Supplementary Figure 3: Comparison of CLIC4 and RYBP mRNA levels in high score and low score treated MDA-MB-231 cells to untreated MDA-MB-231 cells. Real-time PCR of CLIC4 in untreated and high score treated MDA-MB-231 cells normalized to actin. (Mean {plus minus} SEM) (in quadruplicate, ** p < 0.01, Mann-Whitney test). Real-time PCR of RYBP in untreated and low score treated MDA-MB-231 cells normalized to actin. (Mean {plus minus} SEM) (in quadruplicate, ** p < 0.01, Mann-Whitney test).
Funding
Anna Maria and Stephen Kellen Foundation, Emad Zikry and Jeffrey Epstein
Samuel Waxman Cancer Research Foundation
NIH
NIDDK
Irma T. Hirschl Trust
ARTICLE ABSTRACT
Purpose: Despite the fact that interstitial fluid (IF) represents a third of our body fluid, it is the most poorly understood body fluid in medicine. Increased IF pressure is thought to result from the increased deposition of extracellular matrix in the affected tissue preventing its reabsorption. In the cancer field, increased rigidity surrounding a cancerous mass remains the main reason that palpation and radiologic examination, such as mammography, are used for cancer detection. While the pressure produced by IF has been considered, the biochemical composition of IF has not been considered in its effect on tumors.Experimental Design: We classified 135 IF samples from bilateral mastectomy patients based on their ability to promote the invasion of breast cancer cells.Results: We observed a wide range of invasion scores. Patients with high-grade primary tumors at diagnosis had higher IF invasion scores. In mice, injections of high-score IF (IFHigh) in a normal mammary gland promotes ductal hyperplasia, increased collagen deposition, and local invasion. In a mouse model of residual disease, IFHigh increased disease progression and promoted aggressive visceral metastases. Mechanistically, we found that IFHigh induces myofibroblast differentiation and collagen production through activation of CLIC4. IFHigh also downregulates RYBP, leading to degradation of p53. Furthermore, in mammary glands of heterozygous p53-mutant knock-in mice, IFHigh promotes spontaneous tumor formation.Conclusions: Our study indicates that IF can increase the deposition of extracellular matrix and raises the provocative possibility that they play an active role in the predisposition, development, and clinical course of sporadic breast cancers. Clin Cancer Res; 23(18); 5446–59. ©2017 AACR.
