Moore, S. P. G. | Search bioRxiv

bioRxiv2025

VaMiAnalyzer: An open source, python-based application for analysis of 3D in vitro vasculogenic mimicry assays

BackgroundVasculogenic mimicry (VM) is the phenomenon whereby non-vascular tumor cells develop vascular-like structures. VM is linked to more aggressive tumor phenotypes including higher rates of metastasis and invasion and is potentially resistant to anti-angiogenic cancer therapies. VM is investigated in vitro using 3D VM assays with microscopy images capturing the resulting VM structures. The standard method to quantify endpoint data is to count various structural features manually, which is time-consuming and open to bias. At present, no software solutions have been developed to specifically address the analysis and quantification of VM structures. ResultsTo address this limitation, we developed an open source, python-based application, VaMiAnalyzer, allowing straightforward quantification of several VM structural features. The application follows a two-step approach that optionally corrects and enhances the raw input images and then analyzes and quantifies the VM features. ConclusionsVaMiAnalyzer is stand-alone software that allows automated measurement of VM structural features from phase-contrast microscopy images. It produces results that are strongly consistent with manual counts but in a significantly shorter time, allowing speedy, non-biased analysis of VM from microscopy images.

bioinformatics↗

bioRxiv2025

MITF-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

Melanocytes reside in diverse microenvironments that influence their susceptibility to oncogenic transformation; however, investigation of rare melanoma subsets has been limited by the lack of suitable pre-clinical animal models. Here, we developed a primary, immune-competent zebrafish model to study uveal melanoma (UM) using choroidal melanocyte-targeted injection and electroporation of plasmids encoding human GNAQQ209L together with CRISPR/Cas9 cassettes for somatic tumor suppressor gene deletion. Single-cell transcriptional profiling of primary melanocytes and melanoma derived from the eye and skin revealed distinct transcriptional programs, with epithelial-to-mesenchymal transition pathways enriched in ocular tumors. In addition, choroidal fibroblasts from tumor-bearing eyes exhibited marked transcriptional changes, including increased fibronectin and collagen expression, consistent with stromal remodeling. Given prior associations between mitfa loss and accelerated GNAQQ209L tumor onset, the model was applied to determine whether melanocyte differentiation state contributes to the emergence of GNAQ-driven tumors. The increased susceptibility resulted from expansion of Mitfa-independent melanocyte progenitor populations in germline mitfa mutant zebrafish, rather than somatic mitfa loss in differentiated melanocytes, as conditional, melanocyte-specific mitfa deletion in adult zebrafish did not accelerate tumor growth. Furthermore, pax3a-positive melanocyte progenitor cells in mitfa-deficient zebrafish embryos and adult eyes and skin were highly susceptible to transformation induced by GNAQQ209L but not BRAFV600E. Analogous PAX3 positive populations were also identified in mouse and human single-cell transcriptomic datasets. Collectively, these findings establish a critical role for Mitfa-independent melanocyte progenitors in UM pathogenesis. Statement of SignificanceChoroid-targeted GNAQQ209L expression induces anatomically correct uveal melanoma in adult zebrafish, with germline mitfa deletion expanding mitfa-independent melanocyte progenitors with enhanced susceptibility that are transcriptionally distinct from the subpopulation transformed by BRAFV600E.

cancer biology↗