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.

PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer

“…However, this could occur as a result of an altered PD-L1 protein structure [38]. Consistent with this, our recent work showed that atezolizumab treatment alters the structure of PD-L1 on MDA-MB-231 cells by inducing a transition from a random coil and α-helical structure to β-sheet conformation [39]. Independently of the PD-1/PD-L1 signaling pathway, the intracytoplasmic domain of PD-L1 was shown to be functional [16,40,41], therefore, alterations in PD-L1 protein structure by atezolizumab might have a negative impact on its function.…”

Pembrolizumab Interferes with the Differentiation of Human FOXP3+–Induced T Regulatory Cells, but Not with FOXP3 Stability, through Activation of mTOR

“…Our findings could imply that the changes in iTreg transcriptome in response to pembrolizumab treatment are not due to a change in PD-1 expression, but possibly as a result of an altered protein structure (41). Similarly, we have recently reported that atezolizumab (anti-PD-L1) alters the structure of PD-L1 on MDA-MB-231 breast cancer cells by inducing a transition from random coil and a-helical structure to b-sheet conformation (42). Moreover, it has been reported that the action of pembrolizumab is profoundly dependent on the binding of flexible C9D loop of PD-1, rather than blocking PD-1/PD-L1 interactions (43).…”

Fourier Transform Infrared Imaging—A Novel Approach to Monitor Bio Molecular Changes in Subacute Mild Traumatic Brain Injury

“…Total protein is represented by the amide I/amide I + amide II band at 1700–1500 cm −1 . The secondary structure of the protein band (Amide I) consists of α-helix structures at 1655 cm −1 , a β-sheet at ~1645 cm −1 and a random coil at 1630 cm −1 , as shown in Table 1 [ 15 , 17 , 30 , 53 ]. Total lipid was represented by the C–H stretching region—spectral range of 3000–2800 cm −1 which consists of asymmetric and symmetric CH 2 (saturated lipid/lipid acyl) at 2915–2930 and 2860–2840 cm −1 ; CH 3 asymmetric stretching ν as (CH 3 ) (methyl concentration) at 2960–2950 cm −1 ; olefinic=CH at 3027–3000 cm −1 (unsaturated lipid) and lipid ester ν(C=O) (oxidative stress byproducts) at 1755–1715 cm −1 .…”

“…Various functional groups of biomolecules, such as proteins, lipids, carbohydrates and nucleic acids, in samples can be analyzed based on their characteristic vibrational bands in FTIR spectra [ 15 , 16 , 17 ]. FTIR spectroscopy is considered as non-damaging, operator independent and timesaving technique for the characterization of different biological systems [ 18 , 19 , 20 ].…”