Abstract 4021: Cyclooxygenase-2 downregulation reduces activated fibroblasts and modifies the extracellular matrix in MDA-MB-231 breast cancer xenograft
Authors: Krishnamachary Balaji , Stassinopoulos Ioannis , Kakkad Samata M. , Penet Marie-France , Jacob Desmond , Wildes Flonne , Mironchik Yelena , Pathak Arvind , Solaiyappan Meiyappan , Bhujwalla Zaver M. 
Published: August 2015

Abstract
COX-2 is an important mediator of inflammation that significantly influences tumor angiogenesis, invasion and metastasis. Here, we have investigated the role of COX-2 in modifying the number of activated cancer associated fibroblasts (CAFs) and in altering the extracellular matrix (ECM) in a breast cancer model.
To investigate the role of COX-2 in modulating the ECM, we used an MDA-MB-231 cell clone (Clone 13) expressing a short hairpin RNA (shRNA) to downregulate COX-2 [1]. Clone 13 cells were characterized for significantly lower basal and TPA-induced COX-2 and PGE2 expression compared to parental MDA-MB-231 cells using ELISA (PGE2), western blot (COX-2 protein) and q-RT-PCR (COX-2 mRNA). Tumors were derived from parental (n = 5) and Clone 13 (n = 6) MDA-MB-231 cells following inoculation in the mammary fat pad in SCID mice. Tumors were excised at ∼ 500 mm3 and immunohistochemically stained to quantify vessel density (CD31) and activated CAFs (α-smooth muscle actin (SMA)) in 5 μm thick formalin fixed sections. Stained sections were digitally scanned and positive staining quantified using manufacturer supplied software (Aperio Technologies, CA).
Clone 13 tumors showed delayed tumor growth compared to parental MDA-MB-231 tumors. We have previously observed that collagen 1 (Col1) fiber density and fiber volume were significantly lower in COX-2 reduced Clone 13 tumors compared to parental tumors [2]. While cancer cells shape Col1 fiber patterns through the secretion of various enzymes, Col1 fiber is laid down by activated CAFs within or around the tumor. Quantification of activated CAFs by immunohistochemistry for α-SMA in the tumors, and immunoblotting for α-SMA of crude protein extracted from the tumors, revealed significantly fewer CAFs and significantly reduced levels of α-SMA protein in Clone 13 tumors compared to parental MDA-MB-231 tumors. We previously observed a significant decrease in permeability as well as reduced influx and efflux of macromolecular transport in Clone 13 tumors compared to parental tumors, but no difference in vascular volume [2]. Immunohistochemistry for CD31 staining of endothelial cells did not detect a significant difference in CD31 density between Clone 13 and parental tumors further confirming our previous observations about vascular volume. These data reveal the multi-faceted effects of COX-2 in modifying the structure and function of the ECM, and identify the ability to attract and activate fibroblasts as one mechanism by which COX-2 modifies the ECM.
Acknowledgements: We thank Mr. Gary Cromwell for technical assistance. This work was supported by NIH R01CA82337 and P50 CA103175.
References: [1] Stasinopoulos, I., et al., Mol Cancer Res, 2007; [2], Stasinopoulos, I., et al., AACR, 2013 Chicago.
Citation Format: Balaji Krishnamachary, Ioannis Stassinopoulos, Samata M. Kakkad, Marie-France Penet, Desmond Jacob, Flonne Wildes, Yelena Mironchik, Arvind Pathak, Meiyappan Solaiyappan, Zaver M. Bhujwalla. Cyclooxygenase-2 downregulation reduces activated fibroblasts and modifies the extracellular matrix in MDA-MB-231 breast cancer xenograft. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4021. doi:10.1158/1538-7445.AM2015-4021