Possibly, the formation of vessel-like structures and anticoagulation induced by SLPI12 make hypoxic regions of the tumor more accessible to chemotherapy. Conclusion In conclusion, high SLPI expression in MSS CRC is associated with reduced disease recurrence after resection of the primary tumor and adjuvant chemotherapy in stage III patients. is associated with worse outcome, suggesting that SLPI promotes metastasis in human CRC. However, whether SLPI plays a role in CRC before distant metastases have formed is unclear. Therefore, we examined whether SLPI expression is associated with prognosis in CRC patients with localized disease. Using a cohort of 226 stage II and 160 stage III CRC patients we demonstrate that high SLPI protein expression is associated with reduced disease recurrence in patients with stage III micro-satellite stable tumors treated with adjuvant chemotherapy, independently of established clinical risk factors (hazard rate ratio 0.54, mRNA expression is associated with shorter Pax1 overall survival in triple negative breast cancer patients10 and gastric cancer7. In addition, we previously showed that high SLPI protein expression in CRC liver metastases and matched primary tumors was associated with shorter overall survival11. The precise role of SLPI in cancer is, however, unclear. Clones expressing SLPI entered the vasculature more efficiently and formed more metastases than clones that did not express SLPI in a murine model of polyclonal breast cancer12. The role of SLPI in CRC metastasis has not extensively been studied. We previously hypothesized that SLPI expression may be beneficial for metastatic CRC tumor cells via promotion of immune evasion11. However, SLPI has multiple diverse functions and may act differently in various processes related to Protostemonine tumor growth and metastasis. In this study, we assessed whether SLPI expression in CRC at a stage prior to distant metastases formation is associated with disease recurrence. We evaluated the prognostic value of SLPI protein expression in CRC patients classified as stage II (no metastases) and stage III (metastases to regional lymph nodes, but no distant metastases). Both stage II CRC patients and stage III CRC patients have a variable prognosis and therefore the identification of prognostic factors is desired to identify which patients may benefit from adjuvant therapy13,14. In addition, a better understanding of the biology of tumor growth and the formation of metastases may ultimately help to predict survival in CRC patients. Here, we show that high SLPI protein expression in micro-satellite stable (MSS) tumors is associated with reduced disease recurrence in stage III CRC patients treated with adjuvant chemotherapy. Methods Patient cohort and tissue microarray (TMA) generation Between 1996 and 2005, 454 CRC patients classified as stage II or stage III according to the 4th edition of the TNM-classification system underwent medical resection of CRC in the former Kennemer Gasthuis (current Spaarne Gasthuis) hospital in Haarlem, the Netherlands. Individuals with a history of colorectal malignancy, individuals with irradical resection of the primary tumor, individuals who died within 3?weeks after surgery and individuals who were lost for follow-up were excluded from the study cohort (Supplementary Fig.?1). Histologically confirmed, formaldehyde-fixed paraffin-embedded (FFPE) CRC cells samples from 386 individuals were included, as explained previously15. Cells Protostemonine microarrays (TMAs) were generated from the original FFPE cells blocks relating to protocols previously explained16. In short, six tissue core biopsies of 0.6?mm in diameter were punched from morphologically representative cells areas and transferred into recipient TMA paraffin blocks. Tumor samples from this cohort have previously been analyzed for micro-satellite instability (MSI) using a five-marker-based PCR analysis system, as explained previously15. For 48 out of 359 individuals (13%) MSI status could not become determined. These individuals were excluded from your analyses of SLPI manifestation in the subgroup with MSS tumors and the analyses of SLPI manifestation in the subgroup with MSI tumors. SLPI immunohistochemistry Immunohistochemistry for SLPI was performed as previously explained11. In short, sections were stained with either a monoclonal anti-human-SLPI antibody that was raised against human being Protostemonine SLPI purified from sputum (4?g/mL, mouse IgG1, HM2037, clone 31; HycultBiotech, Uden, The Netherlands) or a polyclonal anti-human-SLPI antibody that was raised against micro-satellite stable, micro-satellite instable. Large manifestation of SLPI in stage III micro-satellite stable CRC is associated with reduced disease recurrence Because individuals with micro-satellite instable (MSI) CRC are known to have a better prognosis compared to individuals with micro-satellite stable (MSS) CRC25C27, we evaluated the prognostic value of SLPI in the subgroup of individuals with MSS tumors and in the subgroup of individuals with MSI tumors separately. In stage II individuals with MSS tumors, SLPI manifestation was not associated with disease-free survival (HRR 1.40, micro-satellite stable. In stage III individuals with MSS tumors who did not receive adjuvant chemotherapy, SLPI manifestation was not significantly associated with disease-free survival (HRR 0.80, em P /em -value 0.59, 95%.