High Intra-tumoral and Sera Matrix Metalloproteinase 9 Levels Reduce Glioblastoma and Brain Metastases Patients' Survival

High Intra-tumoral and Sera Matrix Metalloproteinase 9 Levels Reduce Glioblastoma and Brain Metastases Patients' Survival | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High Intra-tumoral and Sera Matrix Metalloproteinase 9 Levels Reduce Glioblastoma and Brain Metastases Patients' Survival Tehila Kaisman-Elbaz, Snir Haddad-Shlaifshtein, Yael Eskira, Vladimir Merkin, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5467623/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Matrix metalloproteinase 9 (MMP-9) has been shown to induce glioblastoma invasion and brain metastases (BM) spread. However, its clinical significance for monitoring disease progression has yet to be established. This study evaluates intra-tumoral and sera MMP-9 levels and their correlation to glioblastoma and BM patients' overall survival (OS). Methods: 69 tumor and pre-operative sera samples were obtained from the brain tumor bank of the neurosurgery department at Soroka University Medical Center from patients who underwent tumor resection between 2015 and 2021. Clinical and imaging data from 27 glioblastoma and 30 BM patients were analyzed, and their MMP-9 levels and activity were measured and compared with 12 meningioma patients and 23 healthy subjects. Survival analyses were performed to examine MMP-9 level, activity, and clinical parameters' correlation with patients' OS. Results: Glioblastoma and BM patients demonstrated increased median intra-tumoral MMP-9 levels (8ng/ml and 4ng/ml, respectively, p<0.001), activity, and pre-operative sera levels (2.8ng/ml and 1.8ng/ml, respectively, p<0.001). MMP-9 was specifically detected within and between glioblastoma cells and tumor endothelia. High intra-tumoral and sera MMP-9 levels, but not its activity, were linked to decreased OS in glioblastoma and BM patients (15.8 versus 8.4 months, p=0.022). MMP-9 was readily measured in patient sera. Conclusions: This study suggests that intra-tumoral and sera MMP-9 can assist in identifying glioblastoma and BM recurrence/progression and that high intra-tumoral and/or sera MMP-9 levels at diagnosis correlate with significantly shorter patient OS. Importantly, sera MMP-9 could be longitudinally and non-invasively monitored in those patients and, once rising, may indicate tumor progression. Figures Figure 1 Figure 2 Figure 3 Introduction Matrix metalloproteinase 9 (MMP-9) has been shown to induce glioblastoma invasion [ 1 ] by altering the extracellular matrix and promoting angiogenesis [ 2 , 3 ]. Despite its potential as a tumor progression biomarker [ 4 , 5 ] and the increasing need for non-invasive modalities to monitor brain tumor patients' disease course, i.e., liquid biopsy [ 6 ], only a few clinical trials have evaluated MMP-9. None have yielded sufficient clinical benefit to be included in patients' treatment and follow-up paradigms [ 7 – 9 ]. MMP-9's role in glioblastoma was highlighted in various studies. Its high level in patients' tumors was correlated with decreased survival and tumor invasiveness, which were reduced once inhibited [ 10 – 13 ]. A similar pattern was detected in metastatic solid cancers, i.e., breast carcinoma and melanoma [ 14 – 16 ]. Notably, once released from tumor cells, MMP-9 was identified in body fluids (i.e., blood, urine, and CSF [ 17 – 19 ]) - even before clinical manifestations occurred [ 5 , 17 ]. Several works showed that MMP-9 sera/plasma levels can predict tumor metastatic propensity and disease recurrence [ 20 – 23 ]. However, the literature describing MMP-9 secretion pattern in glioblastoma patients is less characterized compared to BM, and the works that addressed it describe inconclusive results [ 5 , 23 – 25 ]. Ricci et al. [ 4 ], for instance, found that sera MMP-9 levels differentiated metastatic lesions, gliomas, and meningiomas from healthy controls and correlated it with tumor malignancy grade, similar to Lin et al. [ 26 ]. In turn, Hormigo et al. [ 25 ] reported sera MMP-9 level as an indicator of glioblastoma progression, and Tabouret et al. [ 27 ] related lower MMP-9 levels to enhanced survival of malignant glioma patients. On the other hand, Iwamoto et al. [ 23 ] compared MMP-9 serum levels in 343 glioma patients and found a slim relation to disease state, similar to Crocker et al. [ 28 ]. Notably, works correlating MMP-9 levels with patients' imaging features are scarce. Liu et al. [ 29 ] have previously correlated high-grade gliomas with increased perilesional edema volumes and high MMP-9 levels. Other reports, [ 30 , 31 ], however, associated high MMP-9 levels with smaller edema volumes of glioblastomas and specific brain locations. Liu and Li [ 32 ] linked sera MMP-9 and perfusion-weighted MRI parameters recently to glioblastoma recurrence. This work aims to evaluate the potential of MMP-9 as a biomarker alongside imaging and other parameters in glioblastoma and BM. It seeks to identify disease progression by supplementing routine measures with current patient follow-up paradigms. Methods This study used Soroka University Medical Center neurosurgery department brain tumor bank samples obtained from patients who underwent tumor resection between 2015 and 2021 under the institutional ethical committee approval [0208-16-SOR]. Patients were included if they: 1) signed informed consent to provide tumor tissue and blood samples; 2) underwent tumor resection; 3) had available pre-operative clinical, imagery, and laboratory data. Patients with partially documented data were excluded (n = 17). Glioblastoma (n = 27), BM (n = 30), and meningioma (n = 12) tumor and sera samples were analyzed. Demographics and clinical data were documented per patient, including Karnofsky Performance Status (KPS), oncological and surgical history, diagnosis, birth and exitus dates, imagery, laboratory, and histopathological data. Patients' overall survival (OS) was calculated as the delta between the diagnosis date (date of surgical intervention) and exitus dates. Blood was also withdrawn from healthy volunteers (n = 23) to control cohort patients' MMP-9 levels. Tumor tissue and blood samples analysis Each tumor and blood sample collected was handled according to the institutional ethical committee-approved protocol. Fresh patients’ tumor samples were stored in liquid nitrogen until further analysis. Blood samples were collected in EDTA tubes and serum clot activator with gel separator tubes before surgical intervention. The tubes were then centrifuged for 15 minutes at 2000g at room temperature (RT), and the serum was kept in liquid nitrogen. Imagery tumor data collection and volumetric evaluation Preoperative Magnetic Resonance Imaging scans were retrieved from the institutional Picture Archiving and Communication System. To achieve the most accurate approximation of tumors' mass and edema volume for each patient, Brainlab cranial navigation software (Brainlab®, Germany) was used. Volumetric measurement of the tumor mass (T1W1 + gadolinuim sequence), edema (T2W2 sequence), and the ratio between them were calculated (i.e., edema index (EI) [ 29 ]). Perilesional edema volume was defined by subtracting the tumor mass volume from each patient's combined tumor mass and edema volumes. Protein extraction from tumor tissues Frozen tumor tissue samples were defrosted and added with lysis buffer. Tissues were disrupted with a Dounce homogenizer and incubated for 30 minutes on ice, followed by sonication and centrifugation at 14,000 RPM for 15 minutes under 4°C. Supernatants were collected, and protein concentration was determined using the Bradford method. The samples were kept at -80°C until further testing. Intra-tumoral and sera MMP-9 levels measurement A 4µg/ml purified antibody anti-MMP-9 (BioLegend, USA) was applied on a 96-well plate. The plate was then incubated overnight at 4°C and washed with PBS-Tween solution. Blocking and diluted serum (1:100) or 300 µg protein extract were added, and the plate was incubated. A standard curve using recombinant MMP-9 was prepared according to the manufacturer's instructions (BioLegend, USA). A secondary biotin anti-human MMP-9 antibody and HRP-Avidin (1:1500) were added. To produce a colorimetric reaction, the plates were incubated with TMB and were read using an ELISA reader (MULTISKAN FC Thermo Scientific) at 650nm. Alternatively, immunoblot analysis of MMP-9 level of patients and healthy subjects sera was performed as previously described [ 33 ]. Intra-tumoral and sera MMP-9 activity assessment Total protein extraction from each tumor sample was mixed with a non-reducing sample buffer and applied to acrylamide gels. Gels were run until sufficient band separation was achieved. Following SDS removal, the gels were incubated in Tris-HCl-Triton solution. Gels were then immersed for 30 minutes in 40% methanol, 10% acetic acid, and Coomassie Brilliant Blue G-250 (Merck, Germany) and de-stained in the same solution without the dye for several hours. The gelatinase activity of MMP-9 was evident as a clear white band. Each gel was run with a pre-stained standard protein ladder and the standard curve of purified MMP-9. The gels were then scanned, and the molecular weight was determined according to the standard protein ladder. Band density and gelatinase activity were quantified using GelQuantNET ( www.BiochemLabSolutions.com ). Immunofluorescence of MMP-9 in glioblastoma Tumor tissue was fixated in 4% formaldehyde solution, followed by paraffin embedding, sectioning, and mounting on slides as acceptable. Slides were then deparaffinized and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was blocked, and slides were incubated in a blocking solution and with primary antibody MMP-9 (1:100; Abcam, USA) overnight at 4°C. The next day, slides were washed and incubated with a secondary antibody conjugated to Cy2/Cy3 (the Jackson Laboratory). Following additional rinsing, slides were mounted with a DAPI-containing mounting medium (Immunomount) and viewed in a confocal microscope (Zeiss LSM 510) with appropriate fluorescence barrier and excitation filters. Statistical analysis and OS All statistical analyses were performed using R, version 4.3.0 (R Foundation for Statistical Computing). Chi-squared tests were used for categorical variables with adequate sample sizes, while Fisher's exact test was employed when the criteria for the Chi-squared test were not met. The Shapiro-Wilk test assessed normality for numeric variables, applying the student's t-test for normally distributed data and non-parametric tests when normality was rejected. i.e., Kruskal-Wallis's test. The Mann-Whitney U test was employed for pairwise comparisons when only two groups were compared. Further, univariate and multivariate Cox proportional hazard model analysis was performed. Due to non-normality, Spearman correlation tests were used to assess correlations between continuous numeric variables, and the variance inflation factor was calculated for each variable to avoid multicollinearity issues. Statistical significance was determined when the p-value was less or equal to 0.05. Results Patients' Demographics and Clinical Characterization Table 1 A presents the entire cohort's (n = 69) main characteristics. Glioblastoma patients' median age at diagnosis was 62 (IQR 52.5, 72). Their median KPS was 100, and median OS was 10.1 months (IQR 5.1, 18); 89% of the patients were treated with radiation and chemotherapy as acceptable [ 34 ]. The BM cohort's median age at diagnosis was 63.5 (IQR 56.25, 67), and 77% originated from either melanoma, lung, or breast carcinoma. 