Imaging of PD-L1 in single cancer cells by SERS-based hyperspectral analysis

Abstract

We developed a hyperspectral imaging tool based on surface-enhanced Raman spectroscopy (SERS) probes to determine the expression level and visualize the distribution of PD-L1 in individual cells. Electron-microscopic analysis of PD-L1 antibody - gold nanorod conjugates demonstrated binding the cell surface and internalization into endosomal vesicles. Stimulation of cells with IFN-γ or metformin was used to confirm the ability of SERS probes to report treatment-induced changes. The multivariate curve resolution-alternating least squares (MCR-ALS) analysis of spectra provided a greater signal-noise ratio than single peak mapping. However, single peak mapping allowed a systematic subtraction of background and the removal of non-specific binding and endocytic SERS signals. The mean or maximum peak height in the cell or the mean peak height in the area of specific PD-L1 positive pixels was used to estimate the PD-L1 expression levels in single cells. The PD-L1 levels were significantly up-regulated by IFN-γ and inhibited by metformin in human lung cancer cells from the A549 cell line. In conclusion, the method of analyzing hyperspectral SERS imaging data together with systematic and comprehensive removal of non-specific signals allows SERS imaging to be a quantitative tool in the detection of the cancer biomarker, PD-L1.

Application of Aptamer-SERS Nanotags for Unveiling the PD-L1 Immunomarker Progression Correlated to the Cell Metabolic Bioprocess

“…SERS nanotags, developed by combining SERS-active nanoparticles with a PD-L1 antibody and a Raman reporter, exhibit specific binding to PD-L1 on cell membrane. These nanotags could efficiently unveil information about PD-L1 content on cell membrane through enhanced Raman signals, contributing to precise and sensitive detection in biological studies. − For example, Schlücker et al have proposed using immuno-SERS nanotags to locate PD-L1 on single cancer cell membrane . Feng et al have reported the use of aptamer-combined metal organic frameworks (MOFs) to probe PD-L1 in cells .…”

Electrostimulus-associated PD-L1 expression on cell membrane revealed by immune SERS nanoprobes

“…4,5 Thus, PD-L1 can help tumour cells escape antitumor immune responses and ultimately progress, disseminate, and metastasise. 6 Treatments that inhibit PD-L1 expression have become effective methods in cancer therapy. 4 In this regard, ways to mediate the dynamic expression of PD-L1 on cell membranes and monitor changes in PD-L1 content are essential for cancer treatment.…”

“…4 In this regard, ways to mediate the dynamic expression of PD-L1 on cell membranes and monitor changes in PD-L1 content are essential for cancer treatment. So far, many techniques that provide impactful information in biomedical and bioanalytical research, such as mass spectroscopy, 5 nuclear magnetic resonance, 6 electrochemistry, 7 fluorescence spectroscopy, 5 have been employed for PD-L1 determination. However, these techniques have many disadvantages, such as low spatial resolution, sensitivity, and stability, which limit their application in single-cell analysis.…”

“…So far, immune SERS tags have been developed by decorating SERS-active nanoparticles with a PD-L1 antibody and a Raman reporter, which can specially bind PD-L1 on the cell membranes and disclose the PD-L1 content by SERS. [6][7][8][9][10] For example, Zheng et al developed a charge-transfer-based SERS probe for PD-L1 evaluation at the single-cell level. 9 Schlücker et al proposed immuno-SERS (iSERS) for identifying the locations of PD-L1 on a single cancer cell.…”