Pancreatic Cancer-bearing Mice Research Articles

Autophagy inhibition improves the targeted radionuclide therapy efficacy of 131I-FAP-2286 in pancreatic cancer xenografts

PurposesRadiotherapy can induce tumor cell autophagy, which might impair the antitumoral effect. This study aims to investigate the effect of autophagy inhibition on the targeted radionuclide therapy (TRT) efficacy of 131I-FAP-2286 in pancreatic cancer.MethodsHuman pancreatic cancer PANC-1 cells were exposed to 131I-FAP-2286 radiotherapy alone or with the autophagy inhibitor 3-MA. The autophagy level and proliferative activity of PANC-1 cells were analyzed. The pancreatic cancer xenograft-bearing nude mice were established by the co-injection of PANC-1 cells and pancreatic cancer-associated fibroblasts (CAFs), and then were randomly divided into four groups and treated with saline (control group), 3-MA, 131I-FAP-2286 and 131I-FAP-2286 + 3-MA, respectively. SPECT/CT imaging was performed to evaluate the bio-distribution of 131I-FAP-2286 in pancreatic cancer-bearing mice. The therapeutic effect of tumor was evaluated by 18F-FDG PET/CT imaging, tumor volume measurements, and the hematoxylin and eosin (H&E) staining, and immunohistochemical staining assay of tumor tissues.Results131I-FAP-2286 inhibited proliferation and increased the autophagy level of PANC-1 cells in a dose-dependent manner. 3-MA promoted 131I-FAP-2286-induced apoptosis of PANC-1 cells via suppressing autophagy. SPECT/CT imaging of pancreatic cancer xenograft-bearing nude mice showed that 131I-FAP-2286 can target the tumor effectively. According to 18F-FDG PET/CT imaging, the tumor growth curves and immunohistochemical analysis, 131I-FAP-2286 TRT was capable of suppressing the growth of pancreatic tumor accompanying with autophagy induction, but the addition of 3-MA enabled 131I-FAP-2286 to achieve a better therapeutic effect along with the autophagy inhibition. In addition, 3-MA alone did not inhibit tumor growth.Conclusions131I-FAP-2286 exposure induces the protective autophagy of pancreatic cancer cells, and the application of autophagy inhibitor is capable of enhancing the TRT therapeutic effect.

CCR4 Blockade Diminishes Intratumoral Macrophage Recruitment and Augments Survival of Syngeneic Pancreatic Cancer-Bearing Mice.

Pancreatic cancer is known for its tumor microenvironment (TME), which is rich in stromal and immune cells supporting cancer growth and therapy resistance. In particular, tumor-associated macrophages (TAMs) are known for their angiogenesis- and metastasis-promoting properties, which lead to the failure of conventional therapies for pancreatic cancer. Hence, treatment options targeting TAMs are needed. The C-C chemokine receptor type 4 (CCR4) is critical for immune cell recruitment into the TME, and in this paper we explore the effects of its genetic or immunotherapeutic blockade in pancreatic-cancer-bearing mice. Murine PDA6606 pancreatic cancer cells and murine peritoneal macrophages were used for in vitro migration assays. In vivo, a syngeneic, orthotropic pancreatic cancer model was established. Tumor growth and survival were monitored under prophylactic and therapeutic application of a CCR4 antagonist (AF-399/420/18025) in wildtype (CCR4wt) and CCR4-knockout (CCR4-/-) mice. Immune infiltration was monitored in tumor tissue sections and via flow cytometry of lysed tumors. PDA6606 cells induced less migration in CCR4-/- than in CCR4wt macrophages in vitro. Pancreatic TAM infiltration was higher, and survival was reduced in CCR4wt mice compared to CCR4-/- mice. Antagonizing CCR4 in wildtype mice revealed similar results as in CCR4-/- mice without antagonization. Prophylactic CCR4 antagonist application in wildtype mice was more efficient than therapeutic antagonization. CCR4 seems to be critically involved in TAM generation and tumor progression in pancreatic cancer. CCR4 blockade may help prolong the relapse-free period after curative surgery in pancreatic cancer and improve prognosis.

