Researchers have identified a mechanism called venousization that allows blood vessels to grow without VEGFR signaling, offering a potential new target for treating drug-resistant tumors. According to Nature, this pathway provides an alternative to the standard vascular endothelial growth factor receptor signaling typically required for angiogenesis.
Researchers Used Genetically Modified Mouse Lines for Vessel Development
The study relied on a variety of genetically modified mouse lines to observe vessel development. Researchers used lines including Tg(Cdh5–CreERT2)44, Tg(iSuRe–HadCre)14, Vegfr1/2/3flox, Nr2f2flox, iMb2–Mosaic16, Apln–FlpO15, and the iFlpMosaic lines. To ensure the integrity of the research, all mouse husbandry and experimental protocols were approved by local animal ethics committees and authorities, specifically the Comunidad Autónoma de Madrid and Universidad Autónoma de Madrid, under protocols CAM-PROEX 164.8/20, PROEX 293.1/22, and PROEX-288.7/24.
The mouse colonies were housed in racked individual ventilation cages, utilizing dust- and pathogen-free bedding alongside environmental enrichment to support species-specific behavior. For the generation of a new Vegfr3flox allele, the researchers injected Cas9, guide RNAs, and donor DNAs containing LoxP sites into mouse eggs. Successful deletion was confirmed via PCR and immunostaining for the VEGFR3 protein. To induce CreERT2 activity in adult subjects, the team utilized tamoxifen (Sigma-Aldrich, P5648_1G) dissolved in corn oil at a concentration of 20 mg ml−1.
For experiments involving tumor models, the researchers injected age-matched and sex-matched mice (15–20 weeks old) subcutaneously with 5 × 10^5 tumor cells suspended in Matrigel (Corning, 354234). The tissues were subsequently explanted and postfixed for two hours in 4% PFA (Thermo Scientific, 043368.9M) in PBS at 4 °C. Following postfixation, organs were stored in 30% sucrose, embedded in optimal cutting temperature compound, and frozen at −80 °C. Cryosections measuring 30 µm were prepared on a Leica cryostat for further analysis.
Sequencing and Mapping Techniques
Before loading the samples, cell viability and concentration were precisely determined using a Countess 3 Automated Cell Counter (Thermo Fisher Scientific).
Implications for Tumor Growth
The findings suggest that the ability of vessels to grow independently of VEGFR signaling could have significant clinical relevance. EuropeSays reports that these alternative pathways for blood vessel growth may become targets for combating drug-resistant tumors. The study highlights how blood vessels manage to expand even when one of their primary signaling mechanisms is absent. No randomization or blinding was used during the study, as samples were selected based on genotype, the frequency of Cre- or FlpO-dependent recombination, and the quality of the multiplex immunostaining performed.