Investigation of the Effect of PD-L1 Blockade on Triple Negative Breast Cancer Cells Using Fourier Transform Infrared Spectroscopy

Abstract

Interactions between programmed death-1 (PD-1) with its ligand PD-L1 on tumor cells can antagonize T cell responses. Inhibiting these interactions using immune checkpoint inhibitors has shown promise in cancer immunotherapy. MDA-MB-231 is a triple negative breast cancer cell line that expresses PD-L1. In this study, we investigated the biochemical changes in MDA-MB-231 cells following treatment with atezolizumab, a specific PD-L1 blocker. Our readouts were Fourier Transform Infrared (FTIR) spectroscopy and flow cytometric analyses. Chemometrical analysis, such as principal component analysis (PCA), was applied to delineate the spectral differences. We were able to identify the chemical alterations in both protein and lipid structure of the treated cells. We found that there was a shift from random coil and α-helical structure to β-sheet conformation of PD-L1 on tumor cells due to atezolizumab treatment, which could hinder binding with its receptors on immune cells, ensuring sustained T cell activation for potent immune responses. This work provides novel information about the effects of atezolizumab at molecular and cellular levels. FTIR bio-spectroscopy, in combination with chemometric analyses, may expedite research and offer new approaches for cancer immunology.

Keywords: FTIR; biochemical alterations; breast cancer; chemometric analysis; spectroscopy; tumor cells.

Figure 1

Effect of atezolizumab on PD-L1 expression on MDA-MB-231 cells. MDA-MB-231 cells were cultured in the presence or absence of atezolizumab for up to 24 h to investigate the effect of PD-L1 blockade on the surface and transcriptomic expression of PD-L1. Representative flow cytometric plots show PD-L1 expression on non-treated (NTx) and treated (Tx) MDA-MB-231 cells (A). The bar plot shows differences in PD-L1 transcriptomic expression in MDA-MB-231 cells up to 24 h Tx with atezolizumab (B).

Figure 2

FTIR analysis of MDA-MB-231 untreated and treated cells with atezolizumab. FTIR analyses were performed on non-treated (NTx) and MDA-MB-231 cells treated (Tx) with atezolizumab after 24 h. Plots show FTIR spectra, second derivative and curve fitting of NTx MDA-MB-231 cells (A) and MDA-MB-231 cells Tx with atezolizumab (B). Plots show spectral differences between NTx and Tx MDA-MB-231 cells (C).

Figure 3

Chemometric analysis of FTIR experimental data on the full spectral range (4000–1000 cm⁻¹). Amount of variance captured by PCs (A), PCA score plot (B), loading plots for the spectral data (C), squared correlation for the first three PCs for the spectral collection (D), and hierarchical clustering of the spectra for the different experimental samples (E).

Figure 4

Chemometric analysis of FTIR experimental data on the protein region (1750–1480 cm⁻¹). Amount of variance captured by PCs (A), PCA score plot (B), loading plots for the spectral data (C), and squared correlation for the first 3 PCs for the spectral collection (D).

Figure 5

Hierarchical clustering of the spectral data of the samples. Hierarchical clustering of the spectra for the different experimental samples (A) and PCA biplot explaining which wavenumbers are associated with untreated versus treated samples (B).