Endothelial Cell Cycle Control to Normalize the Tumor Vasculature

Tumor blood vessels are dysfunctional and irregular, impairing immune cell infiltration and drug delivery and creating areas of severe hypoxia that select for aggressive cancer cells. Recent therapeutic strategies aim to normalize the tumor vasculature by targeting angiogenic factors, but this often results in a small window of

efficacy due to resistance or over-pruning. Our lab discovered a mechanism during normal vascular development that may be dysregulated and targetable in tumors. Using mice that express the fluorescent, ubiquitination-based cell cycle indicator (Fucci2) to distinguish cell cycle states (early G1, late G1, S/G2/M), we found that distinct

endothelial cell cycle states (early G1 vs. late G1) are required for endothelial cell specification into venous vs. arterial subtypes, respectively. In addition, we found that endothelial cell cycle control and specification are dysregulated in vascular malformations that resemble tumor vasculature. These malformations were prevented

by treatment with a cell cycle inhibitor, Palbociclib, an FDA-approved CDK4/6 inhibitor for advanced breast cancer treatment. Yet, the effects of Palbociclib, or other cell cycle inhibitors, on tumor vasculature are unknown. We hypothesize that endothelial cell cycle state is dysregulated in the tumor microenvironment, leading

to impaired endothelial cell specification and immature vessels, and that treatment with a cell cycle inhibitor will enable endothelial cell specification and normalize the tumor vasculature. To examine tumor endothelial cell cycle and specification, I induced mammary tumors in Fucci2 mice and found tumor endothelial

cell proliferation is significantly increased with less endothelial cells in late G1. Additionally, tumor endothelial cells exhibit decreased expression of arterial-, venous-, and capillary-enriched genes, indicating immature vessels. Thus, Aim1 will further quantify endothelial cell cycle state and subtype markers in Fucci2 mice with

mammary or melanoma tumors compared to healthy mammary fat pad (MFP) or skin controls. Thus far, I treated Fucci2 mice with mammary tumors with Palbociclib, which significantly decreased the proportion of proliferative endothelial cells and increased the proportion in late G1. Aim 2 will further test the hypothesis that Palbociclib,

and other cell cycle inhibitors, increases tumor endothelial cells in G1 states to resemble healthy tissues and enable arterial-venous specification. We will quantify changes in Fucci2 expression and analyze single-cell RNA sequencing of endothelial and other cells from MFPs and tumors isolated from vehicle- or Palbociclib-treated

mice to identify changes in specification and cell-cell signaling. Finally, we will quantify changes in vessel permeability and maturation and tissue hypoxia after cell cycle inhibitor treatment. I will test delivery of a contrast agent to the tumors to model drug delivery and use microCT to quantify changes in vessel density and structure

before, during, and after treatment. These studies will elucidate how the tumor microenvironment affects endothelial cycle state and specification, and mechanisms by which pharmacological cell cycle inhibition can normalize tumor vasculature, indicating a new use of such drugs for cancer and pathological angiogenesis.