73.3% received adjuvant oncological treatment following surgical resection (i.e., radiation, chemotherapy, and biological therapy). Their median KPS was 90, and they exhibited a median OS of 8 months since their BM diagnosis (IQR 3.7, 20.7). Meningioma patients' median age at diagnosis was 67 (IQR 59.2, 72.5), and their median KPS was 90. Since anemia was previously recognized for its negative influence on glioblastoma [ 35 ] and BM patients' OS [ 36 ], this study cohort was evaluated for it in addition to other parameters. 32% of the patients' cohort were anemic (as defined elsewhere [ 35 ]), and 48% had Red blood Distribution Width (RDW) > 14%. Notably, anemia and RDW > 14% were more prevalent in the BM cohort than in glioblastoma. As follows, anemia or RDW > 14% did not influence OS in this cohort. Tumor and Edema Volumes Analysis A comprehensive imagery assessment was also performed, aiming at correlating it with MMP-9 levels (Fig. 1 A, Table 1 B). Glioblastoma and BM induced significant peritumoral edema of 73.9 cc vs. 85.2 cc, respectively, and their EI was, therefore, significantly elevated compared to meningioma (Figs. 1 B & 1 D). Tumor volumes were not significantly different when comparing tumor types (Fig. 1 C). Glioblastoma and BM Patients Cohort Sub-analysis Further on, glioblastoma and BM cohorts were compared since MMP-9 levels and activity on their OS were mainly assessed (Table 1 C). Notably, age at diagnosis and OS did not differ among the groups. A similar percentage of patients receive complementary oncological treatment. More BM patients, though, were found to have RDW > 14%, and their median KPS was 90. Tumor and edema volumes did not differ. Therefore, these two patient groups were united for further analysis. A sub-analysis of recurrent glioblastoma patients was also performed (Table 1 D). Recurrent glioblastoma patients were younger (53.2 years, SD 13.99), had a high KPS, and exhibited an enhanced OS compared to newly diagnosed glioblastoma patients (18 months, p = 0.005). Intra-tumoral MMP-9 Characterization Patients' intra-tumoral MMP-9 content assessment followed. Glioblastoma and, to a lesser extent, BM tumor samples demonstrated an enhanced intra-tumoral level of MMP-9 (8ng/ml and 4ng/ml, respectively) compared to meningioma (1ng/ml, p < 0.001, Figs. 2 A-C). Intra-tumoral MMP-9 activity was then measured and was comparable in glioblastoma and BM but significantly increased compared to meningioma (p = 0.004, Fig. 2 B-C). Their median values were used to subdivide patients' cohorts according to the intra-tumoral MMP-9 and evaluate its influence on OS, but they were found insignificant (Figs. 2 D-E). Notably, intra-tumoral MMP-9 level and activity were significantly correlated (r = 0.57, Fig. 2 F). The following step was to localize MMP-9 within glioblastoma tumor tissue and aim to identify which cells display the protein. Therefore, two glioblastoma patients' samples were stained. MMP-9-specific staining was detected within endothelial cells of both samples and in the cytoplasm of GFAP-positive stained-astrocytic tumor cells in patient 5. However, for patient 3, scattered staining was noticed between astrocytic cells and not within them, which could indicate a secreted MMP-9 component (Fig. 2 G). Sera MMP-9 level and activity evaluation Subsequently, patients' pre-operative sera MMP-9 levels were measured. The median sera MMP-9 value of healthy subjects was 0.8ng/ml (IQR 0.6, 1), 1.2ng/ml (IQR 0.9, 1.4) for meningiomas and 1.8ng/ml (IQR 1.3, 2.3) for BM. In contrast, glioblastoma patients' sera showed a significantly elevated value of 2.8ng/ml (IQR 2.1, 3.7, p < 0.001). MMP-9 sera activity was also assessed, and a similar enhanced pattern was demonstrated in glioblastoma compared to BM, meningioma patients, and healthy subjects (Fig. 3 B-D). The median sera MMP-9 level and activity values were used to differentiate the cohort into two groups and evaluate the impact on patients' OS. The MMP-9 sera level, not its activity, significantly affected it (Figs. 3 F-E). Notably, glioblastoma and BM patients with low MMP-9 sera levels were found to have an enhanced OS (15.8 compared to 8.4 months, respectively, p = 0.022, Fig. 3 E). Cox Proportional Hazard Model Analysis for OS Univariate analysis indicated that among all parameters examined, high sera MMP-9 levels were shown to decrease glioblastoma and BM patients' OS (p = 0.024, Table 2 A). Intra-tumoral MMP-9 levels and activity and sera MMP-9 activity did not show statistical significance (Table 2 A), though, after including all MMP-9 measurement variables in a multivariate model and adjusting for potential confounders, intra-tumoral MMP-9 levels did show a significant effect on patients' OS (p = 0.023, Table 2 A-B). In univariate and multivariate analyses, KPS level and adjuvant oncological therapy showed a protective effect (p = 0.035 and p 14% did not significantly affect patients' OS. Discussion This study assesses MMP-9's relation to the course of glioblastoma and BM disease. The results demonstrate that glioblastoma and BM patients exhibit higher intra-tumoral and pre-operative sera MMP-9 levels than meningioma patients or healthy individuals, which can be used longitudinally to monitor their disease for progression. Furthermore, the study highlights the significantly poorer OS of the high MMP-9 level patients' group, which may signify that the MMP-9 level potentially reflects the tumor's inherent invasive capability, which later translates into a poorer prognosis. Numerous works have established MMP-9 expression in BM patients' sera and its importance to their treatment selection and prognosis [ 21 , 24 ]. In breast carcinoma, high pre-operative sera MMP-9 levels were correlated with a high disease relapse rate [ 14 ]. Impaired OS and increased metastatic capability were demonstrated in several metanalyses reviewing solid malignancies [ 37 , 38 ]. Therefore, sera MMP-9 may also be essential in BM patients' clinical care. Yet, this has not been thoroughly explored, nor has MMP-9's contribution to glioblastoma care been established thus far [ 5 ]. In previous studies, the high levels of intra-tumoral MMP-9 in glioblastoma have been linked to poor patient outcomes [ 11 , 13 , 14 , 39 ]. Smith et al. [ 17 ] demonstrated that MMP-9 can be detected in patients' urine and cerebrospinal fluid, distinguishing them from healthy individuals [ 25 ]. Recent comprehensive reviews [ 5 , 24 ] highlighted a limited number of studies on plasma/serum MMP-9 levels in brain tumor patients, with inconsistent findings. Hormigo et al. [ 25 ] and Ricci et al. [ 4 ] associated sera MMP-9 levels with malignancy grade and disease activity, suggesting that it could be used to differentiate glioblastoma patients from healthy individuals. Debora et al. [ 40 ] recently demonstrated improved survival for glioblastoma patients with low sera extracellular vesicle MMP-9 levels. However, Iwamoto et al. [ 23 ] found that pre-operative sera MMP-9 levels in 58 newly diagnosed glioblastoma patients were not associated with the detection of disease progression over time. Therefore, so far, literature reports are indecisive [ 41 ], and this study aimed to address several of these uncertainties. The main results presented here show that patients with glioblastoma and BM had the highest detected levels of pre-operative sera MMP-9 (2.8ng/ml and 1.8ng/ml, respectively) compared to meningioma patients and healthy subjects. Furthermore, glioblastoma and BM patients with low sera MMP-9 levels had a significantly longer OS of 15.8 months compared to 8.4 months for those with high levels. This may suggest that high sera MMP-9 represents increased intrinsic tumor activity, which manifests, among other factors, with increased MMP-9 secretion. It highlights MMP-9's potential to be a valuable prognostic biomarker, in addition to age, KPS, and adjuvant oncological therapy, which are well-established prognosis-related parameters and could significantly enhance patient follow-up paradigms. The immunofluorescence analysis of MMP-9 conducted in this study revealed that it is distributed within and between astrocytic tumor cells, supporting MMP-9 secretion and its proposed role in extracellular matrix remodeling, thus promoting tumor cell spread. MMP-9 was also found in and around the tumor vasculature, matching other reports [ 29 ]. It is also well-known that blood-brain-barrier integrity is compromised in glioblastoma, which may correspond with the elevated sera MMP-9 levels detected, as secreted MMP-9 readily crosses the disrupted blood-brain-barrier [ 40 ] and enters the patient's systemic circulation, where its level can be detected and indicate disease progression/recurrence. Although glioblastoma and BMs are distinct, they share similar characteristics that can be compared. Frequent monitoring of these patients' sera MMP-9 is technically feasible. It can serve as a liquid biopsy, providing the ability to perform a proteomic analysis that potentially identifies disease progression or