Experimental Pancreatic Cancer Research Articles

Abstract 5610: Targeting Rb1 in experimental pancreatic cancer remodels immune cell landscape and suppresses tumor progression

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) remains among the cancers with the poorest prognosis. Currently, no effective treatment available for PDA. Immunotherapy that has been shown effective for several cancer types is not successful for PDA treatment overall. The possible reasons of this PDA immunotherapy resistance include lack of neoantigen mutations, low T cell responses and overall immunosuppression state in the tumor microenvironment mediated by myeloid cells and tumor stroma. Recent studies suggest that targeting tumor microenvironment can overcome this immunotherapy resistance and significantly improve the disease outcome. Retinoblastoma (Rb1) protein is a known tumor suppressor protein. In addition, there is accumulating evidence for its role in immune cell response as well, including regulation of apoptosis in myeloid cells. However, the mechanisms of this phenomenon remain not clear. Here we show that targeting Rb1 with a novel molecule AP-3-84 developed in our lab causes apoptosis of tumor-associated macrophages (TAMs) in the model of PDA, induces T cell infiltration of the tumor and decreases tumor burden. Results: Recently our lab has developed the new small molecule AP-3-84 that is able to bind Rb1 and modulate its activity. Originally, we found that this Rb1 modulator can induce cell death of TAMs and thioglycolate-induced macrophages, but not of the other cell types (neither tumor nor T cells). Gene expression changes upon AP-3-84 treatment showed the induction of oxidative and ER stress programs with a clear activation of p53-dependent genes and caspases. We tested the use of AP-3-84 as immunotherapy against PDA cancer growth using 2 PDA cell lines intrinsically characterized by low T cell and high T cell infiltration (T-low-PDA and T-high-PDA, respectively, originally generated and characterized by J. Li et al., Immunity 2018). In accordance with our in vitro observation, AP-3-84 was able to reshape immune cell landscape in mice with T-high-PDA by depleting macrophages and inducing T cell infiltration. Importantly, those changes were accompanied by significant reduction of tumor growth. Addition of AP-3-84 to immunotherapy (anti-CD40/PD1/CTL4) was also able to decrease tumor burden in T-low-PDA model, which is otherwise resistant to this immunotherapy. Thus, AP-3-84 treatment was able to significantly improve the outcome of experimental PDA by altering immune cell subsets and represent a prospective clinical approach for the clinic. Statistical analysis of the data was conducted using unpaired Student t-test with additional false discovery rate adjustment for multiple comparisons for gene expression analysis. Conclusions: Modulation of Rb1 in myeloid cells is a novel promising target for PDA treatment. This approach reduces PDA tumor burden by shifting the immune cell landscape towards greater T cell infiltration upon tumor macrophage depletion. Citation Format: Evgenii N. Tcyganov, Taekyoung Kwak, Adi Narayana Reddy Poli, Laxminarasimha Donthireddy, Joel Cassel, Andrew Kossenkov, Rahul Shinde, Ben Z. Stanger, Joseph Salvino, Luis J. Montaner. Targeting Rb1 in experimental pancreatic cancer remodels immune cell landscape and suppresses tumor progression [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 5610.

The antitumoral activity of TLR7 ligands is corrupted by the microenvironment of pancreatic tumors

Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. Invitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. Invivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.

PCL20-116: Can Antisense Oligonucleotides Targeted to K-Ras Gene Inhibit the Tumor Growth, Invasiveness and Expression of MMP-2 and MMP-9 In Vitro and In Vivo in Hamster Experimental Pancreatic Cancer Model?

PCL20-116: Can Antisense Oligonucleotides Targeted to K-Ras Gene Inhibit the Tumor Growth, Invasiveness and Expression of MMP-2 and MMP-9 In Vitro and In Vivo in Hamster Experimental Pancreatic Cancer Model?

Spatiotemporally controlled induction of gene expression in vivo allows tracking the fate of tumor cells that traffic through the lymphatics.

Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo.

Ocoxin Oral Solution Exerts an Antitumoral Effect in Pancreatic Cancer and Reduces the Stromal-Mediated Chemoresistance.

