Biomarkers are measurable changes associated with physiological or pathophysiological processes. Urine accumulates systematic changes in the body and thus is an ideal sample source for biomarker research. In this study, we performed urinary biomarker discovery in glioma patients using LFQ and PRM-based targeted proteomics quantification. Our results showed that brain disorders could be reflected in the urine and that urine proteins could be used for glioma detection.
In the biomarker discovery phase, the levels of 27 urinary proteins were significantly changed after brain tumor removal. Of these DEPs, several have been reported to be associated with gliomas in previous studies, such as ANXA7, CALR, CD276, PIGR, CTSD, CRYAB, ASAH1, BGLR, and CEACAM1. For example, ANXA7 is a tumor suppressor protein. The expression of this protein is decreased in glioma tissues, and its degradation may contribute to glioma progression . Moreover, loss of ANXA7 is associated with prognosis in glioblastoma patients, and ANXA7 is a strong predictor of patient outcome [33, 34]. Additionally, lower CALR levels have been observed in glioma tissues than in normal brain tissues, and CALR expression is correlated with glioma grade and patient survival . CD276 antigen has been reported to be upregulated in high-grade glioma. The expression of this protein correlates with malignancy grade in gliomas and with poor patient survival [22, 35]. Additionally, expression of PIGR has been identified as a novel predictor of poor glioma patient prognosis after surgical resection . CTSD has been identified as an important protein related to glioma invasion . Biomarkers can be used to predict the prognosis and evaluate the treatment response of glioma patients . These DEPs have potential clinical applications for aiding in the initial diagnosis of gliomas, early recognition of tumor recurrence and monitoring of treatment efficacy. As urinary proteome could be affected by some factors, such as gender, aging, exercise, and smoking. Therefore, the self-controlled method is suitable for clinical study and meets the requirements of urine biomarker research to remove interference.
After functional analysis of DEPs identified by LFQ, several important biological processes were significantly enriched, such as autophagy and angiogenesis. Autophagy is involved in tumorigenesis, and it is reportedly enhanced in glioma compared to normal brain tissue . Moreover, blockade of autophagy has been proposed as an alternative therapeutic option for gliomas. In our study, five differential proteins involved in the autophagy process were significantly changed, including CTSD, NAPSA, LAMP1, LAMP2, and ANXA7. In addition, gliomas are characterized by abundant angiogenesis, and glioma progression is accompanied by extensive neovascularization . Antiangiogenic therapy is also a promising approach to treat glioma and several anti-angiogenic agents have been used for target treatment of glioma [38, 39]. Several of the DEPs identified in this study are involved in angiogenesis, such as CEACAM1, AMPE, AMPN, and PCP. Thus, these proteins might be potential therapeutic targets for gliomas.
In the biomarker validation phase, some promising proteins were used for PRM-based targeted quantification. It was observed that urinary AACT and GELS were significantly upregulated in glioma patients compared to healthy controls, whereas urinary TSP4, MDHM, RINI, CALR, TENA, LEG1, and AHSG were significantly downregulated in glioma patients compared to healthy controls. Interestingly, AACT, GELS, TENA, LEG1, and AHSG had been reported as potential CSF or blood biomarkers of gliomas [40,41,42,43,44], which indicates the validity of our results. Moreover, we found these proteins could also serve as urinary biomarkers for noninvasive detection of gliomas. To obtain the best diagnostic performance, we plotted the ROC curves of different protein combinations. ROC curve analysis showed that the combination of six urinary proteins (AACT, TSP4, MDHM, CALR, LEG1, and AHSG) can effectively discriminate the gliomas patients from healthy controls with the AUC of 0.952. Interestingly, AACT, LEG1, and AHSG are blood or CSF biomarkers of gliomas [40, 43, 44], and CALR is correlated with glioma grade and patient survival .
Compared to other bodily fluids (CSF and blood), urine has been largely ignored during biomarker discovery for brain diseases. The reason may be that urine is located far from the brain, which could raise doubt as to whether pathological changes in the brains will be detected in the urine. Moreover, investigations of gliomas by using urinary proteomics have been rarely reported. In this study, using LFQ and PRM proteome quantification, we identified candidate urinary protein biomarkers with the potential to detect gliomas. Our results also suggested that brain disorders could be reflected in human urine and that urine proteins could be used to distinguish different brain diseases. However, a larger number of clinical urine samples from multi-center patients are needed to verify the specific protein pattern as biomarkers for gliomas detection.
As urine can be collected noninvasively, it is easy to repeatedly collect urine sample from the same individual for longitudinal studies. Thus, in future studies, urine samples can be collected at multiple time points for early diagnosis, early recognition of tumor recurrence, or monitoring of therapeutic efficacy in glioma patients.