Frequent expression of PD-L1 on circulating breast cancer cells

Abstract

Immune checkpoint regulators such as PD-L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In the present paper, we provide evidence that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch(®) system and found PD-L1((+)) CTCs in 11 patients (68.8%). The fraction of PD-L1((+)) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD-L1 on CTCs. The established CTC/PD-L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.

Keywords: Breast cancer; Circulating tumor cells; PD-L1 expression.

Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Figures

Figure 1

Human B7H1 Detection in direct ELISA with monoclonal anti‐human B7‐H1. Antibody: Human B7‐H1 monoclonal Ab (R&D systems). Different proteins have been analyzed: recombinant human B7‐H1, recombinant human B7‐1, recombinant human B7‐2, recombinant B7‐H2, recombinant human B7‐H3, recombinant human B7‐H3b, recombinant human B7‐H4, recombinant B7‐H6, recombinant B7‐H7, recombinant human PD‐L2 and recombinant mouse B7‐H1. The plate was coated with recombinant protein at mass/well cited in the graph. The different concentrations used were: primary Ab (0.5 μg/mL), secondary Ab (goat anti‐mouse IgG polyclonal antibody‐biotinylated at 1:10,000), enzyme (streptavidin‐AP at 1/1000); the substrate was the p‐nitrophenyl phosphate. Abbreviations: rh: recombinant human; rm: recombinant mouse.

Figure 2

Specificity and sensitivity of the anti‐human B7H1 MAb (R&D system). A, Determination of the anti‐human B7H1 MAb recognition profile on test protein mixtures from the assigned cell lines and tumour entities by Western Blot. In parallel, the protein recognition pattern for the mouse isotype control was analyzed. For each lane 20 μg of protein was loaded. Both membranes were exposed to the same X‐ray films. To investigate the band pattern for the two antibodies, a short X‐ray film exposition time (10 s) and an overexposed X‐ray film (4 min) is shown. Alpha Tubulin served as a loading control. B, Flow cytometry analyses. SKBR3 and SU‐DHL‐4 cell lines were incubated with the anti‐human B7‐H1 MAb‐PE (in red) and the corresponding isotypic control (IgG1, in blue) – The flow cytometer used for all the analyses was the Cyan (Beckman Coulter); C, Immunocytochemical analyses. SKBR3 and SU‐DHL‐4 cells were centrifuged gently in a Cytospin™ 4 Cytocentrifuge (Thermo Scientific) on standard glass slides after being stained with the anti‐human B7‐H1‐PE MAb and the corresponding isotypic control. DAPI was added on the preparation to stain nuclei in blue. PDL1(+) tumor cells are stained in red (Fluorescent microscope Axio Imager.M1 Zeiss, software AxioVision Release 4.6.3. – magnification X20). D, CellSearch®system analyses. SKBR3 and SU‐DHL‐4 were analyzed in the CellSearch® system using either the human anti‐human B7‐H1 MAb‐PE or the corresponding isotypic control in the 4th channel (PE). All experiments were repeated 3 times and the figures show representative results.

Figure 3

Detection of PD‐L1 expression on SKBR3 cells with the CellSearch® system. A, Single SKBR3 cells with different levels of PD‐L1 expression (B7H1PE in the 4th channel): score 0 (no PD‐L1 expression), 1 (weak PD‐L1 expression) and 2 (strong PD‐L1 expression); B, SKBR3 clusters with different PD‐L1 immunoscores.

Figure 4

CTCs expressing various degrees of PD‐L1 detected in blood of HR(+) HER2(−) metastatic breast cancer patients with the CellSearch® system. Representative pictures of CTCs classified by scores 0, 1 and 2 for PD‐L1 expression.