Author ORCID Identifier

0000-0002-8154-5533

Date of Graduation

12-2023

Document Type

Dissertation (PhD)

Program Affiliation

Genetics and Epigenetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

George T. Eisenhoffer, PhD

Committee Member

Mikhail Bogdanov, PhD

Committee Member

Catherine Denicourt, PhD

Committee Member

Michael J. Galko, PhD

Committee Member

Guilermina Lozano, PhD

Abstract

Efficient replacement of dead cells in epithelial tissue is crucial for maintaining barrier function and tissue homeostasis. Apoptotic cells can signal to neighboring cells to stimulate proliferation and compensate for cell loss and maintain overall cell numbers in normal physiology and cancer. While dying cells can transmit instructive cues to neighboring cells, the molecular mechanisms that induce cell division are not well understood. Recent evidence suggests that apoptotic bodies (ABs) or apoptotic extracellular vesicles (AEVs) mediate cell-to-cell communication and carry diverse biologically active cellular cargo which can influence cell proliferation. This dissertation visualizes and characterizes AEVs in larval zebrafish and MDA-MB-231 cells to understand apoptosis-induced proliferation mechanisms by assessing proteomic contents and studying real-time dynamics of AEVs. In both systems, I found AEVs to be enriched in molecules that can modulate cell proliferation and immune cell dynamics.

In larval zebrafish, epithelial stem cell-specific ablation induces apoptosis, enabling the characterization of epithelial stem cell-derived AEVs (esAEVs) and their interactions with neighboring cells in vivo. esAEVs exhibit surface localization of macrophage migration inhibitory factor (MIF), stimulating both proliferative and immunogenic effects on recipient cells. MIF on esAEVs promote compensatory proliferation of epithelial stem cells and influence macrophage surveillance. Interestingly, mif is highly expressed in epithelial stem cells, whereas its cognate receptor cd74a and cd74b exhibits higher expression in macrophages, providing an avenue for crosstalk between the two cell populations. Formation of esAEVs upregulates p-ERK signaling in epithelial stem cells, with inhibition of MIF signaling decreasing p-ERK levels, suggesting a role for MIF in cell proliferation.

Clearance of apoptotic debris in early stages is primarily performed by epithelial stem cells, with macrophages displaying enhanced migratory movement but a lesser contribution to clearance. However, ablation or suppression of macrophages during esAEV induction significantly reduces epithelial stem cell proliferation, highlighting the cell non-autonomous role of macrophages in stimulating compensatory proliferation following widespread apoptosis.

Next, I also characterized AEVs derived from MDA-MB-231 cells, referred to as breast cancer-derived AEVs (bcAEVs). The goal of chemotherapy is to induce apoptosis of cancer cells, however the functional significance of cancer derived AEVs is poorly understood. Similar to esAEVs, bcAEVs carry proteins involved in immune regulation and proliferation. Surface proteins such as Wnt3a, MIF, and CD47 are identified in bcAEVs. Functional studies demonstrate that bcAEVs enhance proliferation of recipient MDA-MB-231 cells, with Wnt signaling implicated in this process.

Overall, my results suggest that the contents of apoptotic extracellular vesicles can modulate the behavior of multiple cell populations to drive cell repopulation. The contents found within bcAEVs can help in the development of biomarkers to specifically detect and monitor patient responses to chemotherapy.

Keywords

Epithelia, apoptotic extracellular vesicles, compensatory proliferation, epithelial stem cells, zebrafish

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