Pancreatic carcinoma is one of the most aggressive cancers overcoming chemoresistance. Thus, novel compounds to complement the current antitumor agents are in need. Ocoxin oral solution (OOS) has proven antioxidant, anti-inflammatory, and antistromagenic properties. The aim of this study was to analyze the effect of OOS in an experimental pancreatic cancer model and its implication in stroma-related chemoresistance to paclitaxel and gemcitabine. Murine pancreatic carcinoma 266-6 cells were treated with OOS to analyze cell cycle and to perform a mRNA comparative microarray study. Then the viability was assessed in combination with paclitaxel and/or gemcitabine. Chemoresistance induced by the medium taken from fibroblast cultures was also investigated on 6 human pancreatic carcinoma cell lines. Furthermore, an experimental model of pancreatic cancer was carried out to study the effect of OOS in vivo. Ocoxin oral solution enhances the cytotoxic effect of paclitaxel and gemcitabine, while it ameliorates the chemoresistance induced by fibroblast-derived soluble factors in human pancreatic cancer cells. The OOS also promotes the regulation of the expression of genes that are altered in pancreatic carcinoma and slows down 266-6 cell pancreatic tumor development in vivo. Ocoxin oral solution could be a potential complement to the chemotherapeutic drugs for pancreatic adenocarcinoma.

Experimental pancreatic cancer develops in soft pancreas: novel leads for an individualized diagnosis by ultrafast elasticity imaging.

Rapid, easy and early pancreatic cancer diagnosis and therapeutic follow up continue to necessitate an increasing attention towards the development of effective treatment strategies for this lethal disease. The non invasive quantitative assessment of pancreatic heterogeneity is limited. Here, we report the development of a preclinical imaging protocol using ultrasonography and shear wave technology in an experimental in situ pancreatic cancer model to measure the evolution of pancreatic rigidity.Methods: Intrapancreatic tumors were genetically induced by mutated Kras and p53 in KPC mice. We evaluated the feasiblity of a live imaging protocol by assessing pancreas evolution with Aixplorer technology accross 36 weeks. Lethality induced by in situ pancreatic cancer was heterogeneous in time.Results: The developed method successfully detected tumor mass from 26 weeks onwards at minimal 0.029 cm3 size. Elastography measurements using shear wave methodology had a wide detection range from 4.7kPa to 166.1kPa. Protumorigenic mutations induced a significant decrease of the rigidity of pancreatic tissue before tumors developed in correlation with the detection of senescent marker p16-positive cells. An intratumoral increased rigidity was quantified and found surprisingly heterogeneous. Tumors also presented a huge inter-individual heterogeneity in their rigidity parameters; tumors with low and high rigidity at detection evolve very heterogeneously in their rigidity parameters, as well as in their volume. Increase in rigidity in tumors detected by ultrafast elasticity imaging coincided with detection of tumors by echography and with the detection of the inflammatory protumoral systemic condition by non invasive follow-up and of collagen fibers by post-processing tumoral IHC analysis.Conclusion: Our promising results indicate the potential of the shear wave elastography to support individualization of diagnosis in this most aggressive disease.

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects.

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10–125 μmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.

The ribosome inhibiting protein riproximin shows antineoplastic activity in experimental pancreatic cancer liver metastasis.

Pancreatic ductal adenocarcinoma (PDAC) has one of the poorest prognoses of all malignancy types. To improve the survival of patients with PDAC, the development of novel anticancer agents is warranted. Riproximin (Rpx) is a newly identified plant lectin, which was isolated from Ximenia americana. The ribosome inactivating protein of type II exhibits potent anticancer activity as recently demonstrated. The rat PDAC cell line ASML was used for in vitro and in vivo studies. The antiproliferative effect of Rpx was assessed using an MTT assay. The modulation of proteins involved in apoptosis was evaluated using western blotting. Tumor-bearing nude rats were treated with Rpx, gemcitabine (GEM) or dinaline (DIN) as single agents, or a combination of Rpx with GEM, or DIN. Rpx was administered intraperitoneally at doses of 1.7–5.4 µg/kg, three times/week, GEM was administered intravenously (50 mg/kg/week) and DIN perorally (10 mg/kg, 5 times/week). Rpx inhibited ASML cell proliferation at IC50-values of 0.8–172 pM, caused apoptosis and reduced tumor growth significantly by 90% (P<0.05). The survival rate of rats was significantly increased (21.8 days for Rpx treated vs. 17.6 days for control rats; P=0.05). Higher doses of Rpx caused no further reduction in tumor size when compared with the low dose of Rpx or a combination of Rpx with GEM, or DIN. The standard drug GEM alone was less effective compared with Rpx. In addition, DIN was ineffective, and in combination, reduced the activity of Rpx. These results suggest that Rpx has an evident potential for use in pancreatic cancer treatment. Further experiments are required in order to elucidate its affinity for certain cancer cells and to optimize the combination therapy with other antineoplastic agents.

