Stability of matrix metalloproteinase-9 as biological marker in colorectal cancer

Medical Oncology (2018) 35:50 https://doi.org/10.1007/s12032-018-1109-4 ORIGINAL PAPER Stability of matrix metalloproteinase‑9 as biological marker in colorectal cancer Andreas Jonsson1,3 · Claes Hjalmarsson2 · Peter Falk3 · Marie‑Lois Ivarsson3 Received: 13 February 2018 / Accepted: 26 February 2018 / Published online: 9 March 2018 © The Author(s) 2018. This article is an open access publication Abstract Matrix metalloproteinases (MMPs) are believed to be of importance in the growth and spread of colorectal cancer (CRC). MMP-9 level has been suggested as a biological predictor of prognosis in CRC as well as in other types of cancer such as breast and cervical cancer. The purpose of this study was to investigate the stability over time of MMP-9 in cryopreserved plasma, colorectal tumor tissue extract and macroscopically tumor-free colon mucosa tissue extract samples. Plasma and tissue samples were taken from patients at primary CRC surgery and analyzed for MMP-9. Aliquots of samples from the same patients were stored at – 80 °C pending analysis. These aliquots were analyzed using identical methods after storage periods of nine (plasma) and twelve (tissue) years. No significant difference in plasma MMP-9 concentration was seen between baseline samples and those after 9 years of cryopreservation (median values 9.9 and 9.7 ng/mL, respectively; p > 0.05). MMP-9 levels in the tumor-free tissue extracts had increased to baseline (median values 7.1 and 8.1 ng/mL, respectively; p < 0.01). MMP-9 levels in the tumor tissue extracts had also increased significantly (median values 89.9 and 133.5 ng/mL, respectively; p < 0.01). We have demonstrated that MMP-9 levels in frozen citrated plasma are stable if stored at − 80 °C, whereas MMP-9 levels in extracts from tumor tissue and tumor-free intestinal mucosa appear to increase with time. We conclude that MMP-9 levels in cryopreserved plasma may be considered stable over time and are thus suitable for comparison purposes in consecutive series. Keywords Colorectal cancer · MMPs · MMP-9 · Prognostic marker Introduction The matrix metalloproteinases (MMPs) belong to a family of zinc- and calcium-dependent proteolytic enzymes that are important in the degradation of extracellular matrix (ECM) in many types of cancer [1–4]. More than 25 MMPs have been identified [5, 6], and their regulation is controlled by several mechanisms including transcription, activation, and inhibition. Tumor cells and surrounding stromal cells are all able to synthesize MMPs [5]. * Marie‑Lois Ivarsson 1 Hallands Hospital Varberg, Region Halland, 432 37 Varberg, Sweden 2 Department of Surgery, Sahlgrenska University Hopital, Göteborg, Sweden 3 Fibrinolysis Laboratory, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, 416 85 Göteborg, Sweden MMP-9 is one of the key proteolytic enzymes in the breakdown and reconstruction of ECM in colorectal cancer (CRC) invasion and metastasis. MMP-9 regulates the microenvironment around the tumor and increases the concentration of vascular endothelial growth factor (VEGF) that regulates angiogenesis [7]. MMP-9 is also active in the formation of early metastatic niches [8]. In preclinical models, selective MMP-9 inhibitors have been shown to decrease tumor growth and the incidence of metastases in colorectal cancer and also induce cancer cell apoptosis in pancreatic cancer [9, 10]. Several studies have demonstrated elevated levels of MMP-9 in the tumor tissue and plasma of patients with CRC [11, 12], and MMP-9 level has been suggested as a biological predictor of prognosis in CRC, as well as in other types of cancer such as breast and cervical cancer [13, 14]. Furthermore, in patients with CRC, MMP-9 levels in adjacent tumor-free mucosa are elevated and this could be used as a predictor of 5-year relative survival in colorectal cancer [15]. 13 Vol.:(0123456789) 50 Page 2 of 6 Also fecal MMP-9 measurement has recently come up as a promising marker for CRC [16]. Studies on cancer-related outcome often use consecutive case series where blood and tissue samples are collected and frozen pending analysis. To minimize inter-assay variation, samples are often thawed and analyzed in batches. In this respect, there are concerns regarding the stability of MMP levels in plasma and tissue samples that are cryopreserved for long periods of time [17]. Previous studies have shown that inappropriate handling of specimens may lead to degradation of biomarkers in cancer tissue [18]. This is of greatest concern in longitudinal trials investigating disease progression. It is also important how blood is handled prior to MMP analysis, and whether it is serum or plasma that is used [19–21]. To the best of our knowledge, no data have been published regarding the long-term cryostability of MMP-9 in plasma, or in centrifugal extracts of tumor-free intestinal mucosa or CRC tumor tissue, where samples have been stored for periods longer than 4 years [22–24]. The aim of this study was to investigate the long-term stability of MMP-9 levels in cryopreserved citrated plasma, and centrifugal extracts of tumor tissue and tumor-free intestinal mucosa samples from patients with CRC, thereby exploring the possibility to use cryopreserved samples in future research on consecutive case series. Materials and methods Patients and sample preparation During the period February 1999–2005, blood samples, tumor tissue and biopsies from macroscopically tumor-free mucosa were obtained from patients undergoing surgery for colorectal cancer at the Department of Surgery, Sahlgrenska/ Ostra Hospital, Gothenburg, Sweden. Blood samples were collected in citrate tubes and then centrifuged within 5 min at 10000g for 10 min at 20 °C. The supernatant (citrated plasma) samples were then frozen in several aliquots at − 80 °C pending further analysis. Tissues samples approximately 1 cm2 in area were taken from the area of the tumor, and from tumor-free mucosa approximately 10 cm from the tumor. Tissue samples were immediately frozen in liquid nitrogen in the operating theater and stored at − 80 °C pending homogenization and centrifugation (see below). Tissue extract preparation At the laboratory, tissue samples were thawed, weighed and then homogenized in 1 ml PBS buffer with 0.01% Triton X-100 per 40 mg tissue, and centrifuged at 10000g for 13 Medical Oncology (2018) 35:50 10 min. The supernatant from each of the homogenized tissue sample was then extracted and frozen in several aliquots at − 80 °C pending analysis. MMP‑9 level measurement and data collection Aliquots from both plasma and tissue supernatant samples were analyzed in batches in conjunction with the surgical procedure (baseline), while aliquots from further plasma and tissue supernatant samples were kept frozen at − 80 °C pending later analysis. After 9 years, plasma sample aliquots were thawed and analyzed for MMP-9, and corresponding aliquots of tissue (...truncated)