Abstract 5384: The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts of pancreatic cancer

Abstract

Abstract Pancreatic cancer mostly manifests as pancreatic ductal adenocarcinoma (PDAC) and is characterized by a highly hypoxic microenvironment that is associated with worse patient survival and resistance to therapy. Hypoxia induces angiogenesis and the resulting leaky and unstructured vessels fail to reoxygenate the tumor mass, thus exacerbating hypoxia and its downstream impact on tumorigenesis. Myo-inositol trispyrophosphate (ITPP) is a non-toxic allosteric effector of hemoglobin shown to reduce tumor growth and enhance survival in mice and rat models of different tumor types. Herein the aim is to assess for the first time the ability of ITPP to effectively treat PDAC tumors in vivo in zebrafish xenografts and to investigate its impact on vascular normalization. To that aim, four PDAC cell lines, AsPC1, Capan1, MIA PaCa2, and PANC1 were included, as well as the angiogenic breast cancer cell line, Hs578t. Cells were grown in appropriate conditions and on injection day, labeling was done with deep red. Labeled cells were then injected in the perivitelline space of anesthetized larvae. The following transgenic larvae were used: those having labeled blood vessels only (Tg(kdrl:DsRed)), labeled blood and lymphatic vessels (Tg(fli1:eGFP)), and double transgenics with labeled gata+ erythrocytes (Tg(fli1:eGFP;gata:dsRed)). One day post injection (dpi) xenografts were sorted based on size and treated by immersion for three consecutive days with control medium or ITPP at a concentration selected based on the maximum tolerated dose assay. Treatment was refreshed daily, and at 4dpi live confocal imaging was conducted on select Tg(fli1:eGFP;gata:dsRed) xenografts. All xenografts were then fixed and subjected to whole mount immunofluorescence. Nuclei were stained with DAPI and anti-Caspase3Asp175 was used as a marker for apoptosis. Imaging was performed using the Zeiss LSM confocal microscope and tumor size, apoptosis and mitotic figures were quantified with ImageJ. Vascular function was defined as the presence of gata+ cells within tumor-related vasculature, while vessel density and infiltration were determined in ImageJ by calculating the percentage of GFP area in the tumor. Thus far results from AsPC1-derived xenografts demonstrate that ITPP leads to a significant decrease in mitotic figures (p=0.0132) and an increase in apoptosis (p=0.0025), with no change in tumor size. Preliminary data reveal a trend of increased functional vasculature in both the AsPC1- and Hs578t- derived xenografts upon ITPP treatment. A trend of decreased vasculature infiltration could also be observed in the ITPP-treated Hs578t-derived xenografts. Based on these results, the zebrafish model could be an appropriate alternative for assessing the anti-tumorigenic activity of ITPP in PDAC in a short turnaround time and could potentially enable the visualization of ITPP’s impact on angiogenesis at single cell resolution. Citation Format: Raefa Abou Khouzam, Filipa Amorim, Ayesha Rifath, Husam Nawafleh, Vanda Povoa, Rania Faouzi Zaarour, Jean Marie Lehn, Rita Fior, Perparim Limani, Salem Chouaib. The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts of pancreatic cancer [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 5384.