Author ORCID Identifier

0000-0002-3845-9373

Date of Graduation

5-2020

Document Type

Thesis (MS)

Program Affiliation

Cancer Biology

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Anil K. Sood

Committee Member

Robert Dantzer

Committee Member

Pamela Wenzel

Committee Member

Wei Hu

Committee Member

Peiying Yang

Abstract

Effect of sympathetic nervous system mediators on the tumor microenvironment via small extracellular vesicles in ovarian cancer

Sujanitha Umamaheswaran, BTech

Advisor: Anil K. Sood, MD

Background:Psychological stress can promote progression of gynecological malignancies by increasing secretion of sympathetic nervous system (SNS) mediators, namely catecholamines. However, the mechanisms underlying these effects are poorly explored. Here, we examined the effect of stress hormones on small extracellular vesicles (EVs) release in gynecological malignancies and the biological impact of these stress-conditioned EVs on the tumor microenvironment.

Methods:Supernatants were collected from epithelial ovarian cancer cell lines treated with norepinephrine, epinephrine or hydrocortisone. Small EVs were isolated from the supernatants of these cell lines and assessed for number and particle size. Auto re-uptake studies, invasion assays and tube formation assays were performed on ovarian cancer cells and RF24 endothelial cells exposed to control and stress-conditioned EVs. Tipifarnib, a farnesyl transferase inhibitor, was used to inhibit small EV secretion to study dependence of outcomes of previously performed functional assays on EV number. Mass spectrometry (MS) was performed on control and stress-conditioned small EVs to compare protein content. In vivo Matrigel plug assay was performed to compare the angiogenic potential of control and stress-conditioned EVs.

Results:Addition of 1uM and 10uM of catecholamines and glucocorticoids increased small EV secretion by ~ 4-fold in OVCAR5 and HeyA8 cell lines. Maximum increase in secretion was seen between 8 hr and 24 hr after treatment with stress hormones, after which secretion decreased. EV uptake experiments with ovarian cancer cells showed that the percentage of uptake of stress-conditioned EVs at all timepoints was the same, around 65%, indicating that the capacity of the cell to produce EVs might be decreasing after 24 hours. Stress-conditioned EVs increased invasion of ovarian cancer cells and tube formation capacity of RF24 cells by ~ 2-fold. In vivo analysis of the angiogenic potential of control vs stress-conditioned EVs showed that stress-conditioned EVs induced more angiogenesis than control EVs. MS analysis of EV contents from control and stress-conditioned EVs showed a differential expression of angiogenesis and cell growth-related proteins in the stress-conditioned EVs alone.

Conclusions:Our results demonstrate that addition of stress-hormones increases the angiogenic potential of cancer cell-derived small EVs. Further exploration of the effects of stress-conditioned small EVs on cancer cells and other cell types in the tumor microenvironment and pathway analyses might uncover other mechanisms of stress-induced cancer progression.

Keywords

Small extracellular vesicles, norepinephrine, stress, angiogenesis, ovarian cancer

Available for download on Saturday, May 01, 2021

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