treatment response [ 9 , 39 , 42 ]. It may have added value during routine longitudinal oncological checkups, considering that preoperative elevated MMP-9 levels decline to baseline after surgery [ 9 , 23 , 25 ]. Sera MMP-9 can, therefore, serve as an individual baseline level for follow-up since tumor recurrence should be suspected when it increases. This could help detect tumor recurrence earlier and more definitively when combined with radiological findings, which, once appeared, are often debatable due to pseudoprogression or other possibilities [ 43 ]. In glioblastoma, MMP-9 assists tumor cells to escape from the hypoxic tumor core [ 44 ] and spread further into the infiltrative perilesional edematous zone [ 45 ]. Elevated MMP-9 has previously been found in this area, and a correlation between MMP-9 expression level and EI in malignant glioma was reported [ 29 ], as well as blood-brain barrier disruption [ 40 ]. Therefore, the rationale was to measure MMP-9 levels and to check their correlation with tumor mass and perilesional edema volumes. Despite this hypothesis and the radiological parameters' evaluation conducted in this work, it did not yield a link between either sera or intra-tumoral MMP-9 levels or activity. Recent works show that MMP-9 is differently expressed across glioblastoma locations [ 30 ]. This may explain the significant variability of edema volume observed within this patient cohort, requiring larger patient cohorts to address this question. This retrospective, low-numbered study included selectively sampled patients, and all statistical considerations apply to this type of study. Among other limitations, MMP-9 is typically found in low concentrations in the blood and is subjected to liver degradation [ 28 ] and may be impacted by other parameters [ 40 ]. Furthermore, the standardization of MMP level values and the determination of specific cutoffs are lacking, which makes it difficult to compare or combine results from different studies to achieve a more extensive database and reach more significant statistical implications. This study highlights that higher intra-tumoral and pre-operative sera MMP-9 levels are associated with lower patient OS, suggesting that MMP-9 may indicate the tumor's inherent tendency to spread. Hopefully, advances in diagnostic paradigms, i.e., routine incorporation of liquid biopsy analysis for glioblastoma and BM, will be made soon, and this study's findings can help predict patients' disease recurrence earlier and non-invasively. Declarations Author Contribution T.K.E. planned and supervised the study, analyzed and integrated the collected data, and wrote the main manuscript text. Y.E., S.H.S., T.Z., and N.R.D. conducted laboratory experiments. Y.E. also participated in writing the main manuscript. V.M. and K. L. supervised the study and critically reviewed the manuscript text. G.D. and R.R. conducted the statistical analysis. Y.K. and V.D. reviewed patients' pathological slides and immunofluorescence staining results, and S.H.S., S.T., M.A.V., R.B., and A.A. collected and analysed clinical patient data. M.C., H.A., and M.H. conducted the immunofluorescence staining and assisted and supported the laboratory experiments. I.M. supervised the study and critically reviewed the manuscript text. All authors reviewed the manuscript. References Xie, Q., S. Mittal, and M.E. Berens, Targeting adaptive glioblastoma: an overview of proliferation and invasion. Neuro-oncology, 2014. 16(12): p. 1575-1584. Bergers, G., et al., Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol, 2000. 2(10): p. 737-44. Hadler-Olsen, E., J.O. Winberg, and L. Uhlin-Hansen, Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets. Tumour Biol, 2013. 34(4): p. 2041-51. Ricci, S., et al., Evaluation of matrix metalloproteinase type IV-collagenases in serum of patients with tumors of the central nervous system. Journal of Neuro-oncology, 2017. 131: p. 223-232. Best, M.G., et al., Liquid biopsies in patients with diffuse glioma. Acta neuropathologica, 2015. 129: p. 849-865. Mathios, D. and J. Phallen, Circulating Biomarkers in Glioblastoma: Ready for Prime Time? Cancer J, 2021. 27(5): p. 404-409. Zucker, S. and J. Cao, Measurement of matrix metalloproteinases in serum of patients with melanoma: snarled in technical pitfalls. Clinical Cancer Research, 2005. 11(14): p. 5069-5070. Groves, M.D., et al., Phase II trial of temozolomide plus the matrix metalloproteinase inhibitor, marimastat, in recurrent and progressive glioblastoma multiforme. Journal of Clinical Oncology, 2002. 20(5): p. 1383-1388. Jiguet-Jiglaire, C., et al., Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study. Acta Neuropathol Commun, 2022. 10(1): p. 1. Choe, G., et al., Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype. Clinical Cancer Research, 2002. 8(9): p. 2894-2901. Li, Q., et al., Comparative analysis of matrix metalloproteinase family members reveals that MMP9 predicts survival and response to temozolomide in patients with primary glioblastoma. PLoS One, 2016. 11(3): p. e0151815. Xue, Q., et al., High expression of MMP9 in glioma affects cell proliferation and is associated with patient survival rates. Oncology letters, 2017. 13(3): p. 1325-1330. Wu, Z., et al., Matrix metalloproteinase 9 expression and glioblastoma survival prediction using machine learning on digital pathological images. Scientific Reports, 2024. 14(1): p. 15065. Wu, Z.S., et al., Prognostic significance of MMP‐9 and TIMP‐1 serum and tissue expression in breast cancer. International journal of cancer, 2008. 122(9): p. 2050-2056. Lee, J.-H., J.-W. Choi, and Y.-S. Kim, Serum TIMP-1 predicts survival outcomes of invasive breast carcinoma patients: a meta-analysis. Archives of Medical Research, 2011. 42(6): p. 463-468. Nikkola, J., et al., High serum levels of matrix metalloproteinase-9 and matrix metalloproteinase-1 are associated with rapid progression in patients with metastatic melanoma. Clinical Cancer Research, 2005. 11(14): p. 5158-5166. Smith, E.R., et al., Urinary biomarkers predict brain tumor presence and response to therapy. Clinical cancer research, 2008. 14(8): p. 2378-2386. Loo, H.K., et al., Circulating biomarkers for high-grade glioma. 2019, Taylor & Francis. p. 161-165. Friedberg, M.H., et al., Specific matrix metalloproteinase profiles in the cerebrospinal fluid correlated with the presence of malignant astrocytomas, brain metastases, and carcinomatous meningitis. Cancer: Interdisciplinary International Journal of the American Cancer Society, 1998. 82(5): p. 923-930. Roy, R., J. Yang, and M.A. Moses, Matrix metalloproteinases as novel biomarker s and potential therapeutic targets in human cancer. Journal of clinical oncology, 2009. 27(31): p. 5287-5297. Tabouret, E., et al., MMP2 and MMP9 serum levels are associated with favorable outcome in patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant chemotherapy in the BEVERLY-2 study. Oncotarget, 2016. 7(14): p. 18531. Skerenova, M., et al., Circulating tumor cells and serum levels of MMP-2, MMP-9 and VEGF as markers of the metastatic process in patients with high risk of metastatic progression. 2017. Iwamoto, F.M., et al., Longitudinal prospective study of matrix metalloproteinase-9 as a serum marker in gliomas. Journal of neuro-oncology, 2011. 105: p. 607-612. Roy, R., et al., Metalloproteinases and their roles in human cancer. The Anatomical Record, 2020. 303(6): p. 1557-1572. Hormigo, A.l., et al., YKL-40 and matrix metalloproteinase-9 as potential serum biomarkers for patients with high-grade gliomas. Clinical Cancer Research, 2006. 12(19): p. 5698-5704. Yi, L., et al., Plasma levels of tissue inhibitor of matrix metalloproteinase-1 correlate with diagnosis and prognosis of glioma patients. Chinese medical journal, 2013. 126(22): p. 4295-4300. Tabouret, E., et al., Association of matrix metalloproteinase 2 plasma level with response and survival in patients treated with bevacizumab for recurrent high-grade glioma. Neuro-oncology, 2014. 16(3): p. 392-399. Crocker, M., et al., Serum angiogenic profile of patients with glioblastoma identifies distinct tumor subtypes and shows that TIMP-1 is a prognostic factor. Neuro-oncology, 2010. 13(1): p. 99-108. Liu, Y., et al., Expression of VEGF and MMP-9 and MRI imaging changes in cerebral glioma. Oncology Letters, 2011. 2(6): p. 1171-1175. Fan, X., et al., Regional specificity of matrix metalloproteinase-9 expression in the brain: voxel-level mapping in primary glioblastomas. Clinical Radiology, 2018. 73(3): p. 283-289. Meyer, H.J., et al., Perifocal edema volume is not associated with immunohistochemical features reflecting proliferation potential, microvessel density, neoangiogenesis and invasiveness in brain metastasis. Clin Neurol Neurosurg, 2021. 202: p. 106537. Liu, W. and Z. Li, Diagnostic performance of perfusion-weighted imaging combined with serum MMP-2 and -9 levels in tumor recurrence after postoperative concomitant chemoradiotherapy of glioblastoma. J Clin Ultrasound, 2023. 51(3): p. 563-570. Kaisman-Elbaz, T., et al., Cell death induced by zinc and cadmium is mediated by clusterin in cultured mouse seminiferous tubules. Journal of Cellular Physiology, 2009. 220(1): p. 222-229. Stupp, R., et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England journal of medicine, 2005. 352(10): p. 987-996. Kaisman-Elbaz, T., et al., Hemoglobin levels and red blood cells distribution width highlights glioblastoma patients subgroup with improved median overall survival. Frontiers in Oncology, 2020. 10: p. 432. Berghoff, A.S., et al., Combining standard clinical blood values for improving survival prediction in patients with newly diagnosed brain metastases—development and validation of the LabBM score. Neuro-oncology, 2017. 19(9): p. 1255-1262. Zhang, Q.-W., et al., Matrix metalloproteinase-9 as a prognostic factor in gastric cancer: a meta-analysis. Asian Pacific Journal of Cancer Prevention, 2012. 13(6): p. 2903-2908. Gong, L., et al., Prognostic impact of serum and tissue MMP-9 in non-small cell lung cancer: a systematic review and meta-analysis. Oncotarget, 2016. 7(14): p. 18458. Thanh, H.D., et al., Temozolomide promotes matrix metalloproteinase 9 expression through p38 MAPK and JNK pathways in glioblastoma cells. Scientific Reports, 2024. 14(1): p. 14341. Dobra, G., et al., MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. Cancers (Basel), 2023. 