Multiplexed angiogenic biomarker quantification on single cells

Chen, Si

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https://hdl.handle.net/2142/102454 Copy

Description

Title

Multiplexed angiogenic biomarker quantification on single cells

Author(s)

Chen, Si

Issue Date

2018-12-06

Director of Research (if dissertation) or Advisor (if thesis)

Imoukhuede, Princess

Doctoral Committee Chair(s)

Imoukhuede, Princess

Committee Member(s)

• Bhargava, Rohit
• Harley, Brendan
• Murphy, Catherine
• Smith, Andrew

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Quantitative flow cytometry
• angiogenesis
• VEGF
• PDGF
• Biomarker
• tyrosine kinase receptor
• single cell measurement
• tumor heterogeneity
• glioblastoma
• quantum dot

Abstract

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multi-cell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell (EC) characteristics and response in tumor microenvironments. However, data on other angiogenic receptors, such as platelet derived growth factor receptors (PDGFRs), Tie receptors, are also necessary for the development of an accurate angiogenesis model. To gain insights on the involvement of these angiogenic receptors in angiogenesis, I develop a method to quantify receptor concentrations as well as the cell-by-cell heterogeneity. I establish protocols to measure cell membrane VEGFR, NRP1, Tie2, and PDGFR concentration on several cell and tissue models including human dermal fibroblasts (HDFs) in vitro, a 2D endothelial/fibroblast co-culture model in vitro, and a patient-derived xenograft (PDX) model of glioblastoma (GBM). I demonstrate VEGF-A165-mediated downregulation of membrane PDGFRα (~25%) and PDGFRβ (~30%) on HDFs, following a 24-hour treatment. This supports the idea that VEGF-A165 acts independently of VEGFRs to signal through PDGFRα and PDGFRβ. I uncover high intratumoral heterogeneity within the GBM PDX model, with tumor EC-like subpopulations having high concentrations of membrane VEGFR1, VEGFR2, EGFR, IGFR, and PDGFRs. To gain greater insights into cell heterogeneity and examine angiogenic signaling pathways as a whole, I utilize the unique spectral properties of quantum dots (Qdots), and combines Qdots with qFlow cytometry, to dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, I reduce nonspecific binding between Qdot-conjugated antibodies and cells, identify optimal labeling conditions, and establish that 800 – 20,000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations we demonstrate measurement of 1,100 VEGFR1 and 6,900 VEGFR2 per HUVEC. 24 h VEGF-A165 treatment induce ~90% upregulation of VEGFR1 and ~30% downregulation of VEGFR2 concentration. We further analyze HUVEC heterogeneity and observe that 24 h VEGF-A165 treatment induces ~15% decrease in VEGFR2 heterogeneity. Overall, we demonstrate experimental and analysis strategies for quantifying two or more RTKs at single-level using Qdots, which will provide new insights into biological systems.

Graduation Semester

2018-12

Type of Resource

text

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http://hdl.handle.net/2142/102454

Copyright and License Information

Copyright 2018 Si Chen

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