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

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 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

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

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

Additional file 1: of The anti-fibrotic agent pirfenidone synergizes with cisplatin in killing tumor cells and cancer-associated fibroblasts

MTS assay showing the viability of H358 ( A ), H1299 ( B ), H23 ( C ), H157 ( D ), H2122 ( E ), and PC9 ( F ) after 72-h treatment with a low dose of cisplatin (Cis; 10 μM) and a low dose of pirfenidone (Pirf; 0.5 mg/mL). * P < 0.05. (PDF 1253 kb

Additional file 2: of The anti-fibrotic agent pirfenidone synergizes with cisplatin in killing tumor cells and cancer-associated fibroblasts

Immunoblot analysis shows an increase in phosphorylated ERK when A549 cells are treated with low doses of cisplatin (10 μM) and low doses of pirfenidone (0.5 mg/mL) at early time points (15 and 30 min; A ) when compared to each drug alone. B: At 48 h after treatment, there is a decrease in phosphorylated Akt when compared to each drug alone. (PDF 1211 kb