Could antisense oligonucleotides targeted to K-ras gene inhibit the tumor growth, invasiveness and expression of MMP-2 and MMP-9 in vitro and in vivo in hamster experimental pancreatic cancer model?

Could antisense oligonucleotides targeted to K-ras gene inhibit the tumor growth, invasiveness and expression of MMP-2 and MMP-9 in vitro and in vivo in hamster experimental pancreatic cancer model?

Abstract 276: Enhancing standard chemotherapy response by targeted inhibition of MAPK signaling pathways in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all solid tumors, with an overall survival rate of less than 6%. Gemcitabine (Gem) remained the standard of care agent over the past 16 years despite limited clinical benefits. Nab-paclitaxel (NPT), a water-soluble albumin-bound formulation of paclitaxel, has recently shown greater efficacy in advanced PDAC. Nab-paclitaxel combination with gemcitabine is now standard of care for advanced PDAC. More than 90% of PDAC tumors harbor an activating mutation in the KRAS oncogene. Since to date no therapeutics have effectively targeted this oncogene product, alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. Trametinib (Tra) is a small molecule inhibitor of the RAF-MEK-ERK (MAPK) signaling pathway that is a well-described mediator of KRAS induced transformation and tumorigenesis. In the present preclinical study, we evaluated combination treatment benefits of the standard chemotherapeutics with trametinib to define a novel therapeutic strategy for PDAC. Median animal survival over controls (20 days) in human intraperitoneal PDAC xenografts was significantly improved by NPT (33 days, a 65% increase, p = 0.0004), Gem (26 days, a 30% increase, p = 0.002), NPT+Gem (39 days, a 95% increase, p = 0.0001) and Tra (31 days, a 55% increase, p = 0.0004) therapy. Survival was further increased by addition of trametinib to chemotherapy: NPT+Tra (median: 37 days, a 85% increase, p = 0.0001), Gem+Tra (34 days, a 70% increase, p = 0.0001) and NPT+Gem+Tra (49 days, a 145% increase, p&amp;lt;0.0001). Treatment of subcutaneous tumor-bearing mice with chemotherapy and trametinib resulted in significant tumor growth inhibition. Net tumor growth in different therapy groups over two weeks varied between controls (432.6 mm3), NPT (105.3 mm3), Tra (184 mm3), NPT+Tra (81 mm3), NPT+Gem (37.3 mm3), and NPT+Gem+Tra (-8.1 mm3). Mean tumor weight (in g) in different therapy groups was as follows: controls 0.38±0.06, NPT 0.23±0.06, Tra 0.31±0.03, NPT+Tra 0.23±0.06, NPT+Gem 0.15±0.05 and NPT+Gem+Tra 0.11±0.05. In vitro studies demonstrated inhibition in PDAC cell (AsPC-1, Panc-1, Mia PaCa-2, CFPAC) proliferation by treatment with NPT+Gem, trametinib, and combination. Immunoblot analysis revealed that trametinib effects were accompanied by decrease in phospho-ERK expression and increase in the expression of apoptosis-related cleaved caspase-3 protein. These findings suggest that the effects of the standard chemotherapeutic regimens can be enhanced through specific inhibition of downstream components of the MAPK signaling pathway, which clinically could lead to improved PDAC therapy effects. Citation Format: Niranjan Awasthi, Sheena Monahan, Margaret A. Schwarz, Roderich E. Schwarz. Enhancing standard chemotherapy response by targeted inhibition of MAPK signaling pathways in experimental pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 276.

