Probing metabolic alterations in breast cancer in response to molecular inhibitors with Raman spectroscopy and validated with mass spectrometry

Abstract

Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen. In this work, we demonstrate a rapid, noninvasive, and label-free approach to evaluate treatment response to molecular inhibitors in breast cancer (BC) cells with Raman spectroscopy (RS). Metabolic reprogramming in BC was probed with RS and multivariate analysis was applied to classify the cells into responsive or nonresponsive groups as a function of drug dosage, drug type, and cell type. Metabolites identified with RS were then validated with mass spectrometry (MS). We treated triple-negative BC cells with Trametinib, an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway. Changes measured with both RS and MS corresponding to membrane phospholipids, amino acids, lipids and fatty acids indicated that these BC cells were responsive to treatment. Comparatively, minimal metabolic changes were observed post-treatment with Alpelisib, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, indicating treatment resistance. These findings were corroborated with cell viability assay and immunoblotting. We also showed estrogen receptor-positive MCF-7 cells were nonresponsive to Trametinib with minimal metabolic and viability changes. Our findings support that oncometabolites identified with RS will ultimately enable rapid drug screening in patients ensuring patients receive the most effective treatment at the earliest time point.

Scheme 1. Schematic representation of metabolic changes probed in breast cancer cells with RS after treatment with small molecule inhibitors downregulating the ERK pathway. RS data combined with multivariate analysis and confirmed with mass spectrometry provide an early response to treatment distinguishing responders from nonresponders.

Fig. 1. MDA-MB-231 cells treated with Trametinib and probed with RS. (a) Mean normalized Raman spectra of untreated cells (black) and cells treated with Trametinib at its working concentration 50 nM (red). Spectra were normalized to 1440 cm⁻¹ biological peak. The inset shows a magnified view of the smaller wavenumber region. (b) Difference spectrum obtained from (a) by subtracting the mean normalized Raman spectrum of cells treated with Trametinib (50 nM) from the untreated cells to highlight changes in Raman footprint. Relevant peaks that either increase or decrease with treatment are shown with an arrow. (c) Selective Raman peaks that decreased with treatment including phosphatidylcholine (719 cm⁻¹), DNA (782 cm⁻¹), phenylalanine (1000 cm⁻¹), amide III (1239 cm⁻¹) and lipids & fatty acids (1310 cm⁻¹). Selective Raman peaks that increased after treatment including tyrosine (830 cm⁻¹), sphingomyelin (875 cm⁻¹), and lipids (1057 cm⁻¹). Here, * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001 determined by Student's t-test. (d) Principal component analysis showing clustering of untreated cells relative to those treated with Trametinib represented in a scatter plot. (e) Corresponding PC loading showing both PC1 and PC2. Relevant peaks in PC2 that distinguish the treated from untreated group are shown with arrows.

Fig. 2. RS distinguishing responders from nonresponders as a function of Trametinib dosage. (a) Mean normalized Raman spectra of untreated MDA-MB-231 cells and those treated with various concentrations (1, 50 and 300 nM) of MEKi (Trametinib). Spectra were normalized to 1440 cm⁻¹ biological peak. (b) PC scatter plot showing clustering of cells based on Trametinib concentrations differentiating responders from nonresponders.

Fig. 3. RS distinguishing responders from nonresponders as a function of drug type. (a) PCA scatter plot comparing MDA-MB-231 cells in response to different concentrations (0.5, 1 and 10 μM) of PI3Ki (Alpelisib) showing no distinct clustering among groups. (b) PCA scatter plot comparing untreated cells (black) to those treated with 1 μM of PI3Ki (Alpelisib, blue) and 50 nM of MEKi (Trametinib, red). Clear clustering of cells was observed for those responsive to treatment relative to nonresponders. (c) MTT viability assay of cells treated for 72 h with MEKi (Trametinib) or PI3Ki (Alpelisib) at 0–10 μM concentrations (n = 4 per concentration). Cell viability was measured at 540 nm. All data were presented as mean ± standard deviation. (d) Immunoblotting analysis of cells in response to treatment with MEKi (Trametinib) or PI3Ki (Alpelisib) at increasing concentrations showed reduced ERK1/2 phosphorylation for cells that responded to treatment.

Fig. 4. Mass spectroscopic analysis of MDA-MB-231 cells treated with Trametinib (MEKi) or Alpelisib (PI3Ki) at their working concentrations (MEKi: 50 nM; PI3Ki: 1 μM). The numbers were a ratio of treated cells to untreated cell control where closer to 1 indicated minimal changes in metabolites. All differential features (samples vs. controls) had a p value of <0.05.

Fig. 5. MCF-7 cells treated with Trametinib and interrogated with RS. (a) Mean normalized Raman spectra of untreated cells (black) relative to those treated with MEKi (Trametinib) at 1 nM (blue), 50 nM (red) and 1 μM (cyan). Spectra were normalized to 1440 cm⁻¹ biological peak. Minimal changes in Raman footprint were observed. (b) PCA scatter plot comparing untreated cells with those treated with Trametinib showing no distinct clustering among groups. (c) MTT viability assay of cells treated for 72 h with Trametinib at various concentrations (n = 4 for each concentration). Cell viability was measured at 540 nm. All data were presented as mean ± standard deviation.