Poster R4 - Label-free infrared imaging for metabolic measurements in a three-dimensional in vitro cancer cell culture system

Introduction: The current standard of care for most cancers involves maximum resection of tumor mass surgically, followed by targeted doses of radiation and chemotherapy. Altered cell-metabolism (The Warburg Effect) is the hallmark of cancer cells as the mitochondrial function is diminished in most cancer cells. Popular techniques today for measuring metabolic data include liquid chromatography-mass spectrometry (LC/MS) and the use of isotope labelled carbon for glucose-based or in vivo imaging studies. However, these techniques are either relative in nature (LC/MS) or extremely expensive (isotope tracing) for exploratory studies. This necessitates the need for an alternative approach for localizing and quantifying metabolite uptake at a single cell level. In this study, we propose the use of infrared imaging combined with atomic force microscopy (AFM-IR), to get a high-resolution topographic image of a cell combined with its chemical data. We have further modified this instrument (now called Null-Deflection scanning probe Infrared - NDIR) for enhanced image quality and this innovative approach enables us to perform high resolution imaging and chemical analysis of samples at the single-cell level in a label-free manner. We hypothesize that combining NDIR technique with metabolic data-based machine learning algorithms can help generate metabolic maps for better understanding cancer from a metabolic standpoint.

Materials and

Methods: To be able to localize and quantify metabolites, IR absorbance-concentration calibration curves were established using a non-planar 3D printed microfluidics device for high throughput studies. Cell samples for NDIR imaging were prepared using standard protocol for TEM imaging without any osmium staining. MCF7, MCF10A and U87MG cells were cultured in Matrigel and treated with different glucose concentrations to measure the uptake. Fixed cells were dehydrated in graded ethanol, infiltrated and embedded in epoxy resin, sectioned to 100 nanometer thickness and mounted on gold coated silicon substrates before imaging. Samples were imaged at 1660 cm-1 for protein measurements and imaging at 1033 cm-1 for glucose uptake measurements is now underway.

Results, Conclusions, and Discussions: By combining 3D printing, microfluidics and IR imaging, we have developed an exhaustive library of calibration curves for analyzing key metabolites of interest in cancer studies. We have also optimized a rigorous sample preparation protocol for NDIR imaging of three-dimensional in vitro cell culture systems and have depicted its applications in single cell, acini and spheroid cultures by localizing individual protein structures. Our next attempts will be targeted at localizing and quantifying key metabolite uptake in cancer cells.

Acknowledgements (Optional): This work was supported by NSF grant 2243257, the National Science Foundation Science and Technology Center for Quantitative Cell Biology.