Identification of novel therapeutic strategies through newly developed in vivo Tumor models

Preclinical studies are crucial to clinical research, yet 90% of drug candidates fail in trials. Reducing failure rates requires stringent criteria in preclinical settings, including new therapeutic strategies, better models mimicking in vivo cancer characteristics, and reliable Tumor identification and monitoring methods. This work uses the VevoF2-LAZR-X system, combining photoacoustic and ultrasound imaging, to (a) generate a novel pancreatic cancer (PDAC) model, (b) test an innovative therapeutic strategy, and (c) characterize angiogenesis in an in vivo setting. We developed (a) orthotopic xenograft mice models of PDAC using the ultrasoundguided injection of PANC-1 cell line, (b) which are used for testing a bifunctional antibody (scDb-hERG1/β1) combined with different doses of Gemcitabine (subtherapeutic and therapeutic). Our data show that scDb-hERG1/β1 plus sub-therapeutic gemcitabine has antitumoral effects comparable to therapeutic dose gemcitabine, with increased survival and reduced toxicity. To better mimic in vivo PDAC characteristics, we developed (a) models with co-cultures of PANC-1 and pancreatic stellate cells (RLT-PSC). RLT-PSC contributes to the Tumor microenvironment by supplying nutrients and facilitating metastasis through collagen production. Our models included orthotopic injections of PANC-1, RLT-PSC, and mixed ratios of these cells at different ratio (1:1 and 1:5 PANC-1/RLT-PSC respectively). Ultrasound (US), photoacoustic imaging (PAI), and nonlinear contrast (CE-US) were then performed to compare those models in in vivo settings. It emerges that PANC- 1/RLT-PSC 1:5 exhibited greater hypoxia and reduced vascular perfusion. Noteworthy, Tumor aggressiveness appeared to increase, as metastasis was found in the liver of these models, in contrast to PANC-1 and RLT-PSC alone where it doesn't occur. We then move on to (c) evaluating Tumor angiogenesis focusing on colorectal cancer (CRC), whose management has evolved with anti-angiogenesis therapies like Bevacizumab. Using subcutaneous xenografts models, generated by the injection of HCT-116 wild-type (WT) and Bevacizumab-adapted (Beva/A) cell lines, we characterized Tumor volume, oxygen saturation, hypoxia, and perfusion through PAI and CE-US. The two models, despite the similarities of the expression observed on in vitro conditions for the main angiogenetic-related genes, on in vivo settings exhibit differences. Although both models displayed similar Tumor growth, HCT116-Beva/A tumors showed higher oxygenation (sO2TOT, sO2P) and lower HIF-1α and VEGF-A levels, but no difference were observed for Hbtot, suggesting more efficient oxygenation despite the same vascular density. From the perfusion analysis, little variation can be assessed, suggesting lower blood flow efficiency in the resistant tumors, supportingvariation in the vessels network in the Beva-resistant models. As the hERG1/β1 complex promotes angiogenesis via the PI3K-Akt pathway and VEGF-A secretion, we proceed to test in these two models the impact of scDb-hERG1/β1 in combination with Bevacizumab. This had a much more pronounced effect on sO2TOT and sO2P in the HCT116-Beva/A tumors with both significantly decreasing, thus indicating increased tumour hypoxia due to increased tumour cell death with subsequent abnormal vessels collapse. This is further supported by perfusion parameters that show the effect of combination treatment in Bevacizumab-resistant tumors. Increased levels of both PE and PI, and reduction in mTT and TTP suggests improved perfusion and blood flow following treatment, possibly as a result of vascular remodelling due to combined inhibition of angiogenesis pathways. The following review shows the translational value of different advanced imaging modalities, including ultrasound and photoacoustic imaging techniques, to test novel therapeutic modalities in pancreatic ductal adenocarcinoma and colorectal carcinoma, particularly in their treatment-resistant models.