ITPP in early pancreatic zebrafish xenografts mildly impacts tumor cell death without interfering with vascular normalization.

Abstract

Background: Myo-inositol trispyrophosphate (ITPP) is an anti-hypoxic small molecule that acts as an allosteric effector of hemoglobin in erythrocytes. To capture the early and dynamic effects of ITPP on tumor vasculature and cell viability, we employed a rapid zebrafish xenograft model of pancreatic ductal adenocarcinoma (PDAC) that enables real-time, single-cell-resolution imaging.

Methods: Two PDAC cell lines were used to establish xenografts in wildtype and transgenic zebrafish lines. A double transgenic that permits the visualization of both vessels and erythrocytes was included to determine the impact of ITPP on vascular function. The effects of ITPP alone and in combination with multimodal chemotherapy (FOLFIRINOX) on PDAC tumor proliferation, apoptosis and size were investigated using whole-mount immunostaining and confocal microscopy.

Results: Within a short treatment window, ITPP produced a detectable enhancement in the number of xenografts with erythrocyte-positive tumor-associated vessels, suggesting an early trend toward vessel functionality. This mild effect, however, came with no measurable changes in vessel architecture. While ITPP did not impact the cytotoxic potency of FOLFIRINOX, ITPP-treated xenografts displayed enhanced apoptosis in vivo. This came without any cytotoxicity in vitro, indicating that its mild pro-apoptotic activity is mediated indirectly, presumably through the tumor microenvironment (TME).

Conclusions: Overall, we could not detect major effects of ITPP in the zebrafish xenograft model, possibly due to the lack of a hypoxic TME. This illustrates the model's shortcomings when investigating oxygen-modulating agents. We propose that experiments such as pre-treatment of tumor cells with hypoxic agents could overcome this, potentially expanding the utility and versatility of the zebrafish model.