ColXα1 is a stromal component that colocalizes with elastin in the breast tumor extracellular matrix

Abstract

The tumor microenvironment regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. Increased expression of type X collagen α-1 (ColXα1) in tumor-associated stroma correlates with poor pathologic response to neoadjuvant chemotherapy in estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Evaluation of ColXα1 expression patterns suggests a potential connection with elastin fibers. To investigate the possible interaction between ColXα1 and elastin, we evaluated the expression of ColXα1 in relation to elastin fibers in normal breast tissue, ductal carcinoma in situ, and invasive breast carcinomas at cellular and subcellular levels. Our findings demonstrate that ColXα1 colocalizes with elastin in invasive breast cancer-associated stroma by immunohistochemistry, immunofluorescence, and electron microscopy. In 212 invasive breast carcinomas, this complex was aberrantly and selectively expressed in tumor extracellular matrix in 79% of ER+/HER2-, 80% of ER+/HER2+, 76% of ER-/HER2+, and 58% of triple negative breast cancers. In contrast, ColXα1 was generally absent, while elastin was present perivascularly in normal breast tissue. ColXα1 and elastin were coexpressed in 58% of ductal carcinoma in situ (DCIS) in periductal areas. In mass-forming DCIS with desmoplastic stroma, the complex was intensely expressed in periductal areas as well as within the tumor-associated stroma in all cases. Our data suggest that the breast carcinoma neoplastic process may involve aberrant expression of ColXα1 and elastin in the tumor microenvironment emerging early at the DCIS stage. Enrichment of these complexes in tumor-associated stroma may represent a stromal signature indicative of intrinsic differences between breast cancers. These findings shed light on investigation into the role of aberrant collagen complex expression in tumorigenesis and tumor progression which may be leveraged in therapeutic and theranostic applications.

Keywords: ColXα1; breast cancer; elastin; extracellular matrix; neoadjuvant; tumor microenvironment; type X collagen α-1.

Figure 1

Elastin and ColXα1 immunohistochemistry staining of normal breast and breast tumors. (A,B) In normal breast stroma, ColXα1 was largely negative by IHC apart from occasional perivascular and periductal faint staining. Elastin was present around normal structures including periductal, lobular, and perivascular areas. Scattered elastin staining was also present in normal breast stroma. (C,D) Non‐mass‐forming DCIS, D at high magnification: ColXα1 and elastin expression in a periductal pattern. Elastin highlighted the vessels while ColXα1 was negative. (E) Mass‐forming DCIS: ColXα1 and elastin coexpression in a periductal and stromal pattern of distribution. (F,G) ColXα1 and Elastin were strongly expressed in a similar patchy distribution in an ER+/HER2+ breast cancer (F) and a triple negative breast cancer (G). (H) Invasive ductal carcinoma with DCIS. ColXα1 and elastin expression were present in a periductal pattern and within the stroma. Scale bars = 10 μm.

Figure 2

Immunofluorescence staining of elastin and ColXα1 in tumor‐associated stroma. DAPI highlights the tumor; elastin and ColXα are distributed and colocalized in tumor‐associated stroma. Intratumoral vessels stain with elastin but not ColXα1. Scale bar = 10 μm.

Figure 3

Immunoelectron microscopy of ColXα1 and elastin localization in the tumor microenvironment using double labeling. The micrograph shows a ColXα1 and elastin complex (CE) as patchy amorphous material in the extracellular space near a fibroblast nucleus which is double stained with gold conjugated anti‐ColXα1 antibody (10‐nm gold particles) and gold conjugated anti‐elastin antibody (25‐nm gold particles). The field also contains collagen fibrils (C) and fine cytoplasmic extension of the fibroblasts (F) responsible for elaboration of the extracellular constituents. Original magnification (×25 000). Low (A), intermediate (B), and high (C) magnifications are shown. Larger particles – elastin (long arrows) and smaller particles – ColXα1 (short arrows) are identified in (C).