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.

Grant Number: 5F31CA288057-02
NIH Institute/Center: NIH
Principal Investigator: Shelby Cain