Abstract 3546: DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment

Abstract Existing therapeutics have failed to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes, in part due to tumor associated immune suppression and upregulation of compensatory mechanisms. KRAS and c-MYC are important oncogenes in PDAC linked to tumor immune suppression. We describe the use of difluoromethylornithine (DFMO) and a c-RAF inhibitor (GW5074) to indirectly target c-MYC and KRAS, respectively. DFMO in combination with GW5074 was tested for ability to improve pancreatic cancer outcomes. Pancreatic cancer cells showed decreased cell viability and increased apoptosis in response to a combination of DFMO + GW5074. In vivo orthotopic models of pancreatic tumors had decreased tumor weight in DFMO + GW5074 treated mice, when compared to control group. However, only treatment with single agent DFMO resulted in improved survival of immunocompetent pancreatic tumor bearing mice with respect to control, in contrast to treatment with GW5074 alone or DFMO + GW5074. To understand why the in vivo data was contrary to in vitro results, immunohistochemical analysis of immune cells in the tumor microenvironment was used to reveal increased expression of markers associated with anti-tumor effects such as CD86, CD3, CD4 and CD8 in DFMO treated tumors. Tumors treated with DFMO also displayed decreased expression of MYC, suggesting that DFMO-associated MYC suppression could be linked to decreased immune suppression in the tumor microenvironment and improved survival. In contrast, GW5074 treatment maintained MYC expression in tumors. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based therapeutic strategies in PDAC. Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Jordan M. Beardsley, Veethika Pandey, Michael M. Rohr, Jignesh Parikh, Otto Phanstiel, Deborah A. Altomare. DFMO based improvement in survival of pancreatic cancer-bearing mice is associated with modulation of immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3546.

Solubilization of Hexyl Aminolevulinate by Surfactants for Tumor Fluorescence Detection.

Hexyl aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinic acid (5-ALA) and can induce more protoporphyrin IX (PpIX) formation and stronger fluorescence intensity (FI) than 5-ALA, which will greatly facilitate photodynamic diagnosis and therapy. The main drawback of HAL is its low solubility in neutral aqueous media. In this study, surfactants were used to increase HAL solubility in the cell culture medium and serum, followed by in vitro fluorescence formation measurement in human pancreatic cancer cells (SW1990) and in vivo fluorescence detection in tumor-bearing mice. The results showed that Tween 80 (TW80) and Kolliphor® HS 15 (HS15) increased the solubility of HAL in the selected media. Although TW80 and HS15 exhibited in vitro cytotoxicity at high concentrations (5mgmL-1 ), they facilitated fluorescent signal formation at the early stage of cell incubation. When surfactants were used, the FI should be determined without the routine washing process because surfactant-containing culture medium caused the loss of synthesized PpIX during the washing process. When HAL dissolved in TW80 solution was injected intraperitoneally into pancreatic cancer-bearing mice at a dose of 50mgkg-1 , the tumors exhibited red fluorescence, which indicated that systemic administration of surfactant-solubilized HAL might be applicable for tumor fluorescence detection in vivo.

Abstract C099: Anticancer immunotherapy by MFAP5 blockade inhibits fibrosis and enhances chemosensitivity in ovarian and pancreatic cancer

Abstract Recent studies demonstrate the role of the tumor microenvironment in tumor progression. However, strategies used to overcome the malignant phenotypes of cancer cells modulated by the microenvironment have not been thoroughly explored. In this study, we evaluated the therapeutic efficacy of a newly developed monoclonal antibody targeting microfibril associated protein 5 (MFAP5), which is secreted predominately by cancer associated fibroblasts (CAFs), in ovarian and pancreatic cancer models.MFAP5-targeting monoclonal antibodies were developed using purified full-length human MFAP5 recombinant protein as an antigen in mice and antibodies from hybridoma clones were evaluated for their toxicity, and specificity to human and murine MFAP5. An Octet RED384 system was used to determine the kinetics of binding affinity and the specificity of the antibody clones, which were followed by epitope mapping and functional characterization by in vitroassays. The therapeutic efficacy of a lead anti-MFAP5 antibody clone 130A in tumor suppression was evaluated by ovarian tumor- and pancreatic tumor-bearing mouse models. The results demonstrated that antibody clone 130A recognized a common epitope shared between human and murine MFAP5 protein. Kinetic assays showed that the dissociation constant (Kd) of clone 130A for human and mouse MFAP5 protein were at 1.93nM and 2.51nM respectively, suggesting that clone 130A has high binding affinity to both human MFAP5 and mouse MFAP5 protein. Toxicity studies of clone 130A showed that mice treated with MAbs had no adverse effects in complete blood counts, serum ALT, AST, alkaline phosphatase and urea nitrogen levels, and major organ histology. In vitrofunctional studies showed that clone 130A suppressed ovarian and pancreatic cancer cell, and endothelial cell motility. In vivostudies showed that clone 130A suppressed tumor growth in ovarian and pancreatic cancer-bearing mice, down-regulated MFAP5-induced collagen production in CAFs, suppressed intratumoral microvessel leakiness, and enhanced paclitaxel bioavailability in both ovarian and pancreatic cancer mouse models. These data suggest that MFAP5 blockade using an immunologic approach inhibits fibrosis, induces tumor vessel normalization and enhances chemosensitivity in ovarian and pancreatic cancer, and can be used as a novel therapeutic agent in treating these diseases. Citation Format: Tsz-Lun Yeung, Cecilia Leung, Sammy Ferri-Borgogno, Kay-Pong Yip, Jianting Sheng, Long Vein, Laura Bover, Michael Birrer, Stephen Wong, Samuel C Mok. Anticancer immunotherapy by MFAP5 blockade inhibits fibrosis and enhances chemosensitivity in ovarian and pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C099. doi:10.1158/1535-7163.TARG-19-C099