Chronic stress increases experimental pancreatic cancer growth, reduces survival and can be antagonised by beta-adrenergic receptor blockade.

Chronic stress could promote tumour growth and reduce survival of pancreatic cancer patients via beta-adrenergic receptors of tumour cells. We have tested the impact of chronic acoustic and restraint stress on tumour development in an orthotopic syngeneic murine model of pancreatic cancer. Tumour-bearing C57BL/6 mice exposed to chronic stress had 45% (p=0.0138) higher circulating steroid and 111% (p=0.0052) higher adrenal tyrosine hydroxylase levels. Their immune response was significantly suppressed: The invitro LPS response of splenocytes was significantly reduced regarding Th1- and Th2-cytokines including IFN-gamma, IL-6, IL-10 and MCP-1 (0.0011<p<0.043). Also, tumours of stressed mice showed a tendency towards fewer total CD4 cells, more regulatory T cells (Treg), less T cell/tumour cell contacts and a reduction of CTLA-4 in CD4 cells (p>0.05). TGF-beta invitro was increased by 23.4% using catecholamines (p<0.012) and invivo employing chronic stress (p<0.001). After 5 weeks tumour volumes were 130% (p=0.0061) larger and median survival reduced by 13.5% (p=0.0058). Tumours expressed more VEGF (p=0.0334), had greater microvessel densities (p=0.047), and an increased MMP-9 expression (p=0.0456). Beta-catecholamines increased proliferation in tumour cells by 18% (p<0.0001) and migration by 78% (p=0.0348) whereas the beta-blocker propranolol reduced these effects by 25% (p<0.0001) and 53% (p=0.045), respectively. When stressed tumour-bearing animals were treated with propranolol tumour volumes were reduced by 69% (p=0.0088) and survival improved by 14% (p<0.0058). The potential treatment with beta-blockers of patients with pancreatic cancer or other malignancies should be further evaluated as an adjuvant anti-neoplastic agent in clinical trials.

In vivo experimental research of photodynamic therapy and the fluorescence imaging in the diagnosis of pancreatic cancer

Objective To develop MSNs loaded with ICG and investigate its diagnostic value and photodynamic therapeutic value in the experimental pancreatic cancer. Methods ICG was loaded into MSNs to prepare the ICG/MSNs probe. The probe toxicity was evaluated by MTT assays. Near-infrared stereo fluorescence microscope (NISFM) was applied to investigate whether the ICG/MSNs would be uptaken by the human pancreatic cancer cells. After incubated with PBS, ICG (10 μg/ml), MSNs or ICG/MSNs (10 μg/ml ICG) for 24 h and treated with photodynamic therapy (PDT, (780±25) nm laser, 500 mW/cm2), the human pancreatic cancer cells survival rate was determined by MTT method. The human pancreatic cancer cells were implanted into nude mice to prepare subcutaneous tumor models. The distribution of ICG/MSNs in subcutaneous tumor models was studied with in vivo imaging system (IVIS). With reference to the injection dose of ICG(0.5 mg/kg), the mice in PBS group, ICG group, ICG/MSNs group (4 mice per group) were injected via tail vein with 150 μl PBS, ICG solution and ICG/MSNs solution, respectively. After treated by PDT for 48 h, the mice were observed by IVIS for 2 weeks using BLI to evaluate the therapeutic effect of PDT. NISFM was used to observe the fluorescence in tumor region. One-way analysis of variance was used to analyze the data. Results The diameter of ICG/MSNs was about 100 nm and it could be uptaken by human pancreatic cancer cells. After treated by PDT, the survival rates of human pancreatic cancer cells were (24.5±5.0)%, (81.2±1.6)%, (90.7±2.0)% and (93.4±1.7)% in ICG/MSNs group, ICG group, MSNs group and PBS group, respectively(F=212.289, P<0.05). ICG/MSNs group showed better therapeutic effect than ICG group(P<0.05). After 12 d treated by PDT, the tumor did not recur or grow in ICG/MSNs group, but grew obviously in ICG group and PBS group. The BLI of tumor area in PBS group, ICG group and ICG/MSNs group were (61.2±7.7)×108, (56.7±9.0)×108 and (2.4±1.5)×108, respectively(F=67.098, P<0.05). And the difference between ICG/MSNs and ICG group was statistically significant (P<0.05). Meanwhile, the NISFM showed clearly the tumor location with ICG/MSNs. Conclusions ICG/MSNs have good biocompatibility, good PDT effect on pancreatic cancer cells and pancreatic cancer xenografts. Near-infrared fluorescence imaging with ICG/MSNs could delineate the tumor location. Key words: Pancreatic neoplasms; Photochemotherapy; Optical imaging; Fluorescence; Tumor cells, cultured; Mice, nude

