PD-L1 Expression Is Increased in a Subset of Basal Type Breast Cancer Cells

<div><p>Background</p><p>Tumor cells express programmed death ligand 1 (PD-L1) and is a key immune evasion mechanism. PD-L1 expression in multiple breast cancer cell lines was evaluated to identify intrinsic differences that affect their potential for immune evasion.</p><p>Methods</p><p>PD-L1 expression was analyzed in six breast cancer cell lines: AU565&MCF7 (luminal), BT20&HCC1143 (basal A), MDA231&HCC38 (basal B). Surface and intracellular PD-L1 expression +/− interferon γ for 48 hours was measured by flow cytometry. PD-L1 gene expression data for all breast cancer cell lines in the Comprehensive Cell Line Encyclopedia (CCLE) was analyzed. Correlation between PD-L1 levels and clinicopathologic parameters was analyzed within Oncomine datasets. A tissue microarray containing 61 invasive breast cancer primary tumor cores was stained for PD-L1 expression and analyzed.</p><p>Results</p><p>Basal breast cancer cells constitutively express the highest levels of PD-L1. All cell lines increased PD-L1 expression with interferon γ, but basal B cells (MDA-231 and HCC38) demonstrated the largest increases. There were no differences in protein localization between cell lines. In the CCLE data, basal cell lines demonstrated higher mean PD-L1 expression compared to luminal cell lines. High PD-L1 expressing basal cell lines over-express genes involved in invasion, proliferation, and chemoresistance compared to low PD-L1 basal cell lines. High PD-L1 basal cell lines had lower expression of IRF2BP2 and higher STAT1 levels compared to low PD-L1 expressing cell lines. Within Oncomine datasets PDL1 mRNA levels were higher in basal type tumors. The TMA analysis demonstrated that lymph node positive cases had higher levels of PD-L1 protein expression compared to lymph node negative cases.</p><p>Conclusions</p><p>Basal type breast cancer (especially basal B) express greater levels of PD-L1 constitutively and with IFN γ. High PD-L1 basal cells over-express genes involved in invasion, motility, and chemoresistance. Targeting PD-L1 may enhance eradication of aggressive breast cancer cells by the immune system.</p></div

PD-L1 expression across the six different ATCC cell lines.

<p>Basal subtypes (especially basal B) demonstrated much greater constitutive PD-L1 expression than luminal subtypes. Treatment with IFNγ caused PD-L1 expression to increase in all cell lines but basal subtypes demonstrated much greater inducible levels of PD-L1.</p

Oncomine box plot RNA expression data for PD-L1 (CD274) shown within the TCGA (Fig 5A. TNBC and Fig 5B. nodal status) and Fig 5C. Gluck datasets (PAM50 data).

<p>Oncomine box plot RNA expression data for PD-L1 (CD274) shown within the TCGA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088557#pone-0088557-g005" target="_blank">Fig 5A</a>. TNBC and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088557#pone-0088557-g005" target="_blank">Fig 5B</a>. nodal status) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088557#pone-0088557-g005" target="_blank">Fig 5C</a>. Gluck datasets (PAM50 data).</p

PD-L1 mRNA expression across multiple cell lines in the CCLE database.

<p>Basal cell lines as a group had statistically higher mean PD-L1 expression levels compared to non-basal subtypes.</p

Immunohistochemistry for PD-L1 in a breast cancer TMA.

<p>A) Negative control B) Positive control C) low D) intermediate E) strong.</p

PD-L1 protein localization at baseline and post IFNγ treatment.

<p>PD-L1 protein expression increases with treatment, but there does not appear to be a major shift in protein localization patterns (proportion of surface vs. surface+intracellular) between the different cell lines and molecular subtypes.</p

ATCC cell lines used in the flow cytometry analysis.

<p>ATCC cell lines used in the flow cytometry analysis.</p

High PD-L1 expressing basal breast cancer cell lines (N = 12) demonstrate higher levels of STAT1 expression and lower levels of IRF2BP2 compared to low PD-L1 expressing cell lines (N = 12).

<p>Error bars are 95% CI.</p

Additional file 1: of Neoadjuvant radiotherapy of early-stage breast cancer and long-term disease-free survival

Relative survival and bootstrap analyses. Relative survival for ER-positive patients who underwent partial mastectomy (stratified by radiation sequencing) and bootstrap analyses for neoadjuvant and adjuvant RT cohorts matched for size and year of diagnosis. (DOCX 749 kb

Additional file 1: of Evaluation of invasive breast cancer samples using a 12-chemokine gene expression score: correlation with clinical outcomes

Outcomes (OS, RFS for entire cohort and RFS for HER2+) plus multivariate regression analysis of overall survival within the TCC dataset. (DOCX 119 kb