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Supplementary Data File from Alterations in Pericyte Subpopulations Are Associated with Elevated Blood–Tumor Barrier Permeability in Experimental Brain Metastasis of Breast Cancer

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posted on 2023-03-31, 18:28 authored by L. Tiffany Lyle, Paul R. Lockman, Chris E. Adkins, Afroz Shareef Mohammad, Emily Sechrest, Emily Hua, Diane Palmieri, David J. Liewehr, Seth M. Steinberg, Wojciech Kloc, Ewa Izycka-Swieszewska, Renata Duchnowska, Naema Nayyar, Priscilla K. Brastianos, Patricia S. Steeg, Brunilde Gril

Supplemental Tables. Supplemental Tables S1-6. Table S1. Antibodies used for quantification and concentrations. Table S2. Antibodies that could not be quantified in the model systems under fixation conditions used to identify Texas Red dextran permeability. Table S3. Summary statistics for analysis of brain metastases vs. uninvolved brain for each analyzed immunofluorescence marker. Table S4. Summary statistics for analysis of highly permeable vs. poorly permeable brain metastases for each analyzed immunofluorescence marker. Table S5. Antibodies that could not be quantified in human specimens. Table S6: Summary of immunofluorescence analysis of human brain metastasis specimens Supplemental Figures. Supplemental Figures S1-9. Figure S1. Homogenous HER2 staining in a SUM190-BR3 brain metastasis. Figure S2. No significant trend in Claudin-5 expression in three models of brain metastasis. Figure S3. Increased VEGF expression in brain metastasis. Figure S4. Decreased ZO-1 expression in brain metastasis. Figure S5. Decreased AQP4 expression and an alteration in the AQP4 expression pattern in brain metastasis. Figure S6: Heterogeneous trends in Collagen IV expression in three models of brain metastasis. Figure S7: Colocalization of Desmin+ pericytes and CD13+ pericytes with PDGFR-β. Figure S8: Desmin immunofluorescent staining in human specimens. Figure S9. Examples of immunofluorescent staining in human specimen #5 Supplemental Experimental Procedures. Detailed experimental procedures that were not included in the body of the manuscript. These include SUM190-BR3 cell line derivation, animal experiments, immunofluorescence, image analysis, statistical analysis, and human craniotomy specimen preparation.

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ARTICLE ABSTRACT

Purpose: The blood–brain barrier (BBB) is modified to a blood–tumor barrier (BTB) as a brain metastasis develops from breast or other cancers. We (i) quantified the permeability of experimental brain metastases, (ii) determined the composition of the BTB, and (iii) identified which elements of the BTB distinguished metastases of lower permeability from those with higher permeability.Experimental Design: A SUM190-BR3 experimental inflammatory breast cancer brain metastasis subline was established. Experimental brain metastases from this model system and two previously reported models (triple-negative MDA-231-BR6, HER2+ JIMT-1-BR3) were serially sectioned; low- and high-permeability lesions were identified with systemic 3-kDa Texas Red dextran dye. Adjoining sections were used for quantitative immunofluorescence to known BBB and neuroinflammatory components. One-sample comparisons against a hypothesized value of one were performed with the Wilcoxon signed-rank test.Results: When uninvolved brain was compared with any brain metastasis, alterations in endothelial, pericytic, astrocytic, and microglial components were observed. When metastases with relatively low and high permeability were compared, increased expression of a desmin+ subpopulation of pericytes was associated with higher permeability (231-BR6 P = 0.0002; JIMT-1-BR3 P = 0.004; SUM190-BR3 P = 0.008); desmin+ pericytes were also identified in human craniotomy specimens. Trends of reduced CD13+ pericytes (231-BR6 P = 0.014; JIMT-1-BR3 P = 0.002, SUM190-BR3, NS) and laminin α2 (231-BR6 P = 0.001; JIMT-1-BR3 P = 0.049; SUM190-BR3 P = 0.023) were also observed with increased permeability.Conclusions: We provide the first account of the composition of the BTB in experimental brain metastasis. Desmin+ pericytes and laminin α2 are potential targets for the development of novel approaches to increase chemotherapeutic efficacy. Clin Cancer Res; 22(21); 5287–99. ©2016 AACR.

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    Clinical Cancer Research

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    Breast CancerProgression, Invasion & MetastasisMetastasis

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