Adding targeted inhibition of PI3K and MAPK signaling pathways to standard chemotherapy in experimental pancreatic cancer.

278 Background: Treatment of pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge as gemcitabine (Gem) plus nab-paclitaxel (NPT) represent the current standard for systemic therapy of advanced PDAC. More than 90% of PDAC tumors harbor an activating mutation in the KRAS oncogene. Currently no therapeutics exist that can effectively target this oncogene product, but alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. The RAF-MEK-ERK (MAPK) and the AKT-PI3K signaling pathways are well-described mediators of KRAS induced transformation and tumorigenesis and can be explored as targets for combination therapy. Methods: We evaluated combination treatment benefits of NPT+Gem with the MEK inhibitor trametinib (Tra) and the AKT inhibitor MK-2206 (MK) in experimental xenografts to test their therapeutic potential against PDAC. Results: Median animal survival in human intraperitoneal PDAC xenografts in mice revealed that the median survival was 21 days in controls; this was significantly improved by the NPT+Gem combination (35 days, a 67% increase over controls, p = 0.0007). Median survival was further increased by addition of MK-2206 or trametinib to NPT+Gem chemotherapy group: NPT+Gem+MK (39 days, a 86% increase over controls, p = 0.0003), NPT+Gem+Tra (43 days, a 105% increase over controls, p = 0.0003) and NPT+Gem+MK+Tra (48 days, a 129% increase over controls, p = 0.002). In human subcutaneous PDAC xenografts, MK-2206 or trametinib were able to enhance NPT+Gem effects. Compared to controls (100±34.8), the percent net local tumor growth in different therapy groups was: NPT+Gem 21.8±5.9 (p = 0.003), NPT+Gem+MK 17.2±4.8 (p = 0.002), NPT+Gem+Tra 7.1±11.2 (p = 0.002) and NPT+Gem+MK+Tra 5.7±9.1 (p = 0.001). In vivo effects of Tra and MK correlated with reduced expression of phospho-ERK and phospho-AKT in Western blots of tumor samples. Conclusions: These findings suggest that the effects of NPT+Gem can be enhanced through combined inhibition of MAPK and PI3K signaling pathways, which clinically could yield in improved antitumor results.

