Type IV collagen in breast and colorectal cancer : a potential biomarker of metastatic disease

Abstract: Background: Metastatic colorectal cancer (mCRC) and metastatic breast cancer (mBC) are two leading causes of cancer-related mortality worldwide. Early detection of metastatic disease is critical, and sensitive, easily accessed and cost-effective biomarkers that can diagnose mBC and mCRC at an early stage would have high clinical value. The best circulating markers: CEA and CA 15-3 are suboptimal, but a combination with other proteins can improve their potential to detect metastatic disease. One potential source of new biomarkers is the tumor stroma, including the extracellular matrix (ECM), vasculature, and stromal cells like immune cells and fibroblasts. Both the tumor cell and stromal compartment are vital for cancer progression. A combination of biomarkers from both compartments could likely best reflect the heterogeneous nature of the metastatic disease. Stromal type IV collagen (COL IV) is the main constituent of the basement membrane of healthy tissues. COL IV is upregulated with some cancers, including colorectal liver metastases (CLM), and is considered a potential biomarker for CLM. The origin of elevated levels of COL IV in CLM is not known but may result from both increased ECM production and ECM degradation associated with cancer-related tumor stroma remodeling. Aims: In this thesis, COL IV and its potential to be used as a biomarker for mCRC and mBC is studied, with a specific emphasis on liver metastases. The aims are to compare levels of circulating COL IV (cCOL IV) in mCRC and mBC patients with controls and evaluate its diagnostic and prognostic value; to evaluate the combination of cCOL IV with other proteins; to determine the cellular origin of COL IV in CLM and study COL IV expression in cell lines; to study the expression of COL IV degrading proteases in CLM and to evaluate tissue expression of COL IV in bone and liver metastases from BC patients. Methods: Plasma levels of cCOL IV, CA 15-3, CEA, and other cancer-related proteins were analyzed with ELISA, ECLIA and Multiplex assay in mCRC and mBC patients, healthy controls and patients with primary CRC or BC as controls. The cellular origin of COL IV expression in CLM was examined with in situ hybridization, and the expression of COL IV in mBC tissue and COL IV degrading proteases (MMP -2, -7, -9 and -13) in CLMs were studied with immunohistochemistry. COL IV expression in CRC and fibroblast cell lines was analyzed with immunofluorescence.Results: cCOL IV is elevated in mBC patients and correlates with poor survival. The combination of cCOL IV with CA 15-3 and cCOL IV alone are superior to CA 15-3 at detecting mBC. COL IV is highly expressed in the tissue of liver- and bone BC metastases. Circulating COL IV, CEA, OPN, CYFRA 21-1, IL-8, HGF, and MIF are elevated, and TRAIL is lower in mCRC patients compared with controls. COL IV, CEA, OPN, CYFRA 21-1, and IL-8 were higher, and TRAIL was lower in mCRC patients with liver metastases compared to patients with extrahepatic disease. Circulating CEA, OPN, and HGF are very good, and cCOL IV is acceptable at distinguishing mCRC patients from patients with primary CRC. The combination of OPN + CEA is superior to CEA alone at detecting mCRC. High HGF and cCOL IV (one cohort) in mCRC patients correlate to poor prognosis. cCOL IV is elevated in CLM patients compared to healthy controls and is very good at discriminating between healthy controls and CLM patients. COL IV is expressed in CLM by cancer-associated fibroblasts, and COL IV degrading proteases are expressed primarily by stromal cells in CLM. COL IV is expressed by fibroblasts, not tumor cells, in vitro. Conclusion: cCOL IV is a promising tumor marker of metastatic BC and CRC and circulating HGF and OPN are potential biomarkers of mCRC. Our results show that the metastatic site can impact the circulating levels of numerous cancer-related proteins, which aligns with our hypothesis that combining biomarkers instead of using one might be best for detecting metastatic cancer through blood analysis. COL IV is expressed by stromal cells, not tumor cells, in CLM tissue and in vitro.