15(3). Linhares, P., et al., Glioblastoma: Is There Any Blood Biomarker with True Clinical Relevance? Int J Mol Sci, 2020. 21(16). Fontanilles, M., et al., Metabolic remodeling in glioblastoma: a longitudinal multi-omics study. Acta Neuropathol Commun, 2024. 12(1): p. 162. Hotchkiss, K.M., et al., A brave new framework for glioma drug development. Lancet Oncol, 2024. 25(10): p. e512-e519. Zhou, W., et al., Increased expression of MMP-2 and MMP-9 indicates poor prognosis in glioma recurrence. Biomedicine & Pharmacotherapy, 2019. 118: p. 109369. Schuler, P.J., et al., Urokinase plasminogen activator, uPAR, MMP-2, and MMP-9 in the C6-glioblastoma rat model. in vivo, 2012. 26(4): p. 571-576. Tables Tables 1 to 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files TablesFinal09112024.docx Table 1. Patients’ Main Demographics and Clinical Characteristics. A. Age at diagnosis and KPS were similar among the glioblastoma and BM patient groups. Adjuvant oncological treatment was applied to most patients included in this study. Anemia and RDW>14% were more commonly found in patients with BM. OS was measured and displayed within the table. Patients’ main imagery characteristics are presented in B. Edema volume and Edema Index were increased in patients with glioblastoma and BM, and the measured values of tumor volume were also presented. Glioblastoma and BM Patients’ Cohort Sub-analysis. C. Since glioblastoma and BM were primarily highlighted in this study, their characteristics were compared. Both glioblastoma and BM patients' functional statuses were high, more BM patients had RDW>14%, and recurrent disease was increasingly demonstrated in the glioblastoma group. Newly diagnosed glioblastoma patients were then compared to recurrent glioblastoma patients. D. Recurrent glioblastoma patients were younger, and their OS was improved. Table 2 - Patients’ Cohort Cox Proportional Hazard Model Survival Analysis. Univariate analysis of patient's clinical parameters. A. KPS and oncological treatment were significantly related to patient OS. B. Univariate analysis of high intra-tumoral and serum MMP-9 levels and activity showed that serum MMP-9 levels significantly influenced OS. The multivariate analysis then indicated that high intra-tumoral MMP-9 and sera levels, KPS, and adjuvant oncological treatment were the most important factors impacting OS in glioblastoma and BM patients. C. Glioblastoma and BM patients with low intra-tumoral and sera MMP-9 levels demonstrated significantly enhanced OS compared to patients with high intra-tumoral and sera MMP-9 levels. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5467623","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":384820571,"identity":"97119c8d-a40e-4e83-a83e-ecb75faf3adc","order_by":0,"name":"Tehila Kaisman-Elbaz","email":"data:image/png;base64,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","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Tehila","middleName":"","lastName":"Kaisman-Elbaz","suffix":""},{"id":384820573,"identity":"b77ad7c2-f2dc-437d-8868-1b3fafbb4c10","order_by":1,"name":"Snir Haddad-Shlaifshtein","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Snir","middleName":"","lastName":"Haddad-Shlaifshtein","suffix":""},{"id":384820575,"identity":"9d707b3d-f86b-4d2a-a48a-456335f92244","order_by":2,"name":"Yael Eskira","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yael","middleName":"","lastName":"Eskira","suffix":""},{"id":384820577,"identity":"c2586e75-6c85-450c-ab94-49dab2768f5a","order_by":3,"name":"Vladimir Merkin","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Merkin","suffix":""},{"id":384820581,"identity":"bc8586e2-9a8a-4bf8-ab51-5c0e188dbb9a","order_by":4,"name":"Guy Dumanis","email":"","orcid":"","institution":"Ariel University","correspondingAuthor":false,"prefix":"","firstName":"Guy","middleName":"","lastName":"Dumanis","suffix":""},{"id":384820583,"identity":"375b7948-ef2c-4260-b031-26eec9b7ddfc","order_by":5,"name":"Sivan Turiel","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sivan","middleName":"","lastName":"Turiel","suffix":""},{"id":384820585,"identity":"0f275de8-df87-4aaf-89c0-38d77d184c28","order_by":6,"name":"Maya Atar-Vardi","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Atar-Vardi","suffix":""},{"id":384820586,"identity":"90b734e3-dd71-46b8-baa6-0a8fe66921a6","order_by":7,"name":"Romi Bari","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Romi","middleName":"","lastName":"Bari","suffix":""},{"id":384820587,"identity":"4ee8f500-2338-49fb-bb93-b2cff533efda","order_by":8,"name":"Adi Alt","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Adi","middleName":"","lastName":"Alt","suffix":""},{"id":384820588,"identity":"2fd30c38-5f70-404d-bf75-0a92a741c78f","order_by":9,"name":"Tali Zamed","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Tali","middleName":"","lastName":"Zamed","suffix":""},{"id":384820589,"identity":"c2d9564d-0dea-43f5-a1f0-cdf2cefd5afa","order_by":10,"name":"Noa Rotem-Dai","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Noa","middleName":"","lastName":"Rotem-Dai","suffix":""},{"id":384820591,"identity":"84be4094-4d10-457a-8019-688bcfdf916d","order_by":11,"name":"Konstantin Lavrenkov","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Lavrenkov","suffix":""},{"id":384820592,"identity":"ee342e47-6a75-4093-8787-b3c501c70952","order_by":12,"name":"Yarden Kezerle","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yarden","middleName":"","lastName":"Kezerle","suffix":""},{"id":384820593,"identity":"7579338a-0666-460a-8e5d-472865dd371f","order_by":13,"name":"Victor Dyomin","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"","lastName":"Dyomin","suffix":""},{"id":384820594,"identity":"48a779c1-22fa-441e-9acf-87231f736325","order_by":14,"name":"Ronit Razon","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Ronit","middleName":"","lastName":"Razon","suffix":""},{"id":384820595,"identity":"9600bfe5-cb32-4193-9bb6-36f9380e00bb","order_by":15,"name":"Moumita Chakraborty","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Moumita","middleName":"","lastName":"Chakraborty","suffix":""},{"id":384820596,"identity":"d6c664d2-9841-4046-918c-0fe53f90408f","order_by":16,"name":"Hila Asraf","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Hila","middleName":"","lastName":"Asraf","suffix":""},{"id":384820597,"identity":"c20f3286-eddb-4362-bb1a-37129c223c1b","order_by":17,"name":"Michal Hershfinkel","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Hershfinkel","suffix":""},{"id":384820598,"identity":"ba0c599b-8777-49a2-8569-3b8a590d4ea7","order_by":18,"name":"Israel Melamed","email":"","orcid":"","institution":"Soroka University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Israel","middleName":"","lastName":"Melamed","suffix":""}],"badges":[],"createdAt":"2024-11-16 22:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5467623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5467623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72285102,"identity":"04b6ed11-be6b-416b-a29f-10e2f8f403f3","added_by":"auto","created_at":"2024-12-24 16:52:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1100424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeasurement of patients’ tumor volume and edema. A.\u003c/strong\u003e An example of patients' MRI imagery analysis using Brainlab® cranial navigation software. The purple marking on the left image panel demonstrates the tumor mass delineation performed on the T1W1+gadolinium MRI sequence in each case included in this study. The light blue marking on the right panel demonstrates the edema volume measured on the T2W1 MRI sequence. \u003cstrong\u003eImagery parameters analysis is shown in B-D.\u003c/strong\u003e Edema volume and Edema Index significantly differed between glioblastoma and BM and meningioma groups, whereas tumor mass volume was not.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5467623/v1/a9f7871032a730380bd00460.png"},{"id":72285104,"identity":"7d9ff4d8-b0f4-43ff-9e6f-9bb895e535f2","added_by":"auto","created_at":"2024-12-24 16:52:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3819141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntra-tumoral MMP-9 level and activity assessment. A-C. \u003c/strong\u003eIntra-tumoral MMP-9 levels and activity were measured and found to be the highest in glioblastoma, then in BM, compared to meningioma patients. The median levels were used to divide the groups according to high and low MMP-9 levels or activity and assess their link to patients' OS, which were insignificant. \u003cstrong\u003eD-E.\u003c/strong\u003e Intra-tumoral MMP-9 levels and activity were found to be correlated. \u003cstrong\u003eF.\u003c/strong\u003e Immunofluorescence straining of tumor sample from glioblastoma patient number 5 showed that intra-tumoral MMP-9 (represented by the red staining) was then localized to tumor astrocytes (GFAP-positive cells, stained with green) and endothelial cells (marked with white arrow). However, alternatively, MMP-9 was diffusely scattered within the tumor tissue sample of glioblastoma patient number 3 between astrocytic tumor cells and not within them, as demonstrated in patient number 5 tumor tissue sample. \u003cstrong\u003eG.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5467623/v1/67ad136c1abef6efe81184a1.png"},{"id":72285103,"identity":"41c4b649-39b4-404b-9ecb-c552935aa279","added_by":"auto","created_at":"2024-12-24 16:52:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":948171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification and activity of sera MMP-9. \u003c/strong\u003eSignificant MMP-9 levels and activity were detected in glioblastoma and BM patients' sera compared to meningioma or healthy control sera. A-B. Western blot analysis visually shows the higher sera MMP-9 level detected in glioblastoma and BM, while the MMP-2 level did not exhibit a similar pattern. Using an MMP-9 inhibitor (TIMP-1 complex) also highlights the specific results obtained. \u003cstrong\u003eC.\u003c/strong\u003e The median MMP-9 sera levels and activity of glioblastoma and BM patients were divided into high and low sera MMP-9 levels groups. High sera MMP-9 levels significantly worsened patients' OS, whereas sera MMP-9 activity did not show the same effect. \u003cstrong\u003eD-F.