Abstract A25: Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses

Abstract The aim of this study was to evaluate the efficacy of LOAd immunotherapy to treat pancreatic cancer in a xenograft model as well as to determine the capacity of LOAds to stimulate the immune system in in vitro models. LOAd viruses are oncolytic adenoviruses (5/35) armed with immunostimulatory genes in order to shift the tumor milieu towards immune activation at the same time providing release and spread of tumor antigens due to oncolysis. In this manner, LOAd virus is an antigen-independent immunotherapy for various solid tumors. LOAd armed with CD40 ligand (LOAd700) was constructed from the ICOVIR system by changing the virus shaft and knob to that of serotype 35 to broaden virus binding and entry into cells (pending patent EP14163704). Oncolysis in OCOVIRs is restricted to cells with a disrupted Rb pathway. Viruses were produced using A549 cells. The pancreatic cancer cell lines BxPC3, Panc01, MiaPaCa2 and PaCa3 were used for in vitro evaluation and Panc01 was used in the xenograft model in Nu/Nu mice. Human dendritic cells (DCs) were obtained by differentiation of CD14+ monocytes with GM-CSF and IL4 for 7 days. LOAd viruses infected both the panel of pancreatic tumor cell lines and human DCs with high efficacy and both the cell lines and the DCs expressed the CMV driven transgene/s post transduction. The oncolytic LOAd virus did not kill DCs nor healthy pancreatic cells present in donor islet cell isolation surplus material. In contrast, oncolysis was restricted to the tumor cell lines. The LOAd-transduced DCs increased markers of maturation such as MHC II, CD86, CD70 and CD83 and produced high levels of IL12. LOAd stimulated DCs were able to activate and expand antigen-specific T cells and NK cells as demonstrated in a CMV in vitro system. LOAd viruses could control tumor growth in a xenograft immunodeficient model due to oncolysis alone. In conclusion, LOAd700 can kill pancreatic tumor cells in vitro and control growing tumor in mice due to oncolysis alone. Further, upon DC transduction, LOAd viruses mature DCs to efficient antigen presenters and stimulators of Th1 effector cells such as T cells and NK cells. LOAd is an interesting new immunotherapy for solid malignancies including pancreatic cancer. Citation Format: Emma Svensson, Rafael Moreno, Ioanna Milenova, Lisa Christiansson, Ramon Alemany, Angelica Loskog. Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A25.

Abstract 3491: Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, and is expected to become the second-leading cause of cancer death by 2030. Gemcitabine, the standard of care agent over the past 16 years, has limited clinical benefits. Recently, nab-paclitaxel has shown greater efficacy against advanced PDAC in combination with gemcitabine. Our lab has demonstrated superior antitumor activity of nab-paclitaxel compared with gemcitabine or docetaxel in preclinical PDAC studies. Insulin like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC and correlate with aggressive tumor phenotype and poor prognosis indicating that the IGF system is an important therapeutic target in PDAC patients. BMS-754807, a small molecule inhibitor of IGF-type 1 receptor (IGF-1R) and insulin receptor (IR), has shown efficacy in PDAC. We evaluated combination treatment benefits of nab-paclitaxel with BMS-754807 in experimental PDAC. In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Heterotopic tumor growth and animal survival experiments were performed in murine xenografts. In vitro analysis of four PDAC cell lines (AsPC-1, BxPC-3, MIA PaCa-2, Panc-1) revealed that single agent nab-paclitaxel and BMS-754807 both inhibited cell proliferation in a dose-dependent manner. To evaluate the combination treatment benefits of nab-paclitaxel and BMS-754807, the IC25 dose of BMS-754807 was combined with increasing doses of nab-paclitaxel. Addition of BMS-754807 decreased the nab-paclitaxel IC50 from 7.2 μM to 490 nM for AsPC-1, 430 nM to 50 nM for BxPC-3, 740 nM to 540 nM for MIA PaCa-2, and 690 nM to 280 nM for Panc-1 cell lines. These data suggest that BMS-754807 combines well with nab-paclitaxel and efficiently decreases the nab-paclitaxel IC50 in PDAC cell lines. In a heterotopic AsPC-1 PDAC model, relative to controls (100±21.3), percent net local tumor growth was 17.1±21.1 with nab-paclitaxel (p = 0.002), 37.6±16.2 with BMS-754807 (p = 0.01) and 0.76±9.1 with nab-paclitaxel plus BMS-754807 (p = 0.0002). In Panc-1 subcutaneous xenografts, compared with controls (100±31.5), percent net tumor growth was 7.8±27.2 with nab-paclitaxel (p = 0.002), 40.2±24.1 with BMS-754807 (p = 0.02) and -29.7±16.8 with nab-paclitaxel plus BMS-754807 (p = 0.0001). In an animal survival study, median animal survival was increased compared to controls (21 days) after nab-paclitaxel therapy (40 days, a 90% increase, p = 0.002), BMS-754807 therapy (27 days, a 29% increase, p = 0.01), and nab-paclitaxel plus BMS-754807 therapy (47 days, a 124% increase, p = 0.005). These results highlight measurable antitumor activity of BMS-754807 in experimental PDAC and support the potential of BMS-754807 in combination with nab-paclitaxel as promising targeted therapy for clinical pancreatic cancer therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz. Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2015-3491