Contrast-enhanced ultrasound imaging of acute changes in pancreatic cancer following targeted hyaluronan treatment.

The purpose of this study was to monitor acute changes in pancreatic tumor perfusion with contrast-enhanced ultrasound (CEUS) imaging following targeted hyaluronan (HA) treatment. Intratumoral accumulation of HA is one of contributing factors that can lead to an increased tumor interstitial pressure (TIP). These elevated TIP levels can hinder delivery of chemotherapeutic drugs and cause treatment failure. For this study, pancreatic cancer-bearing mice were imaged at baseline and again at 2 h after intravenous administration of physiological saline (control group) or PEGPH20, which targets HA (therapy group). CEUS data were collected for 5 min and the temporal sequence was first analyzed using a singular value filter (SVF) to remove any background clutter signal. Given the time history of contrast agent flow, a tumor perfusion parametric analysis was performed. A series of morphological image operations was applied to quantify structural features of the tumor angiogenic network including vessel count, density, length, diameter, tortuosity, and branching points. After imaging, animals were euthanized, and tumors excised for histological processing. Acute microvascular changes were found at 2 h after drug administration as confirmed by CEUS imaging. Further, histologic analysis of tumor sections revealed lower HA accumulation in the therapy group animals. Overall, these findings suggest that CEUS imaging of acute changes in tumor perfusion may help identify an optimal window whereby follow-up chemotherapeutic drug dosing would be more effective.

Abstract 1231: Improving survival in pancreatic cancer using Doxorubicin in combination with L-DOS47

Abstract BACKGROUND: The tumor microenvironment is acidic, which inhibits the activity of weakly basic drugs such as doxorubicin. In order to test whether neutralization of tumor acidity can improve survival in pancreatic cancer-bearing mice treated with doxorubicin, we tested doxorubicin in combination with L-DOS47. L-DOS47 is a novel therapy comprised of urease conjugated to an antibody against CEACAM6, which raises the pH in the tumor microenvironment by converting endogenous urea to NH3 and CO2. Our findings demonstrate that raising tumor pH with L-DOS47 can improve responses to chemotherapy. As L-DOS47 is currently in clinical testing for NSCLC, current study is clinically relevant. METHODS: Panc02 pancreatic cancer cell line (obtained from Emmanual Zervos, East Carolina University, Greenville, NC) was infected to express CEACAM6. 1 x 106 CEACAM6 Panc02 Clone were injected subcutaneously in the right flank of C57BL/6 mice. Four days later, tumor sizes were measured with calipers and mice were forcibly randomized into 3 groups (10 mice/ group). The 3 groups were treated with Doxorubicin only; L-DOS47 4 hours before Doxorubicin; and L-DOS47 24 hours before Doxorubicin. The rationale for different timings were that MRS measurements of tumor pH indicate that tumor alkalization has been observed both 4 and 24 hours following injection of L-DOS47. Treatments were begun immediately after randomization. Doxorubicin (2.5 mg/kg) was injected every 3-4 days and L-DOS47 (90 μg/kg) was injected either 4 or 24 hour prior to Doxorubicin i.v. RESULTS: All mice were humanely euthanized when tumors either reached 2000 mm3 (endpoint) or had serious ulcerations. For the tumors hadn’t reached to the endpoint due to serious ulcerations, times to reach the endpoint were estimated using a mathematical model that describes exponential tumor growth. Kaplan Meier survival curves were generated for each treatment group. According to Log-rank Mantel-Cox, Log-rank for trend, Gehan-Breslow-Wilcoxon test observed mice group survival was significantly different from each other (p<0.008 for all 3 tests). The probability of survival of mice group treated with L-DOS47 either 4 or 24 hours before doxorubicin was significantly better than doxorubicin only group. CONCLUSION: In this study we have observed that neutralizing tumor pH can improve the effect of chemotherapy in vivo. This is consistent with prior observations where neutralization with bicarbonate improved the response to chemotherapy via reversal of an ion-trapping effect [1]. The current work is significant, however, as L-DOS47 can be used clinically, whereas bicarbonate cannot.

