PD-L1 and epithelial-mesenchymal transition in circulating tumor cells from non-small cell lung cancer patients: A molecular shield to evade immune system?

Figures & data

Figure 1. In representative images, nuclei are displayed in blue. Pseudo-colors were changed for each of the four channels to optimize the visualization of the different signals. (Panel A) Phenotype of circulating tumor cells (CTCs) in filters obtained by ScreenCell Cyto kit. Hematoxylin and eosin (H&E) stains and immunofluorescence for wide spectrum screening cytokeratin (K), PD-L1, and N-Cadherin (N-Cad). Large (upper) or small (lower) clusters of K+ cells are shown. Subpopulations of K+ cells co-expressing PD-L1 and N-Cad (yellow arrow) and lacking PD-L1 expression (green arrow) are displayed. (Panel B) Immunofluorescence for thyroid transcription factor 1 (TTF1), PD-L1, and Vimentin (VIM) in filters. Clusters of TTF1+ cells (A) and single TTF1+ cells co-expressing PD-L1 and Vimentin. (Panel C) Immunofluorescence for thyroid transcription factor 1 (TTF1) and PD-L1. PD-L1 expression at cytoplasmic/membranous level (empty arrows) and at nuclear level (full arrows) in TTF1+ cells.

Table 1. Percentage of PD-L1 positive CTCs and of double PD-L1/EMT positive CTCs in patients with NSCLC.

			PD-L1+ CTCs			EMT+ CTCs
Patient	CTCs (Score)	PD-L1+ CTCs	EMT+	EMT−	EMT+CTCs	PD-L1+	PD-L1−	Stained filters (n.)
#1	2	40%	70%	30%	30%	95%	<5%	2
#2	N.D.	/	/	/	/	/	/	2
#3	2.5	40%	65%	35%	30%	95%	<5%	2
#4	1.5	5%	50%	50%	10%	30%	70%	2
#5	N.D.	/	/	/	/	/	/	2
#6	1.5	15%	70%	30%	15%	95%	<5%	2
#7	2.5	55%	78%	22%	45%	95%	<5%	2
#8	2	15%	50%	50%	10%	75%	25%	2
#9	0.5	30%	60%	40%	25%	95%	<5%	2
#10	2.5	60%	70%	30%	50%	90%	10%	2
#11	N.D.	/	/	/	/	/	/	1
#12	N.D.	/	/	/	/	/	/	1
#13	3	80%	70%	30%	50%	95%	<5%	1

The presence of CTC clusters or single CTCs was assessed by using a semi-quantitative score, as following: 0 < no clusters/single cells; 1 = 1–3 clusters/single cells; 2 = 4–5 clusters/single cells; 3 = > 5 clusters/single cells. The percentage of PD-L1+ CTCs (with and without co-expression of EMT markers) and EMT+ CTCs (with and without co-expression of PD-L1) are also indicated.