Abstract 1025: Antitumor activity of nintedanib (BIBF 1120), a triple angiokinase inhibitor, in combination with gemcitabine in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States with extremely poor prognosis. Gemcitabine (Gem), the standard of care agent over the past 15 years, has limited clinical benefits. There is an urgent requirement for novel therapeutic strategies to improve overall survival of patients with pancreatic cancer. Angiogenesis, an essential process for tumor growth and metastasis, remains a sensible target for PDAC therapy. VEGF, PDGF, FGF and their receptors are all overexpressed and have been correlated with poor prognosis in human PDAC. Nintedanib (BIBF 1120, Nin) is a triple angiokinase inhibitor that targets VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β signaling. Nintedanib is currently under clinical investigation for several solid tumors. The present study investigated the combination treatment benefits of gemcitabine and nintedanib in experimental PDAC. Cell proliferation, migration and protein expression were analyzed by WST-1 assay, wound healing assay and Western blotting. Tumor growth and animal survival experiments were performed in murine xenografts. Percent inhibition of cell proliferation in the Gem, Nin and Gem+Nin groups was 22%, 25% and 53% (AsPC-1 PDAC cells, Gem 50 nM, Nin 100 nM); 55%, 11% and 80% (endothelial cells HUVECs, Gem 100 nM, Nin 100 nM); 91%, 68% and 94% (fibroblast WI-38 cells, Gem 100 nM, Nin 100 nM). Nintedanib increased apoptosis, indicated by the cleavage of caspase-3 protein in all three cell-types tested. In PDAC cells, antitumor activity was accompanied by higher level of cell cycle inhibitor protein p27. Nintedanib (10 μM) inhibited in vitro wound closure by 84% in endothelial cells. In a heterotopic PDAC model, compared to the percent net local tumor growth in controls (100±29), a reduced growth of 60.8±10.5 (p=0.02) was observed in the Gem group, while a net reduction of -2.1±9.9 (p=0.0001) and -12.4±16.2 (p=0.0001) was observed in Nin and Gem+Nin groups, respectively. Compared with controls (16 days) in an AsPC-1 intraperitoneal model, median animal survival in Gem, Nin and Gem+Nin treatment groups was 25 days, 31 days and 38 days. The strong antitumor activity of nintedanib in experimental PDAC supports the potential of nintedanib-controlled mechanisms as targets for improved clinical PDAC therapy. Citation Format: Niranjan Awasthi, Stefan Hinz, Rolf A. Brekken, Margaret A. Schwarz, Roderich E. Schwarz. Antitumor activity of nintedanib (BIBF 1120), a triple angiokinase inhibitor, in combination with gemcitabine in experimental pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1025. doi:10.1158/1538-7445.AM2014-1025

Treatment of experimental pancreatic cancer with 213-Bismuth-labeled chimeric antibody to single-strand DNA

Background: Novel approaches to treatment of pancreatic cancer (PCa) are urgently needed. A chimeric monoclonal antibody (mAb) chTNT3 binds to single-strand DNA (ssDNA) and RNA released from the non-viable cells in fast growing tumors. Here the authors investigated whether radioimmunotherapy (RIT) using chTNT3 mAb radiolabeled with 213-Bismuth (213Bi) could be effective in treatment of experimental PCa. Methods: Two human PCa cell lines, Panc1 and MiaPaCa-2, were used for in vitro experiments. The xenografts in mice were established using MiaPaCa-2 cells. Therapy compared 213Bi-chTNT3 (700 μCi) to gemcitabine or cisplatin, untreated controls and ‘cold’ chTNT3. Results: RIT abrogated the tumors growth while tumors in control groups grew aggressively. Chemotherapy was less effective than RIT and toxic to mice while RIT did not have any side effects. Conclusions: RIT with 213Bi-chTNT3 was safe and effective in the treatment of experimental PCa in comparison with chemotherapy. This makes α-RIT targeting ssDNA a promising modality for further development.