Long non-coding RNA GAS5 antagonizes the chemoresistance of pancreatic cancer cells through down-regulation of miR-181c-5p

ObjectiveTo explore the core mechanism of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in the regulation of multidrug resistance of pancreatic cancer cells. MethodsmRNA levels of GAS5, miR-181c-5p and Hippo pathway related genes were detected by quantitative real-time PCR (qRT-PCR). Protein levels of MDR-1, MST1, YAP and TAZ were measured by western blot. Cell viability was detected by MTT assay. The combination between GAS5 and miR-181c-5p was confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assay. We also established pancreatic cancer-bearing mice model and analyzed tumor volumes. ResultsOur data showed GAS5 expression was significantly down-regulated, miR-181c-5p expression was significantly up-regulated in pancreatic cancer cells. Besides, Overexpresson of GAS5 obviously inhibited cell viability, while GAS5 knockdown showed the opposite outcome. Additionally, we also found that GAS5 negatively regulated miR-181c-5p, and miR-181c-5p dramatically promoted pancreatic cancer cell chemoresistance through inactivating the Hippo signaling. GAS5 regulated chemoresistance and Hippo pathway of pancreatic cancer cells via miR-181c-5p/Hippo. Finally, we confirmed that overexpression of GAS5 inhibited tumor growth in pancreatic cancer-bearing mice model. ConclusionGAS5 regualtes Hippo signaling pathway via miR-181c-5p to antagonize the development of multidrug resistance in pancreatic cancer cells.

Preliminary data of the antipancreatic tumor efficacy and toxicity of long-circulating and pH-sensitive liposomes containing cisplatin.

Pancreatic cancer is the fourth most common cause of cancer-related death in the USA. This is mainly because of the chemoresistance of this type of tumor; thus, the development of novel therapeutic modalities is needed. Long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP) were administered systemically into pancreatic tumor-bearing mice for a period of 14 days. The antitumor efficacy and toxicity of this new treatment method on the basis of cisplatin-loaded liposomes was compared with the classical free-CDDP method. Tc-HYNIC-βAla-bombesin(7-14) tumor uptake and histopathologic findings were used to monitor and compare the two treatment modalities. The antitumor activity of SpHL-CDDP treatment was shown by (a) decrease in tumor volume, (b) development of tumor necrotic areas, and (c) decrease in Tc-HYNIC-βAla-bombesin(7-14) tumor uptake. Toxicity was evaluated by the development of inflammation and necrotic areas in the kidneys, liver, spleen, and intestine: toxic effects were greater with free-CDDP than SpHL-CDDP. SpHL-CDDP showed significant antitumor activity in pancreatic cancer-bearing mice, with lower toxicity in comparison with free-CDDP.

Targeted activation of melanoma differentiation-associated protein 5 (MDA5) for immunotherapy of pancreatic carcinoma

The RIG-I-like helicase melanoma differentiation-associated protein 5 (MDA5) is an innate immune receptor for double-stranded viral RNA (dsRNA) that, upon activation, induces a Type I interferon (IFN)-driven immune response. In the present study, we demonstrate that human und murine pancreatic cancer cells express functional MDA5 and are highly sensitive to MDA5-induced cell death. Activation of MDA5 by cytosolic delivery of the synthetic dsRNA analog poly(I:C) led to phosphorylation of the transcription factor IRF3, IFNβ production and upregulation of MHC-I expression. MDA5 signaling also induced tumor cell apoptosis via the intrinsic pathway and sensitized tumor cells toward extrinsic, Fas-mediated apoptosis. Systemic treatment of orthotopic pancreatic cancer-bearing mice with the MDA5 ligand resulted in activated CD8+ T cell tumor infiltration, an increased frequency of tumor antigen-specific CD8+ T cells and an immunogenic cytokine milieu in the tumor microenvironment. These effects were paralleled by MDA5-induced pronounced tumor cell death in situ and significantly prolonged survival in two different mouse models for pancreatic cancer, an immunotherapeutic response dependent on CD8+ T cells. Treated mice were further protected from subsequent tumor challenge. In summary, we identified MDA5 as a novel therapeutic target for overcoming apoptosis resistance and tumor-mediated immunosuppression in pancreatic cancer. MDA5 ligands link innate with adaptive immune mechanisms for effective tumor control.

