Figure 2.

VM vessels co-localize with REST⁺ non-NE cells. (A) IHC in serial CDX21 tissue sections for VM vessels (PAS⁺/CD31⁻, pink), endothelial vessels (PAS⁺/CD31⁺, pink+brown), REST, SYP and NCAM (brown). Scale bars, 50 μm. (B) Quantification of SYP, NCAM, and REST expression in VM-positive (black circles) and VM-negative (open circles) regions of CDX identified in (A). Each circle represents a region that was defined and quantified, and two independent mice were analyzed (n = 2 mice, n = 5 regions). Data are mean (± SEM). (C, D) Multiplex IHC showing VM vessels (PAS⁺/CD31⁻, yellow arrows), endothelial vessels (PAS⁺/CD31⁺, brown, green arrows), and REST (blue, in C) or SYP (blue, in D). Scale bars, 100 μm. (E) Percentage of VM vessels co-localized with REST (REST⁺ VM vessels) (n = 2 mice per CDX). Where the total REST expression for each CDX was derived from.⁸ Tumor sections from three independent mice per CDX were analyzed and the mean REST expression plotted. (F) Multiplex IHC illustrating VM vessels (PAS⁺/CD31⁻, yellow arrows), endothelial vessels (PAS⁺/CD31⁺, brown, green arrows), and REST (blue) in SCLC patient biopsy samples. Scale bars, 20 μm. (G) Percentage of VM vessels co-localized with REST in SCLC patient biopsy samples. n = 5 (<4% VM vessel score and <20 VM vessels per section; VM vessel scores are 9% (light blue), 5% (black), 4% (dark blue), 6% (white), and 9% (pink). (H) Pearson correlation of VM score versus REST transcript expression in CDX (R = 0.1753, p = 0.0468). The average REST expression is illustrated for three independent tumors per CDX.⁸ (I) Pearson correlation of VM score versus SYP transcript expression in CDX (R = 0.2822, p = 0.0091). The average SYP expression is illustrated for three independent tumors per CDX.⁸ CDX13 was removed from the analyses in H and I as there was a high level of VM vessels and REST expression in the tumor, which may bias the analysis. **p < 0.01, ****p < 0.0001 two-tailed unpaired Student’s t test. CDX, circulating tumor cell-derived explant; IHC, immunohistochemistry; NE, neuroendocrine; PAS, Periodic Acid Schiff stain; SEM, standard error of the mean; VM, vasculogenic mimicry.