Enhancement of Nab-Paclitaxel Antitumor Activity through Addition of Multitargeting Antiangiogenic Agents in Experimental Pancreatic Cancer

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel, NPT) has recently shown efficacy in pancreatic ductal adenocarcinoma (PDAC). Targeting tumor angiogenesis is a sensible combination therapeutic strategy for cancer, including PDAC. We tested the hypothesis that NPT response in PDAC can be enhanced by the mechanistically different antiangiogenic agents bevacizumab (Bev) or sunitinib (Su), despite its inherently increased tumor penetration and drug delivery. Compared with controls (19 days), median animal survival was increased after NPT therapy (32 days, a 68% increase, P = 0.0008); other regimens with enhanced survival were NPT+Bev (38 days, a 100% increase, P = 0.0004), NPT+Su (37 days, a 95% increase, P = 0.0004), and NPT+Bev+Su (49 days, a 158% increase, P = 0.0001) but not bevacizumab, sunitinib, or Bev+Su therapy. Relative to controls (100 ± 22.8), percentage net local tumor growth was 28.2 ± 23.4 with NPT, 55.6 ± 18 (Bev), 38.8 ± 30.2 (Su), 11 ± 7.2 (Bev+Su), 32.8 ± 29.2 (NPT+Bev), 6.6 ± 10.4 (NPT+Su), and 13.8 ± 12.5 (NPT+Bev+Su). Therapeutic effects on intratumoral proliferation, apoptosis, microvessel density, and stromal density corresponded with tumor growth inhibition data. In AsPC-1 PDAC cells, NPT IC(50) was reduced >6-fold by the addition of sunitinib (IC(25)) but not by bevacizumab. In human umbilical vein endothelial cells (HUVEC), NPT IC(50) (82 nmol/L) was decreased to 41 nmol/L by bevacizumab and to 63 nmol/L by sunitinib. In fibroblast WI-38 cells, NPT IC(50) (7.2 μmol/L) was decreased to 7.8 nmol/L by sunitinib, but not by bevacizumab. These findings suggest that the effects of one of the most active cytotoxic agents against PDAC, NPT, can be enhanced with antiangiogenic agents, which clinically could relate to greater responses and improved antitumor results.

Non-invasive quantification of anti-angiogenic therapy by contrast-enhanced MRI in experimental pancreatic cancer

Currently, early changes of tumor vasculature after angiogenesis inhibition can only be evaluated by histopathology, a method not suitable in a clinical setting. To quantify effects of different angiogenesis inhibitors on the microvasculature of orthotopically implanted pancreatic cancers by contrast-enhanced magnetic resonance imaging (MRI) in order to establish a non-invasive technique for monitoring antiangiogenic cancer treatment. DSL-6A/C1 pancreatic cancers were implanted in the pancreas of 109 Lewis rats. Three weeks later, antiangiogenic treatment was initiated by administration of Bevacizumab (n = 38) or Suramin (n = 27) while the control group (n = 44) remained untreated. Dynamic MRI was performed 24 h, 1 week, and 4 weeks after treatment initiation. Fractional tumor plasma volume (fPV, %) and vascular permeability (K(PS), mL/min/100 cc) were calculated based on the MRI data by using a pharmacokinetic model. Twenty-four hours after the initial dose, a significant decline in K(PS) was observed in the Bevacizumab group compared to the control and Suramin group (0.002 ± 0.008; 0.057 ± 0.046 and 0.064 ± 0.062 (mean ± SD); P < 0.05). At 1 week, fPV was significantly smaller in Bevacizumab and Suramin treated tumors compared to control tumors (6.25 ± 2.74, 7.47 ± 3.44, and 15.10 ± 9.97, respectively; P < 0.05). Differences in tumor volumes were first observed after 4 weeks of treatment with significantly larger control tumors (4380.3 ± 1590.6 vs. 869.6 ± 717.2 and 1676.5 ± 2524.1 mm(3); P < 0.05). Dynamic MRI can quantify antiangiogenic effects on tumor microvasculature before changes in tumor volumes are detectable. Thus, this technique is a reasonable addition to morphological MRI and may be applied as an alternative to histopathology.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g-L-1 [S. platensis], 60 μΜ [PCB], and 125 μΜ [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.