Characterizing intercellular heterogeneity in tumor microenvironments via infrared spectroscopic imaging

Hsieh, Pei-Hsuan

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https://hdl.handle.net/2142/122101 Copy

Description

Title

Characterizing intercellular heterogeneity in tumor microenvironments via infrared spectroscopic imaging

Author(s)

Hsieh, Pei-Hsuan

Issue Date

2023-11-01

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

Bhargava, Rohit

Doctoral Committee Chair(s)

Bhargava, Rohit

Committee Member(s)

• Prasanth, Kannanganattu
• Smith, Andrew
• Dobrucki, Wawrzyniec

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Infrared spectroscopic imaging
• 3D cell culture
• Drug quantification
• Breast cancer

Abstract

Differential responses to drug among individual cells have been a major issue due to the intercellular heterogeneity, which are driven by two main factors: the cell cycle status and spatial organization. In this context, infrared (IR) spectroscopic imaging has emerged as a promising technique. It shows advantages on its minimal sample preparation steps and non-destructive detection capabilities, allowing for qualitative and quantitative observations of cell dynamics with a high-throughput manner. The thesis primarily focuses on delving into biological systems by Fourier transform infrared (FT-IR) spectroscopic imaging. First, this study presents a computational and quantitative approach for cell phase analysis in tissue-like 3D structures without any biomarker staining. We report the use of FT-IR spectroscopic imaging to identify subtle biochemical changes within cells, indicative of the G1/S and G2/M phases of the cell cycle. The cells in two-dimensional (2D) cell cultures were synchronized and their states were confirmed by flow cytometry and DNA quantification. Two critical wavenumbers (1059 and 1219 cm−1) were identified as spectral indicators of the cell cycle. These markers were subsequently applied to distinguish cell cycle stages within a 3D cell culture model. Temporal dependence of spectral biomarkers during acini maturation validated the hypothesis that the cells are more proliferative in the early stages of acini development; later stages of the culture showed stability in the overall composition but unique spatial differences in cells in the two phases. Continuing onward, we employ a hydrogel-based compartmentalized system to study the interactions between probiotic bacteria and A498 human cancer cells. The L. lactis strain, situated in the permeable hydrogel shell, secretes a polypeptide known as nisin. Our results demonstrate that nisin induces cell apoptosis, a process we further characterize by an unbiased measurement of chemical content (i.e., nucleic acids, protein secondary structures and lipid conformations) using IR spectroscopy. These features then become the basis for establishing a quantitative relationship. By utilizing a multivariate regression model, we predict cellular dynamics within the co-culture system, providing a comprehensive understanding of nisin’s influence on the cultured cells. Lastly, to maximize the information from the IR spectrum, a doxorubicin derivative (DOX-IR label) was developed as a bioprobe by introducing a metal-carbonyl moiety ((Cp)Fe(CO)2) onto doxorubicin. This modification results in the emergence of two distinguishable peaks (1996 and 2046 cm-1) within the bio-silent region (1800-2700 cm-1). The conjugation and properties of DOX-IR label were confirmed by NMR, mass, ATR-IR, UV-vis spectroscopy and elemental analysis. Subsequently, the biological efficacy of DOX-IR label was evaluated on a 2D cell culture platform. The subcellular localization of DOX-IR label was visualized using fluorescence microscopy, capitalizing the inherent fluorescent property of doxorubicin. Furthermore, the amount of DOX-IR label taken up by each cell was quantified by referencing the carbonyl peaks in IR spectrum and this quantification was correlated with an image-based calibration curve. In summary, the thesis takes advantages of the quantitative, non-destructive and high-throughput capabilities of IR spectroscopic imaging to explore the influence of the intrinsic and extrinsic factors towards intercellular heterogeneity. This approach enables a comprehension of cell status and drug-cell interactions across multiple platforms and has the potential to expedite future drug development efforts.

Graduation Semester

2023-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Pei-Hsuan Hsieh

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