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5467623/v1/cbc046ee1edcedc88a34622a.png"},{"id":72288378,"identity":"2d8d9fc9-109e-4522-a48b-c8803d9a0858","added_by":"auto","created_at":"2024-12-24 17:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6118379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5467623/v1/b4d52a05-468e-4f3c-8d28-a2b2441fa71f.pdf"},{"id":72285105,"identity":"18223e28-7b61-4804-a66d-a6a6d7b05abb","added_by":"auto","created_at":"2024-12-24 16:52:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1223053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Patients’ Main Demographics and Clinical Characteristics. A. \u003c/strong\u003eAge at diagnosis and KPS were similar among the glioblastoma and BM patient groups. Adjuvant oncological treatment was applied to most patients included in this study. Anemia and RDW\u0026gt;14% were more commonly found in patients with BM. OS was measured and displayed within the table. \u003cstrong\u003ePatients’ main imagery characteristics are presented in B.\u003c/strong\u003e Edema volume and Edema Index were increased in patients with glioblastoma and BM, and the measured values of tumor volume were also presented. \u003cstrong\u003eGlioblastoma and BM Patients’ Cohort Sub-analysis. C. \u003c/strong\u003eSince glioblastoma and BM were primarily highlighted in this study, their characteristics were compared. Both glioblastoma and BM patients' functional statuses were high, more BM patients had RDW\u0026gt;14%, and recurrent disease was increasingly demonstrated in the glioblastoma group. \u003cstrong\u003eNewly diagnosed glioblastoma patients were then compared to recurrent glioblastoma patients. D. \u003c/strong\u003eRecurrent glioblastoma patients were younger, and their OS was improved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 - Patients’ Cohort Cox Proportional Hazard Model Survival Analysis. Univariate analysis of patient's clinical parameters. A.\u003c/strong\u003e KPS and oncological treatment were significantly related to patient OS. \u003cstrong\u003eB.\u003c/strong\u003e Univariate analysis of high intra-tumoral and serum MMP-9 levels and activity showed that serum MMP-9 levels significantly influenced OS. The multivariate analysis then indicated that high intra-tumoral MMP-9 and sera levels, KPS, and adjuvant oncological treatment were the most important factors impacting OS in glioblastoma and BM patients. \u003cstrong\u003eC. \u003c/strong\u003eGlioblastoma and BM patients with low intra-tumoral and sera MMP-9 levels demonstrated significantly enhanced OS compared to patients with high intra-tumoral and sera MMP-9 levels.\u003c/p\u003e","description":"","filename":"TablesFinal09112024.docx","url":"https://assets-eu.researchsquare.com/files/rs-5467623/v1/41f842a3ae76a97022c094b4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High Intra-tumoral and Sera Matrix Metalloproteinase 9 Levels Reduce Glioblastoma and Brain Metastases Patients' Survival","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMatrix metalloproteinase 9 (MMP-9) has been shown to induce glioblastoma invasion [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] by altering the extracellular matrix and promoting angiogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite its potential as a tumor progression biomarker [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and the increasing need for non-invasive modalities to monitor brain tumor patients' disease course, i.e., liquid biopsy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], only a few clinical trials have evaluated MMP-9. None have yielded sufficient clinical benefit to be included in patients' treatment and follow-up paradigms [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMMP-9's role in glioblastoma was highlighted in various studies. Its high level in patients' tumors was correlated with decreased survival and tumor invasiveness, which were reduced once inhibited [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A similar pattern was detected in metastatic solid cancers, i.e., breast carcinoma and melanoma [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, once released from tumor cells, MMP-9 was identified in body fluids (i.e., blood, urine, and CSF [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]) - even before clinical manifestations occurred [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Several works showed that MMP-9 sera/plasma levels can predict tumor metastatic propensity and disease recurrence [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the literature describing MMP-9 secretion pattern in glioblastoma patients is less characterized compared to BM, and the works that addressed it describe inconclusive results [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Ricci et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], for instance, found that sera MMP-9 levels differentiated metastatic lesions, gliomas, and meningiomas from healthy controls and correlated it with tumor malignancy grade, similar to Lin et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In turn, Hormigo et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] reported sera MMP-9 level as an indicator of glioblastoma progression, and Tabouret et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] related lower MMP-9 levels to enhanced survival of malignant glioma patients. On the other hand, Iwamoto et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] compared MMP-9 serum levels in 343 glioma patients and found a slim relation to disease state, similar to Crocker et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, works correlating MMP-9 levels with patients' imaging features are scarce. Liu et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] have previously correlated high-grade gliomas with increased perilesional edema volumes and high MMP-9 levels. Other reports, [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], however, associated high MMP-9 levels with smaller edema volumes of glioblastomas and specific brain locations. Liu and Li [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] linked sera MMP-9 and perfusion-weighted MRI parameters recently to glioblastoma recurrence.\u003c/p\u003e \u003cp\u003eThis work aims to evaluate the potential of MMP-9 as a biomarker alongside imaging and other parameters in glioblastoma and BM. It seeks to identify disease progression by supplementing routine measures with current patient follow-up paradigms.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study used Soroka University Medical Center neurosurgery department brain tumor bank samples obtained from patients who underwent tumor resection between 2015 and 2021 under the institutional ethical committee approval [0208-16-SOR]. Patients were included if they: 1) signed informed consent to provide tumor tissue and blood samples; 2) underwent tumor resection; 3) had available pre-operative clinical, imagery, and laboratory data. Patients with partially documented data were excluded (n\u0026thinsp;=\u0026thinsp;17).\u003c/p\u003e\n\u003cp\u003eGlioblastoma (n\u0026thinsp;=\u0026thinsp;27), BM (n\u0026thinsp;=\u0026thinsp;30), and meningioma (n\u0026thinsp;=\u0026thinsp;12) tumor and sera samples were analyzed. Demographics and clinical data were documented per patient, including Karnofsky Performance Status (KPS), oncological and surgical history, diagnosis, birth and exitus dates, imagery, laboratory, and histopathological data. Patients\u0026apos; overall survival (OS) was calculated as the delta between the diagnosis date (date of surgical intervention) and exitus dates. Blood was also withdrawn from healthy volunteers (n\u0026thinsp;=\u0026thinsp;23) to control cohort patients\u0026apos; MMP-9 levels.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eTumor tissue and blood samples analysis\u003c/h2\u003e\n \u003cp\u003eEach tumor and blood sample collected was handled according to the institutional ethical committee-approved protocol. Fresh patients\u0026rsquo; tumor samples were stored in liquid nitrogen until further analysis. Blood samples were collected in EDTA tubes and serum clot activator with gel separator tubes before surgical intervention. The tubes were then centrifuged for 15 minutes at 2000g at room temperature (RT), and the serum was kept in liquid nitrogen.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eImagery tumor data collection and volumetric evaluation\u003c/h3\u003e\n\u003cp\u003ePreoperative Magnetic Resonance Imaging scans were retrieved from the institutional Picture Archiving and Communication System. To achieve the most accurate approximation of tumors\u0026apos; mass and edema volume for each patient, Brainlab cranial navigation software (Brainlab\u0026reg;, Germany) was used. Volumetric measurement of the tumor mass (T1W1\u0026thinsp;+\u0026thinsp;gadolinuim sequence), edema (T2W2 sequence), and the ratio between them were calculated (i.e., edema index (EI) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]). Perilesional edema volume was defined by subtracting the tumor mass volume from each patient\u0026apos;s combined tumor mass and edema volumes.\u003c/p\u003e\n\u003ch3\u003eProtein extraction from tumor tissues\u003c/h3\u003e\n\u003cp\u003eFrozen tumor tissue samples were defrosted and added with lysis buffer. Tissues were disrupted with a Dounce homogenizer and incubated for 30 minutes on ice, followed by sonication and centrifugation at 14,000 RPM for 15 minutes under 4\u0026deg;C. Supernatants were collected, and protein concentration was determined using the Bradford method. The samples were kept at -80\u0026deg;C until further testing.