Overexpression of proCOL11A1 as a stromal marker of breast cancer

Background: Our previous studies demonstrated the expression of procollagen11A1 in fibroblasts of pancreatic cancer desmoplasia and the lack of expression in fibroblasts of pancreatitis by means of the polyclonal antibody (anti-proCOL11A1 pAb) we generated. In a similar way, we decided to compare the expression of procollagen11A1 in fibroblasts of infiltrating ductal carcinoma of the breast and fibroblasts of benign sclerosing lesions of the breast, in order to validate the anti-proCOL11A1 pAb in this setting and to study how proCOL11A1 expression relates to other prognostic and predictive factors, as well as to survival. Methods: 45 core biopsies of sclerosing adenosis and 50 core biopsies of infiltrating ductal carcinoma of the breast were stained with anti-proCOL11A1 pAb, a polyclonal antibody highly specific to the less homologous fraction of proCOL11A1 (in comparison with proCOL5A1 and proCOL11A2). In addition, the expression of the proCOL11A1 gene was measured by RT-qPCR. On the other hand, the expression of proCOL11A1 was compared to the expression of estrogenic receptors, progestagen receptors, the state of the epidermal growth factor receptor 2 (HER2), the histologic grade and the stage of the disease. We also compared the immunohistochemical expression of proCol11A1 to the disease-free interval, and to overall survival. Results: The immunohistochemical analysis showed that proCOL11A1 was expressed in 100% of infiltrating ductal carcinomas, but only focally expressed in 2.2% (1 case) of sclerosing adenosis, in agreement with RT-qPCR results. ProCOL11A1 expression did not prove to have a prognostic value in relation to the disease-free interval or to overall survival in infiltrating ductal carcinoma. Conclusion: The anti-proCOL11A1 pAb is a stromal marker for breast cancer and the expression of proCOL11A1 does not seem to have a prognostic value in infiltrating ductal carcinoma of the breast

Normalized gene expression levels (log₂-transformed fold change) in pancreatic ductal adenocarcinoma (PDAC) samples relative to normal pancreas (NP) and chronic pancreatitis (CP).

<p>Normalized gene expression levels (log₂-transformed fold change) in pancreatic ductal adenocarcinoma (PDAC) samples relative to normal pancreas (NP) and chronic pancreatitis (CP).</p

Plot of all data of immunostaining score with anti-proCOL11A1 mAb in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC).

<p>Bars: ± 1 SD.</p

Confocal microscopy of pancreatic cultured CAFs.

<p>Double fluorescence stain illustrates the presence of cells proCOL11A1+/CK7+, proCOL11A1+/αSMA+ and proCOL11A1+/VIM+. <i>Red</i>: proCOL11A1; <i>green</i>: CK7, αSMA and VIM, respectively; <i>blue</i>: nuclei. Insets: randomly taken high power fields of culture. Scale bar 100 μm (X200) and 20 μm (X630). </p

Overexpression of COL11A1 by Cancer-Associated Fibroblasts: Clinical Relevance of a Stromal Marker in Pancreatic Cancer

<div><p>Background</p><p>The collagen11A1 (COL11A1) gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available.</p> <p>Methods and findings</p><p>A total of 54 pancreatic ductal adenocarcinomas (PDAC), 23 chronic pancreatitis (CP) samples, and cultured peritumoral stromal cells of PDAC (passages 3-6) were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1) Validation of COL11A1 gene overexpression by q-RT-PCR. </p> <p>Findings</p><p>the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2) Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. </p> <p>Findings</p><p>anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs) of PDAC but it does not stain chronic benign condition (chronic pancreatitis) stromal cells, epithelial cells, or normal fibroblasts. 3) Evaluation of the discrimination ability of the antibody. </p> <p>Findings</p><p>anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981). 4) Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. </p> <p>Findings</p><p>ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype) in different proportions.</p> <p>Conclusions/Significance</p><p>Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for CAFs, and thus, anti-proCOL11A1 is a powerful new tool for cancer research and clinical diagnostics.</p> </div

Co-staining of anti-proCOL11A1 mAb with different fibroblastic markers and CK 7, in Chronic Pancreatitis.

<p><b>A</b>, anti-proCOL11A1 (brown) <i>vs</i>. desmin (magenta); <b>B</b>, Anti-proCOL11A1 (brown) <i>vs</i>. αSMA (magenta); <b>C</b>, anti-proCOL11A1 (brown) <i>vs</i>. VIM (magenta); <b>D</b>, anti-proCOL11A1 (brown) <i>vs</i>. GFAP (not staining); <b>E</b>, anti-proCOL11A1 (brown) vs. CK7 (magenta); <b>F</b>, CK7 (epithelial tumor cells: <i>brown</i>) vs. VIM (magenta).(all photomicrographs at ×200, Scale bar 200 μm). .</p

Co-staining of anti-proCOL11A1 mAb with different fibroblastic markers and CK 7, in Pancreatic Ductal Adenocarcinoma.

<p><b>A</b>, anti-proCOL11A1 (brown) <i>vs</i>. desmin (magenta); <b>B</b>, anti-proCOL11A1 (brown) <i>vs</i>. αSMA (magenta); <b>C</b>, anti-proCOL11A1 (brown) <i>vs</i>. VIM (magenta); <b>D</b>, anti-proCOL11A1 (brown) <i>vs</i>. GFAP (not staining); <b>E</b>, anti-proCOL11A1 (brown) vs. CK7 (magenta); <b>F</b>, CK7 (epithelial tumor cells: <i>brown</i>) vs. VIM (magenta) (all photomicrographs at ×200, Scale bar 200 μm; inset X1000). </p

Comparative immunohistochemical profile of stromal cells of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) in serial sections.

<p>Stromal cells in CP were negative for anti-proCOL11A1 mAb (A) and desmin (B) whereas a substantial number of stromal cells of PDAC expressed anti-proCOL11A1 mAb (E) and desmin (F). In CP and PDAC the stain of stromal cells for αSMA (C and G, respectively) and VIM (D and H, respectively) were diffuse and non-selective. Anti-proCOL11A1 mAb (A and E), desmin (B and F), αSMA (C and G) and VIM (D and H) (all photomicrographs at ×400, Scale bar 50 μm).</p