Fatima Charles

I. Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including variable success rates, contamination risk, and extended culture timelines. We demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent superior alternatives for biobanking across three mammalian genera ( Canis , Bison , and Equus ). Blood-derived cells exhibited 2-3 fold faster doubling rates (15-20 hours versus >35 hours for fibroblasts) and reduced time to generate banked lines from 3-4 weeks to 1.5-2 weeks. Proteomic profiling of 32 canonical markers confirmed EPCs and pericytes represent distinct populations with lineage-specific molecular signatures. Optical genome mapping demonstrated equivalent genomic stability across all cell types with no detectable structural variants or aneuploidies. Critically, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable embryos with efficiency meeting or exceeding fibroblasts. Gray wolf blood-derived cells produced six viable fetuses with 15% implantation rate, while bison EPCs showed higher blastocyst formation (7%) than fibroblasts (3%) from the same individual. Blood collection during routine veterinary procedures offers minimally invasive sampling with reduced contamination compared to skin biopsies. These findings support integrating blood-derived cell banking into conservation programs, enabling opportunistic genetic preservation during standard management activities and expanding options for genetic rescue through assisted reproductive technologies.

VISTA is a key immune checkpoint receptor under investigation as a target for cancer immunotherapy. However, a better understanding of the signaling mechanisms of VISTA is needed to optimize the therapeutic potential. Here, we identified a conserved four amino acid (NPGF) intracellular motif in VISTA that suppresses cell proliferation by constraining cell-intrinsic growth receptor signaling. A class of triple-negative breast cancers (TNBC) with high VISTA expression and low proliferative index was identified and characterized. The NPGF motif bound to the adapter protein NUMB and recruited Rab11 endosomal recycling machinery. The NPGF motif sequestered NUMB at endosomes, which interfered with EGFR trafficking and signaling to suppress tumor growth. These tumor suppressive effects did not require canonical VISTA ligands or a functioning immune system. Mutation of the VISTA NPGF domain reverted VISTA-induced growth suppression in multiple breast cancer mouse models. The NPGF motif was also required for response of VISTA+ TNBCs to VISTA-blocking antibodies. These results define a mechanism by which VISTA recruits adapter proteins to control malignant epithelial cell growth and signaling. They also define distinct intracellular residues that are critical for response to therapeutic antibodies that could be exploited to improve immunotherapy.