\u003c/p\u003e\n\u003ch3\u003eIntra-tumoral and sera MMP-9 levels measurement\u003c/h3\u003e\n\u003cp\u003eA 4\u0026micro;g/ml purified antibody anti-MMP-9 (BioLegend, USA) was applied on a 96-well plate. The plate was then incubated overnight at 4\u0026deg;C and washed with PBS-Tween solution. Blocking and diluted serum (1:100) or 300 \u0026micro;g protein extract were added, and the plate was incubated. A standard curve using recombinant MMP-9 was prepared according to the manufacturer\u0026apos;s instructions (BioLegend, USA). A secondary biotin anti-human MMP-9 antibody and HRP-Avidin (1:1500) were added. To produce a colorimetric reaction, the plates were incubated with TMB and were read using an ELISA reader (MULTISKAN FC Thermo Scientific) at 650nm. Alternatively, immunoblot analysis of MMP-9 level of patients and healthy subjects sera was performed as previously described [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIntra-tumoral and sera MMP-9 activity assessment\u003c/h3\u003e\n\u003cp\u003eTotal protein extraction from each tumor sample was mixed with a non-reducing sample buffer and applied to acrylamide gels. Gels were run until sufficient band separation was achieved. Following SDS removal, the gels were incubated in Tris-HCl-Triton solution. Gels were then immersed for 30 minutes in 40% methanol, 10% acetic acid, and Coomassie Brilliant Blue G-250 (Merck, Germany) and de-stained in the same solution without the dye for several hours. The gelatinase activity of MMP-9 was evident as a clear white band. Each gel was run with a pre-stained standard protein ladder and the standard curve of purified MMP-9. The gels were then scanned, and the molecular weight was determined according to the standard protein ladder. Band density and gelatinase activity were quantified using GelQuantNET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.BiochemLabSolutions.com\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence of MMP-9 in glioblastoma\u003c/h2\u003e\n \u003cp\u003eTumor tissue was fixated in 4% formaldehyde solution, followed by paraffin embedding, sectioning, and mounting on slides as acceptable. Slides were then deparaffinized and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was blocked, and slides were incubated in a blocking solution and with primary antibody MMP-9 (1:100; Abcam, USA) overnight at 4\u0026deg;C. The next day, slides were washed and incubated with a secondary antibody conjugated to Cy2/Cy3 (the Jackson Laboratory). Following additional rinsing, slides were mounted with a DAPI-containing mounting medium (Immunomount) and viewed in a confocal microscope (Zeiss LSM 510) with appropriate fluorescence barrier and excitation filters.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eStatistical analysis and OS\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using R, version 4.3.0 (R Foundation for Statistical Computing). Chi-squared tests were used for categorical variables with adequate sample sizes, while Fisher\u0026apos;s exact test was employed when the criteria for the Chi-squared test were not met. The Shapiro-Wilk test assessed normality for numeric variables, applying the student\u0026apos;s t-test for normally distributed data and non-parametric tests when normality was rejected. i.e., Kruskal-Wallis\u0026apos;s test. The Mann-Whitney U test was employed for pairwise comparisons when only two groups were compared.\u003c/p\u003e\n\u003cp\u003eFurther, univariate and multivariate Cox proportional hazard model analysis was performed. Due to non-normality, Spearman correlation tests were used to assess correlations between continuous numeric variables, and the variance inflation factor was calculated for each variable to avoid multicollinearity issues. Statistical significance was determined when the p-value was less or equal to 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatients' Demographics and Clinical Characterization\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e1\u003c/span\u003eA presents the entire cohort's (n\u0026thinsp;=\u0026thinsp;69) main characteristics. Glioblastoma patients' median age at diagnosis was 62 (IQR 52.5, 72). Their median KPS was 100, and median OS was 10.1 months (IQR 5.1, 18); 89% of the patients were treated with radiation and chemotherapy as acceptable [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe BM cohort's median age at diagnosis was 63.5 (IQR 56.25, 67), and 77% originated from either melanoma, lung, or breast carcinoma. 73.3% received adjuvant oncological treatment following surgical resection (i.e., radiation, chemotherapy, and biological therapy). Their median KPS was 90, and they exhibited a median OS of 8 months since their BM diagnosis (IQR 3.7, 20.7). Meningioma patients' median age at diagnosis was 67 (IQR 59.2, 72.5), and their median KPS was 90.\u003c/p\u003e \u003cp\u003eSince anemia was previously recognized for its negative influence on glioblastoma [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and BM patients' OS [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], this study cohort was evaluated for it in addition to other parameters. 32% of the patients' cohort were anemic (as defined elsewhere [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]), and 48% had Red blood Distribution Width (RDW)\u0026thinsp;\u0026gt;\u0026thinsp;14%. Notably, anemia and RDW\u0026thinsp;\u0026gt;\u0026thinsp;14% were more prevalent in the BM cohort than in glioblastoma. As follows, anemia or RDW\u0026thinsp;\u0026gt;\u0026thinsp;14% did not influence OS in this cohort.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTumor and Edema Volumes Analysis\u003c/h2\u003e \u003cp\u003eA comprehensive imagery assessment was also performed, aiming at correlating it with MMP-9 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Glioblastoma and BM induced significant peritumoral edema of 73.9 cc vs. 85.2 cc, respectively, and their EI was, therefore, significantly elevated compared to meningioma (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Tumor volumes were not significantly different when comparing tumor types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGlioblastoma and BM Patients Cohort Sub-analysis\u003c/h2\u003e \u003cp\u003eFurther on, glioblastoma and BM cohorts were compared since MMP-9 levels and activity on their OS were mainly assessed (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, age at diagnosis and OS did not differ among the groups. A similar percentage of patients receive complementary oncological treatment. More BM patients, though, were found to have RDW\u0026thinsp;\u0026gt;\u0026thinsp;14%, and their median KPS was 90. Tumor and edema volumes did not differ. Therefore, these two patient groups were united for further analysis. A sub-analysis of recurrent glioblastoma patients was also performed (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Recurrent glioblastoma patients were younger (53.2 years, SD 13.99), had a high KPS, and exhibited an enhanced OS compared to newly diagnosed glioblastoma patients (18 months, p\u0026thinsp;=\u0026thinsp;0.005).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIntra-tumoral MMP-9 Characterization\u003c/h2\u003e \u003cp\u003ePatients' intra-tumoral MMP-9 content assessment followed. Glioblastoma and, to a lesser extent, BM tumor samples demonstrated an enhanced intra-tumoral level of MMP-9 (8ng/ml and 4ng/ml, respectively) compared to meningioma (1ng/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Intra-tumoral MMP-9 activity was then measured and was comparable in glioblastoma and BM but significantly increased compared to meningioma (p\u0026thinsp;=\u0026thinsp;0.004, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Their median values were used to subdivide patients' cohorts according to the intra-tumoral MMP-9 and evaluate its influence on OS, but they were found insignificant (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). Notably, intra-tumoral MMP-9 level and activity were significantly correlated (r\u0026thinsp;=\u0026thinsp;0.57, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe following step was to localize MMP-9 within glioblastoma tumor tissue and aim to identify which cells display the protein. Therefore, two glioblastoma patients' samples were stained. MMP-9-specific staining was detected within endothelial cells of both samples and in the cytoplasm of GFAP-positive stained-astrocytic tumor cells in patient 5. However, for patient 3, scattered staining was noticed between astrocytic cells and not within them, which could indicate a secreted MMP-9 component (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSera MMP-9 level and activity evaluation\u003c/h2\u003e \u003cp\u003eSubsequently, patients' pre-operative sera MMP-9 levels were measured. The median sera MMP-9 value of healthy subjects was 0.8ng/ml (IQR 0.6, 1), 1.2ng/ml (IQR 0.9, 1.4) for meningiomas and 1.8ng/ml (IQR 1.3, 2.3) for BM. In contrast, glioblastoma patients' sera showed a significantly elevated value of 2.8ng/ml (IQR 2.1, 3.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). MMP-9 sera activity was also assessed, and a similar enhanced pattern was demonstrated in glioblastoma compared to BM, meningioma patients, and healthy subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). The median sera MMP-9 level and activity values were used to differentiate the cohort into two groups and evaluate the impact on patients' OS. The MMP-9 sera level, not its activity, significantly affected it (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-E). Notably, glioblastoma and BM patients with low MMP-9 sera levels were found to have an enhanced OS (15.8 compared to 8.4 months, respectively, p\u0026thinsp;=\u0026thinsp;0.022, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCox Proportional Hazard Model Analysis for OS\u003c/h2\u003e \u003cp\u003eUnivariate analysis indicated that among all parameters examined, high sera MMP-9 levels were shown to decrease glioblastoma and BM patients' OS (p\u0026thinsp;=\u0026thinsp;0.024, Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Intra-tumoral MMP-9 levels and activity and sera MMP-9 activity did not show statistical significance (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), though, after including all MMP-9 measurement variables in a multivariate model and adjusting for potential confounders, intra-tumoral MMP-9 levels did show a significant effect on patients' OS (p\u0026thinsp;=\u0026thinsp;0.023, Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In univariate and multivariate analyses, KPS level and adjuvant oncological therapy showed a protective effect (p\u0026thinsp;=\u0026thinsp;0.035 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). MMP-9 activity, tumor type, age at diagnosis, gender, edema volume, anemia, and RDW\u0026thinsp;\u0026gt;\u0026thinsp;14% did not significantly affect patients' OS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assesses MMP-9's relation to the course of glioblastoma and BM disease. The results demonstrate that glioblastoma and BM patients exhibit higher intra-tumoral and pre-operative sera MMP-9 levels than meningioma patients or healthy individuals, which can be used longitudinally to monitor their disease for progression. Furthermore, the study highlights the significantly poorer OS of the high MMP-9 level patients' group, which may signify that the MMP-9 level potentially reflects the tumor's inherent invasive capability, which later translates into a poorer prognosis.