Fibroblast subtypes define a metastatic matrisome in breast cancer
Heather M. Brechbuhl, Alexander S. Barrett, Etana Kopin, Jaime C. Hagen, Amy L. Han, Austin E. Gillen, Jessica Finlay-Schultz, Diana M. Cittelly, Philip Owens, Kathryn B. Horwitz, Carol A. Sartorius, Kirk Hansen, Peter Kabos
Heather M. Brechbuhl, Alexander S. Barrett, Etana Kopin, Jaime C. Hagen, Amy L. Han, Austin E. Gillen, Jessica Finlay-Schultz, Diana M. Cittelly, Philip Owens, Kathryn B. Horwitz, Carol A. Sartorius, Kirk Hansen, Peter Kabos
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Research Article Oncology

Fibroblast subtypes define a metastatic matrisome in breast cancer

Abstract

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient — that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146+) or lack (CD146–) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146– fibroblasts promoted increased metastasis compared with CD146+ fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146+ CAFs produced an environment rich in basement membrane proteins, while CD146– CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146– CAFs predicted for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146– CAFs require aggressive treatments.

Authors

Heather M. Brechbuhl, Alexander S. Barrett, Etana Kopin, Jaime C. Hagen, Amy L. Han, Austin E. Gillen, Jessica Finlay-Schultz, Diana M. Cittelly, Philip Owens, Kathryn B. Horwitz, Carol A. Sartorius, Kirk Hansen, Peter Kabos

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Figure 5

CD146– CAFs promote metastasis in vivo.

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CD146– CAFs promote metastasis in vivo. MCF-7 tumors mixed with either C...
MCF-7 tumors mixed with either CD146– or CD146+ CAFs were grown in the mammary fat pad of mice. (A and B) Representative image and quantification of metastasis to the lung of mice with MCF-7/CD146– or MCF-7/CD146+ tumors in whole tissue (green fluorescence). Scale bar: 250 mm. Contingency analysis used Fisher’s exact test. (C) Lesions were detected by IHC staining with human-specific pan-cytokeratin (blue) and counted in all lobes. Blue arrows show single cell, micrometastasis (≤20 cells), or macrometastasis (>20 cells). (D) Analysis of the total number of metastatic lesions showed that mice with MCF-7/CD146– tumors had significantly more lesions than those with MCF-7/CD146+ tumors. Total analysis used Mann-Whitney U test. (E) Lesions were grouped according to the number of cells per lesion. Mice bearing MCF-7/CD146– tumors had significantly more micrometastases compared with MCF-7/CD146+ tumors. Lung lesion by size analysis used a 2-way ANOVA. Scale bars: 100 mm. (F) Tumors with CD146– CAFs grew significantly larger than tumors with CD146+ CAFs. Analysis was by 2-way ANOVA followed by Tukey’s multiple-comparisons test. (G) The number of metastatic lung lesions did not correlate with the size of the tumor when the tumors were combined and analyzed as a total group (P = 0.6616, R2 = 0.01806) or separated by CD146– (red; P = 0.3574, R2 = 0.1563) or CD146+ (blue; P = 0.4953, R2 = 0.1231) CAF influence. Correlation was tested using Pearson’s correlation analysis. (H) Gene signature based on proteomic analysis is predictive of the presence of lymph node metastasis at diagnosis in breast cancer patients with small tumors. n = 7 mice with CD146–, and n = 6 mice with CD146+; *P < 0.05; **P < 0.01; ****P < 0.0001.