Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy

Abstract

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC−MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC−MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC−MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC−MS to investigate pharmacokinetics at the single-cell level.

Raman Microscopy: Progress in Research on Cancer Cell Sensing

“…However, it is complicated to quantify the drug uptake or metabolism response at a single cell level using the RM alone. Recently, it has been demonstrated that correlating the Raman signal with the destructive mass spectrometry analysis renders the drug and metabolite quantification at a single-cell level possible [ 62 ]. Indeed, the combined platform is capable of providing more information about drug uptake, metabolism, and effects at the single-cell level, demonstrating the potential to investigate pharmacokinetics at the single-cell level.…”

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

“…In the future, due to recent increasing interest in combining datasets from imaging modalities 19 , it may also be of interest to study the systematic variations between Raman signals and mass spectrometry results (as well as other omics methods) by using integrative computational approaches. Interestingly, we have already found several weak but significant linear correlations between the intensities of Raman peaks identified by VIP scores and the abundance of tamoxifen or its metabolite at the single-cell level as identified by MS 4 . This data may suggest a metabolic relationship between MS profiles and Raman spectra and the possibility to predict these values.…”

“…Minimize the laser intensity at the sample to ~2.4 mW/µm 2 so that cells survive laser exposure. 4. Set up the microchamber at 5% CO 2 and 37 °C.…”

“…Trim the spectral range to 600-1,700 cm -1 to select the fingerprint region and ensure that there is no unwanted edge effects on the spectra due to bad polynomial fitting. 4. Perform a normalization step such as vector normalization (intensity at each wavenumber is divided by the global l2 norm or maximum singular value of a spectrum) to normalize the spectral intensity 10 , although other normalizations can be considered.…”

“…To achieve this, we have designed a unique system in which living single cells are screened using label-free Raman spectroscopy then further characterized using mass spectrometry 4 . Raman spectroscopy provides a molecular fingerprint of the cellular state, a complex spectrum resulting from the contributions of many molecules inside the cell.…”