Antitumor activity of mHSP65-TTL enhanced by administration of low dose cyclophosphamide in pancreatic cancer-bearing mice

Pancreatic cancer remains a lethal malignancy. Despite chemotherapy or/and radiotherapy after the surgery, the improvement on the overall survival of the patients has still been minimal. To develop novel therapeutic approaches, we tried to prepare mHSP65-TTL, a candidate vaccine prepared by mixing the recombinant mycobacterial heat shock protein 65 (mHSP65) with tumor tissue lysate (TTL) of Panc02 pancreatic cancer tissue. The mHSP65-TTL were used to immune the C57BL/6 mice implanted with the Panc02 cancer cells, in combination with or without low dose cyclophosphamide (CY). The results showed that mHSP65-TTL significantly prolonged the survival of the pancreatic cancer bearing mice and low dose CY enhanced the efficacy of the mHSP65-TTL. In addition, we detected mRNA expression of RORγt and IL-17A in spleen cells of mice received mHSP65-TTL or mHSP65-TTL plus CY, and found that mHSP65-TTL up-regulated mRNA expressions of RORγt and IL-17A, CY alone or mHSP65-TTL plus CY up-regulated mRNA expressions of RORγt. The work could provide an insight into a combinational approach for the immunotherapy of pancreatic cancer.

Nafamostat mesilate can prevent adhesion, invasion and peritoneal dissemination of pancreatic cancer thorough nuclear factor kappa‐B inhibition

Constitutive activation of nuclear factor kappa-B (NF-κB) contributes to the aggressive behavior of pancreatic cancer. Over-expression of downstream target genes of NF-κB such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) leads to the promotion of cell adhesion, angiogenesis, invasion and metastasis. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, blocks NF-κB activation in pancreatic cancer. We hypothesized that nafamostat mesilate may inhibit cell adhesion, angiogenesis, invasion and metastases in peritoneal dissemination of pancreatic cancer. In vitro, we assessed inhibition of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9 activity by nafamostat mesilate using human pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Changes in adhesion and invasion abilities of cancer cells were then evaluated by nafamostat mesilate treatment. In vivo, the efficacy of nafamostat mesilate treatment was assessed using peritoneal dissemination of pancreatic cancer in mice. In vitro, nafamostat mesilate inhibited activities of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9. Moreover, nafamostat mesilate not only inhibited cell adhesion and invasion but also increased the sensitivity of anoikis. In vivo, tumor growth using AsPC-1 cells of the treatment group was significantly slower, and survival rate was significantly better, than those in control group (p < 0.05). Nafamostat mesilate reduced peritoneal metastasis and prolonged survival of pancreatic cancer-bearing mice.

Effect of transplantation of pro-angiogenic monocytes to pancreatic cancer-bearing mice on resistance to chemotherapy/radiotherapy.

212 Background: Hypoxic tumors are usually resistant to conventional chemotherapy and radiotherapies, which typically target actively dividing cells. The tumor vasculatures are unorganized and lack adequate pericyte coverage, which compromises delivery of drugs to tumors. How best to normalize the aberrant tumor vasculature to maximize anticancer drug delivery comprises an area of intensive investigation. Methods: We tested out hypothesize that bone marrow (BM) cells may be able to restore appropriate vessel function in tumor vasculature using nude mice bearing pancreatic cancer xenografts and genetically engineered mice that develop pancreatic adenocarcinoma. Results: Culturing BM mononuclear cells with endothelial growth medium resulted in the early outgrowth of spindle-shaped attached monocytic cells expressing CD11b/CXCR4 with a significant vessel stabilizing activity. Intravenous administration of these cultured pro- angiogenic cells into mice bearing pancreatic cancer significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Consistent with a marked reduction in gene expressions involved in drug resistance such as MDR1 and ABCG2 in monocytes-injected tumors, a combination of the transplantation and chemotherapeutic agents reduced tumor size and significantly increased areas of necrosis as compared to chemotherapy alone. Conclusions: Our findings offer an alternate approach to improve delivery and efficacy of anticancer drugs to hypoxic tumors through a remodeling of the abnormal tumor vessels. No significant financial relationships to disclose.