A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

Brian M. Burkel; David R. Inman; María Virumbrales-Muñoz; Erica J. Hoffmann; Suzanne M. Ponik

doi:10.3791/63413

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Method Article

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

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DOI:

10.3791/63413

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May 24th, 2022

Brian M. Burkel¹ , David R. Inman¹ , María Virumbrales-Muñoz¹^,² , Erica J. Hoffmann¹ , Suzanne M. Ponik¹^,³

¹Department of Cell and Regenerative Biology, University of Wisconsin-Madison, ²Department of Pathology, University of Wisconsin-Madison, ³Carbone Cancer Center, University of Wisconsin-Madison

Summary

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

Abstract

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

Introduction

The extracellular matrix (ECM) in the tumor microenvironment is known to be dynamically deposited and remodeled by multiple cell types to further facilitate disease progression¹^,²^,³. These matrix alterations provide both mechanical and biological cues that alter cell behavior and often result in a continuing cycle of matrix remodeling⁴. Investigation into the dynamic, reciprocal interplay between tumor cells and the extracellular matrix is often conducted using three-dimensional (3D) in vitro culture or microfluidic systems. While these bottom....