\u003c/p\u003e \u003cp\u003eNumerous works have established MMP-9 expression in BM patients' sera and its importance to their treatment selection and prognosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In breast carcinoma, high pre-operative sera MMP-9 levels were correlated with a high disease relapse rate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Impaired OS and increased metastatic capability were demonstrated in several metanalyses reviewing solid malignancies [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, sera MMP-9 may also be essential in BM patients' clinical care. Yet, this has not been thoroughly explored, nor has MMP-9's contribution to glioblastoma care been established thus far [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn previous studies, the high levels of intra-tumoral MMP-9 in glioblastoma have been linked to poor patient outcomes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Smith et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] demonstrated that MMP-9 can be detected in patients' urine and cerebrospinal fluid, distinguishing them from healthy individuals [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Recent comprehensive reviews [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] highlighted a limited number of studies on plasma/serum MMP-9 levels in brain tumor patients, with inconsistent findings. Hormigo et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Ricci et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] associated sera MMP-9 levels with malignancy grade and disease activity, suggesting that it could be used to differentiate glioblastoma patients from healthy individuals. Debora et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] recently demonstrated improved survival for glioblastoma patients with low sera extracellular vesicle MMP-9 levels. However, Iwamoto et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] found that pre-operative sera MMP-9 levels in 58 newly diagnosed glioblastoma patients were not associated with the detection of disease progression over time. Therefore, so far, literature reports are indecisive [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and this study aimed to address several of these uncertainties.\u003c/p\u003e \u003cp\u003eThe main results presented here show that patients with glioblastoma and BM had the highest detected levels of pre-operative sera MMP-9 (2.8ng/ml and 1.8ng/ml, respectively) compared to meningioma patients and healthy subjects. Furthermore, glioblastoma and BM patients with low sera MMP-9 levels had a significantly longer OS of 15.8 months compared to 8.4 months for those with high levels. This may suggest that high sera MMP-9 represents increased intrinsic tumor activity, which manifests, among other factors, with increased MMP-9 secretion. It highlights MMP-9's potential to be a valuable prognostic biomarker, in addition to age, KPS, and adjuvant oncological therapy, which are well-established prognosis-related parameters and could significantly enhance patient follow-up paradigms.\u003c/p\u003e \u003cp\u003eThe immunofluorescence analysis of MMP-9 conducted in this study revealed that it is distributed within and between astrocytic tumor cells, supporting MMP-9 secretion and its proposed role in extracellular matrix remodeling, thus promoting tumor cell spread. MMP-9 was also found in and around the tumor vasculature, matching other reports [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It is also well-known that blood-brain-barrier integrity is compromised in glioblastoma, which may correspond with the elevated sera MMP-9 levels detected, as secreted MMP-9 readily crosses the disrupted blood-brain-barrier [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and enters the patient's systemic circulation, where its level can be detected and indicate disease progression/recurrence.\u003c/p\u003e \u003cp\u003eAlthough glioblastoma and BMs are distinct, they share similar characteristics that can be compared. Frequent monitoring of these patients' sera MMP-9 is technically feasible. It can serve as a liquid biopsy, providing the ability to perform a proteomic analysis that potentially identifies disease progression or treatment response [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It may have added value during routine longitudinal oncological checkups, considering that preoperative elevated MMP-9 levels decline to baseline after surgery [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Sera MMP-9 can, therefore, serve as an individual baseline level for follow-up since tumor recurrence should be suspected when it increases. This could help detect tumor recurrence earlier and more definitively when combined with radiological findings, which, once appeared, are often debatable due to pseudoprogression or other possibilities [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn glioblastoma, MMP-9 assists tumor cells to escape from the hypoxic tumor core [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and spread further into the infiltrative perilesional edematous zone [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Elevated MMP-9 has previously been found in this area, and a correlation between MMP-9 expression level and EI in malignant glioma was reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as well as blood-brain barrier disruption [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, the rationale was to measure MMP-9 levels and to check their correlation with tumor mass and perilesional edema volumes. Despite this hypothesis and the radiological parameters' evaluation conducted in this work, it did not yield a link between either sera or intra-tumoral MMP-9 levels or activity. Recent works show that MMP-9 is differently expressed across glioblastoma locations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This may explain the significant variability of edema volume observed within this patient cohort, requiring larger patient cohorts to address this question.\u003c/p\u003e \u003cp\u003eThis retrospective, low-numbered study included selectively sampled patients, and all statistical considerations apply to this type of study. Among other limitations, MMP-9 is typically found in low concentrations in the blood and is subjected to liver degradation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and may be impacted by other parameters [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, the standardization of MMP level values and the determination of specific cutoffs are lacking, which makes it difficult to compare or combine results from different studies to achieve a more extensive database and reach more significant statistical implications.\u003c/p\u003e \u003cp\u003eThis study highlights that higher intra-tumoral and pre-operative sera MMP-9 levels are associated with lower patient OS, suggesting that MMP-9 may indicate the tumor's inherent tendency to spread. Hopefully, advances in diagnostic paradigms, i.e., routine incorporation of liquid biopsy analysis for glioblastoma and BM, will be made soon, and this study's findings can help predict patients' disease recurrence earlier and non-invasively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.K.E. planned and supervised the study, analyzed and integrated the collected data, and wrote the main manuscript text. Y.E., S.H.S., T.Z., and N.R.D. conducted laboratory experiments. Y.E. also participated in writing the main manuscript. V.M. and K. L. supervised the study and critically reviewed the manuscript text. G.D. and R.R. conducted the statistical analysis. Y.K. and V.D. reviewed patients\u0026apos; pathological slides and immunofluorescence staining results, and S.H.S., S.T., M.A.V., R.B., and A.A. collected and analysed clinical patient data. M.C., H.A., and M.H. conducted the immunofluorescence staining and assisted and supported the laboratory experiments. I.M. supervised the study and critically reviewed the manuscript text. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eXie, Q., S. Mittal, and M.E. Berens, Targeting adaptive glioblastoma: an overview of proliferation and invasion. Neuro-oncology, 2014. 16(12): p. 1575-1584.\u003c/li\u003e\n \u003cli\u003eBergers, G., et al., Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol, 2000. 2(10): p. 737-44.\u003c/li\u003e\n \u003cli\u003eHadler-Olsen, E., J.O. Winberg, and L. Uhlin-Hansen, Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets. Tumour Biol, 2013. 34(4): p. 2041-51.\u003c/li\u003e\n \u003cli\u003eRicci, S., et al., Evaluation of matrix metalloproteinase type IV-collagenases in serum of patients with tumors of the central nervous system. Journal of Neuro-oncology, 2017. 131: p. 223-232.\u003c/li\u003e\n \u003cli\u003eBest, M.G., et al., Liquid biopsies in patients with diffuse glioma. Acta neuropathologica, 2015. 129: p. 849-865.\u003c/li\u003e\n \u003cli\u003eMathios, D. and J. Phallen, Circulating Biomarkers in Glioblastoma: Ready for Prime Time? Cancer J, 2021. 27(5): p. 404-409.\u003c/li\u003e\n \u003cli\u003eZucker, S. and J. Cao, Measurement of matrix metalloproteinases in serum of patients with melanoma: snarled in technical pitfalls. Clinical Cancer Research, 2005. 11(14): p. 5069-5070.\u003c/li\u003e\n \u003cli\u003eGroves, M.D., et al., Phase II trial of temozolomide plus the matrix metalloproteinase inhibitor, marimastat, in recurrent and progressive glioblastoma multiforme. Journal of Clinical Oncology, 2002. 20(5): p. 1383-1388.\u003c/li\u003e\n \u003cli\u003eJiguet-Jiglaire, C., et al., Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study. Acta Neuropathol Commun, 2022. 10(1): p. 1.