Characterizing the Trafficking of Fibronectin Type III Proteins Bound to 𝛂v𝞫3 Integrin Receptors Across the Blood Brain Barrier in vitro

Author

Sheher-Bano Ahmed, Smith CollegeFollow

Publication Date

2024-5

First Advisor

Sarah J. Moore

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Neuroscience

Keywords

receptor-mediated transcytosis, alphavBeta3 integrins, fibronectin type III proteins, glioblastoma

Abstract

Neurological illnesses such as brain cancer remain difficult to treat, despite varied efforts by researchers. The biggest challenge to treating these illnesses is the presence of the blood brain barrier, a physical and chemical barrier made primarily of endothelial and astrocytic cell types which prevent larger molecules, such as chemotherapeutic drugs, from entering the brain. In glioblastoma, or primary brain cancer, the blood brain barrier can be infiltrated by cancerous cell types, becoming the blood tumor barrier. The blood tumor barrier is less predictable than the blood brain barrier, posing an even greater challenge to the treatment of glioblastoma. One strategy currently being explored to deliver chemotherapeutic drugs to cancer cells is through the use of protein-drug conjugates, which selectively bind to receptors that are overexpressed on cancerous cells. The proteins have drug payloads bound to them which can then be delivered directly to cancerous cells, reducing side effects from chemotherapy drugs. However, crossing the blood tumor barrier remains a challenge with these proteins. Receptor-mediated transcytosis is one method for proteins to cross the blood tumor barrier. 𝛂v𝞫3 integrin receptors are extracellular proteins overexpressed in cancer cells that are implicated in tumor angiogenesis, and in particular in glioblastoma. There is increasing evidence that 𝛂v𝞫3 integrin receptors can perform receptor-mediated transcytosis with proteins bound to them. An Fn3 protein has been previously engineered to bind with high affinity to the 𝛂v𝞫3 integrin receptor. The goal of this thesis is to verify whether the engineered Fn3 protein can undergo receptor-mediated transcytosis when bound to 𝛂v𝞫3 integrin receptors. To do this, I first focused on developing an in vitro model of the blood tumor barrier. I used transwell plates to develop several co-culture models. Primarily, I worked with U87MG cells, which are derived from astrocyte cells. I also worked with the primary brain endothelial cell line, HBEC-5i, in order to better mimic the conditions of the blood tumor barrier. I was able to identify the experimental conditions necessary for the co-culture of U87MG-U87MG co-cultures as well as HBEC 5i-U87MG co-cultures on transwell plates. I identified the most appropriate imaging techniques for these transwell inserts. I developed an experimental protocol to test the tight junctions of the transwell inserts. Although I had planned further experiments with these co-cultures, I was not able to complete them due to persistent contamination issues. I verified that the U87MG cells express 𝛂v𝞫3 integrin receptors. I was also able to identify that Fn3 proteins that bind 𝛂v𝞫3 integrins are internalized within U87MG cells.

Rights

©2024 Sheher-Bano Ahmed. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

80 pages: color illustrations. Includes bibliographical references (pages 77-80).

Recommended Citation

Ahmed, Sheher-Bano, "Characterizing the Trafficking of Fibronectin Type III Proteins Bound to 𝛂v𝞫3 Integrin Receptors Across the Blood Brain Barrier in vitro" (2024). Honors Project, Smith College, Northampton, MA.
https://scholarworks.smith.edu/theses/2594