Further immune profiling revealed that the loss of H13 triggered potent immune response which was featured by a substantial increase in effective CD8+ T cells and reduction in immunosuppressive macrophages in both subcutaneous and orthotopic models of pancreatic cancer. These findings are in accordance with our clinically relevant analysis showing that low expression of H13 is correlated with improved patient survival in pancreatic cancer and augmented responsiveness to immune checkpoint therapies in several cancer types. Conclusions: Our AsCpf1-based LOF screening platform enables unbiased exploration of in vivo functional players at whole-genome level, which can be applied to diverse disease context and models. In our lead screen, we have identified H13 as a promising immunomodulatory target for patients with pancreatic cancer. Citation Format: Xiaofei Wang, Jintan Liu, Giulio Draetta, Haoqiang Ying, Wantong Yao. Genome-wide CRISPR screen identifies novel immunotherapeutic targets in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C034.

Drug-repurposing by virtual and experimental screening of PFKFB3 inhibitors for pancreatic cancer therapy

Pancreatic cancer (PC) is the most frequently occurring cancer, with few effective treatments and a 5-year survival rate of only about 11%. It is characterized by stiff interstitium and pressure on blood vessels, leading to an increased glycolytic metabolism. PFKFB3 plays an important role in glycolysis, and its products (fructose-2,6-bisphosphate), which are allosteric PFK1 activators, limit the glycolytic rate. In this study, 14 PFKFB3 inhibitors were obtained by virtually screening the FDA-approved compound library. Subsequently, the in-vitro investigations confirmed that Lomitapide and Cabozantinib S-malate exhibit the excellent potential to inhibit PFKFB3. The combined administration of Lomitapide and Gemcitabine at a certain molar ratio indicated an enhanced anti-tumor effect in Orthotopic Pancreatic Cancer (OPC) models. This investigation provides a new treatment strategy for PC therapy.

Photo-Triggered Cascade Therapy: A NIR-II AIE Luminogen Collaborating with Nitric Oxide Facilitates Efficient Collagen Depletion for Boosting Pancreatic Cancer Phototheranostics.

The dense extracellular matrix (ECM) in the pancreatic cancer severely hampers the penetration of nanodrugs, which causes inferior therapeutic efficacy. To address this issue, a multifunctional liposome, namely, Lip-DTI/NO, integrating a type-I photosensitizer DTITBT with glutathione (GSH) or heat-responsive nitric oxide (NO) donor S-nitroso-N-acetyl-D-penicillamine (SNAP) is constructed to deplete the tumor ECM, leading to enhanced drug delivery and consequently improved phototherapy. The loaded DTITBT possesses multiple functions including NIR-II fluorescence imaging, efficient superoxide radical (O2 •-) generation and excellent photothermal conversion efficiency, making it feasible for precisely pinpointing the tumor in the phototherapy process. Responding to the intracellular overexpressed glutathione or heat produced by photothermal effect of DTITBT, NO can be released from SNAP. Upon 808nm laser irradiation, Lip-DTI/NO could selectively induce in situ generation of peroxynitrite anion (ONOO-) in tumor after cascade processes including O2 •- production, GSH or heat-triggered NO release, and rapid reaction between O2 •- and NO. The generated ONOO- could activate the expression of endogenous matrix metalloproteinases which could efficiently digest collagen of tumor ECM, thus facilitating enhanced penetration and accumulation of Lip-DTI/NO in tumor. In vivo evaluation demonstrates the notable therapeutic efficacy via ONOO--potentiated synergistic photodynamic-photothermal therapies on both subcutaneous and orthotopic pancreatic cancer model.