\u003c/li\u003e\n \u003cli\u003eChoe, G., et al., Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype. Clinical Cancer Research, 2002. 8(9): p. 2894-2901.\u003c/li\u003e\n \u003cli\u003eLi, Q., et al., Comparative analysis of matrix metalloproteinase family members reveals that MMP9 predicts survival and response to temozolomide in patients with primary glioblastoma. PLoS One, 2016. 11(3): p. e0151815.\u003c/li\u003e\n \u003cli\u003eXue, Q., et al., High expression of MMP9 in glioma affects cell proliferation and is associated with patient survival rates. Oncology letters, 2017. 13(3): p. 1325-1330.\u003c/li\u003e\n \u003cli\u003eWu, Z., et al., Matrix metalloproteinase 9 expression and glioblastoma survival prediction using machine learning on digital pathological images. Scientific Reports, 2024. 14(1): p. 15065.\u003c/li\u003e\n \u003cli\u003eWu, Z.S., et al., Prognostic significance of MMP‐9 and TIMP‐1 serum and tissue expression in breast cancer. International journal of cancer, 2008. 122(9): p. 2050-2056.\u003c/li\u003e\n \u003cli\u003eLee, J.-H., J.-W. Choi, and Y.-S. Kim, Serum TIMP-1 predicts survival outcomes of invasive breast carcinoma patients: a meta-analysis. Archives of Medical Research, 2011. 42(6): p. 463-468.\u003c/li\u003e\n \u003cli\u003eNikkola, J., et al., High serum levels of matrix metalloproteinase-9 and matrix metalloproteinase-1 are associated with rapid progression in patients with metastatic melanoma. Clinical Cancer Research, 2005. 11(14): p. 5158-5166.\u003c/li\u003e\n \u003cli\u003eSmith, E.R., et al., Urinary biomarkers predict brain tumor presence and response to therapy. Clinical cancer research, 2008. 14(8): p. 2378-2386.\u003c/li\u003e\n \u003cli\u003eLoo, H.K., et al., Circulating biomarkers for high-grade glioma. 2019, Taylor \u0026amp; Francis. p. 161-165.\u003c/li\u003e\n \u003cli\u003eFriedberg, M.H., et al., Specific matrix metalloproteinase profiles in the cerebrospinal fluid correlated with the presence of malignant astrocytomas, brain metastases, and carcinomatous meningitis. Cancer: Interdisciplinary International Journal of the American Cancer Society, 1998. 82(5): p. 923-930.\u003c/li\u003e\n \u003cli\u003eRoy, R., J. Yang, and M.A. Moses, Matrix metalloproteinases as novel biomarker s and potential therapeutic targets in human cancer. Journal of clinical oncology, 2009. 27(31): p. 5287-5297.\u003c/li\u003e\n \u003cli\u003eTabouret, E., et al., MMP2 and MMP9 serum levels are associated with favorable outcome in patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant chemotherapy in the BEVERLY-2 study. Oncotarget, 2016. 7(14): p. 18531.\u003c/li\u003e\n \u003cli\u003eSkerenova, M., et al., Circulating tumor cells and serum levels of MMP-2, MMP-9 and VEGF as markers of the metastatic process in patients with high risk of metastatic progression. 2017.\u003c/li\u003e\n \u003cli\u003eIwamoto, F.M., et al., Longitudinal prospective study of matrix metalloproteinase-9 as a serum marker in gliomas. Journal of neuro-oncology, 2011. 105: p. 607-612.\u003c/li\u003e\n \u003cli\u003eRoy, R., et al., Metalloproteinases and their roles in human cancer. The Anatomical Record, 2020. 303(6): p. 1557-1572.\u003c/li\u003e\n \u003cli\u003eHormigo, A.l., et al., YKL-40 and matrix metalloproteinase-9 as potential serum biomarkers for patients with high-grade gliomas. Clinical Cancer Research, 2006. 12(19): p. 5698-5704.\u003c/li\u003e\n \u003cli\u003eYi, L., et al., Plasma levels of tissue inhibitor of matrix metalloproteinase-1 correlate with diagnosis and prognosis of glioma patients. Chinese medical journal, 2013. 126(22): p. 4295-4300.\u003c/li\u003e\n \u003cli\u003eTabouret, E., et al., Association of matrix metalloproteinase 2 plasma level with response and survival in patients treated with bevacizumab for recurrent high-grade glioma. Neuro-oncology, 2014. 16(3): p. 392-399.\u003c/li\u003e\n \u003cli\u003eCrocker, M., et al., Serum angiogenic profile of patients with glioblastoma identifies distinct tumor subtypes and shows that TIMP-1 is a prognostic factor. Neuro-oncology, 2010. 13(1): p. 99-108.\u003c/li\u003e\n \u003cli\u003eLiu, Y., et al., Expression of VEGF and MMP-9 and MRI imaging changes in cerebral glioma. Oncology Letters, 2011. 2(6): p. 1171-1175.\u003c/li\u003e\n \u003cli\u003eFan, X., et al., Regional specificity of matrix metalloproteinase-9 expression in the brain: voxel-level mapping in primary glioblastomas. Clinical Radiology, 2018. 73(3): p. 283-289.\u003c/li\u003e\n \u003cli\u003eMeyer, H.J., et al., Perifocal edema volume is not associated with immunohistochemical features reflecting proliferation potential, microvessel density, neoangiogenesis and invasiveness in brain metastasis. Clin Neurol Neurosurg, 2021. 202: p. 106537.\u003c/li\u003e\n \u003cli\u003eLiu, W. and Z. Li, Diagnostic performance of perfusion-weighted imaging combined with serum MMP-2 and -9 levels in tumor recurrence after postoperative concomitant chemoradiotherapy of glioblastoma. J Clin Ultrasound, 2023. 51(3): p. 563-570.\u003c/li\u003e\n \u003cli\u003eKaisman-Elbaz, T., et al., Cell death induced by zinc and cadmium is mediated by clusterin in cultured mouse seminiferous tubules. Journal of Cellular Physiology, 2009. 220(1): p. 222-229.\u003c/li\u003e\n \u003cli\u003eStupp, R., et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England journal of medicine, 2005. 352(10): p. 987-996.\u003c/li\u003e\n \u003cli\u003eKaisman-Elbaz, T., et al., Hemoglobin levels and red blood cells distribution width highlights glioblastoma patients subgroup with improved median overall survival. Frontiers in Oncology, 2020. 10: p. 432.\u003c/li\u003e\n \u003cli\u003eBerghoff, A.S., et al., Combining standard clinical blood values for improving survival prediction in patients with newly diagnosed brain metastases\u0026mdash;development and validation of the LabBM score. Neuro-oncology, 2017. 19(9): p. 1255-1262.\u003c/li\u003e\n \u003cli\u003eZhang, Q.-W., et al., Matrix metalloproteinase-9 as a prognostic factor in gastric cancer: a meta-analysis. Asian Pacific Journal of Cancer Prevention, 2012. 13(6): p. 2903-2908.\u003c/li\u003e\n \u003cli\u003eGong, L., et al., Prognostic impact of serum and tissue MMP-9 in non-small cell lung cancer: a systematic review and meta-analysis. Oncotarget, 2016. 7(14): p. 18458.\u003c/li\u003e\n \u003cli\u003eThanh, H.D., et al., Temozolomide promotes matrix metalloproteinase 9 expression through p38 MAPK and JNK pathways in glioblastoma cells. Scientific Reports, 2024. 14(1): p. 14341.\u003c/li\u003e\n \u003cli\u003eDobra, G., et al., MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. Cancers (Basel), 2023. 15(3).\u003c/li\u003e\n \u003cli\u003eLinhares, P., et al., Glioblastoma: Is There Any Blood Biomarker with True Clinical Relevance? Int J Mol Sci, 2020. 21(16).\u003c/li\u003e\n \u003cli\u003eFontanilles, M., et al., Metabolic remodeling in glioblastoma: a longitudinal multi-omics study. Acta Neuropathol Commun, 2024. 12(1): p. 162.\u003c/li\u003e\n \u003cli\u003eHotchkiss, K.M., et al., A brave new framework for glioma drug development. Lancet Oncol, 2024. 25(10): p. e512-e519.\u003c/li\u003e\n \u003cli\u003eZhou, W., et al., Increased expression of MMP-2 and MMP-9 indicates poor prognosis in glioma recurrence. Biomedicine \u0026amp; Pharmacotherapy, 2019. 118: p. 109369.\u003c/li\u003e\n \u003cli\u003eSchuler, P.J., et al., Urokinase plasminogen activator, uPAR, MMP-2, and MMP-9 in the C6-glioblastoma rat model. in vivo, 2012. 26(4): p. 571-576.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5467623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5467623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Matrix metalloproteinase 9 (MMP-9) has been shown to induce glioblastoma invasion and brain metastases (BM) spread. However, its clinical significance for monitoring disease progression has yet to be established. This study evaluates intra-tumoral and sera MMP-9 levels and their correlation to glioblastoma and BM patients' overall survival (OS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e69 tumor and pre-operative sera samples were obtained from the brain tumor bank of the neurosurgery department at Soroka University Medical Center from patients who underwent tumor resection between 2015 and 2021.\u003cstrong\u003e \u003c/strong\u003eClinical and imaging data from 27 glioblastoma and 30 BM patients were analyzed, and their MMP-9 levels and activity were measured and compared with 12 meningioma patients and 23 healthy subjects. Survival analyses were performed to examine MMP-9 level, activity, and clinical parameters' correlation with patients' OS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eGlioblastoma and BM patients demonstrated increased median intra-tumoral MMP-9 levels (8ng/ml and 4ng/ml, respectively, p\u0026lt;0.001), activity, and pre-operative sera levels (2.8ng/ml and 1.8ng/ml, respectively, p\u0026lt;0.001). MMP-9 was specifically detected within and between glioblastoma cells and tumor endothelia. High intra-tumoral and sera MMP-9 levels, but not its activity, were linked to decreased OS in glioblastoma and BM patients (15.8 versus 8.4 months, p=0.022). MMP-9 was readily measured in patient sera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study suggests that intra-tumoral and sera MMP-9 can assist in identifying glioblastoma and BM recurrence/progression and that high intra-tumoral and/or sera MMP-9 levels at diagnosis correlate with significantly shorter patient OS. Importantly, sera MMP-9 could be longitudinally and non-invasively monitored in those patients and, once rising, may indicate tumor progression.\u003c/p\u003e","manuscriptTitle":"High Intra-tumoral and Sera Matrix Metalloproteinase 9 Levels Reduce Glioblastoma and Brain Metastases Patients' Survival","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-24 16:52:26","doi":"10.21203/rs.3.rs-5467623/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c93d96c2-8011-4601-a2ed-fc56c06af456","owner":[],"postedDate":"December 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-24T16:52:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-24 16:52:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5467623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5467623","identity":"rs-5467623","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}