Tumor-initiating cells with reprogramming plasticity are thought to be essential for cancer development and metastatic regeneration in many cancers; however, the molecular mechanisms are not fully understood. We have previously identified that CD44, a breast tumor-initiating cell marker, drives mammosphere self-renewal and multicellular aggregation of circulating tumor cell (CTC) clusters, thereby enhancing tumorigenesis and metastatic colonization in triple negative breast cancer (TNBC). This study reports that CD81, a tetraspanin protein normally enriched in small extracellular vesicles (exosomes), functions as a binding partner of CD44 and facilitates mammosphere formation. Using machine learning-assisted protein structure modeling, co-immunoprecipitation, and mutagenesis approaches, we further demonstrate that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. In-depth global and phosphoproteomic analyses of clustering tumor cells unveils endocytosis-related signature pathways of proteins and phosphorylation patterns regulated by CD81 and CD44 specifically or shared between the two. Notably, CRISPR Cas9-mediated depletion of either CD44 or CD81 results in loss of both proteins in cancer cell-secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells for mammosphere formation. CD81 is expressed in >80% of human CTCs and specifically enriched in clustered CTCs and co-expressed along with CD44 in both breast tumors and CTCs of breast cancer patients. Mimicking the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cluster aggregation, tumorigenesis, and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights the novel role of CD81 and its partnership with CD44 in cancer exosomes, self-renewal, CTC clustering, and metastasis initiation of TNBC. Finally, we also demonstrate the use of a micro-flowcytometer for high throughput single circulating exosome profiling as a promising approach to assess novel and dynamic biomarkers in cancer.

Creator

• Ramos, Erika Kathryn

DOI

• https://doi.org/10.21985/n2-t96g-br91

Subject

• Molecular biology
• Biology
• Cellular biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:16152
• etdadmin_upload_916551

Keyword

• exosomes
• CD44
• cancer stem cells
• extracellular vesicles
• CD81
• breast cancer

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright