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Altered CSF Proteomic Profiling of Paediatric Acute Lymphocytic Leukemia Patients with CNS Infiltration

Altered CSF Proteomic Profiling of Paediatric Acute Lymphocytic Leukemia Patients with CNS... Hindawi Journal of Oncology Volume 2019, Article ID 3283629, 8 pages https://doi.org/10.1155/2019/3283629 Research Article Altered CSF Proteomic Profiling of Paediatric Acute Lymphocytic Leukemia Patients with CNS Infiltration Fei Mo, Xuelei Ma , Xiaobei Liu, Ruofan Zhou, Yunuo Zhao, and Hui Zhou Department of Biotherapy, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, China Correspondence should be addressed to Xuelei Ma; drmaxuelei@gmail.com Received 5 January 2019; Accepted 9 April 2019; Published 2 May 2019 Academic Editor: om Th as E. Adrian Copyright © 2019 Fei Mo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. For childhood acute lymphocytic leukemia (ALL), central nervous system leukemia (CNSL) is still the main reason of treatment failure. Changes of cerebrospinal uid fl (CSF) proteome are deemed to occur aer ft intrathecal chemotherapy. Objective. To find critical CSF biomarkers, which could be utilized to increase diagnostic and prognostic accuracy of CNSL. Methods.We performed proteomic profiling of CSF before and after the treatment of six sporadic paediatric patients diagnosed as ALL with central nervous system (CNS) involvement. CSF samples were properly processed and analyzed through the use of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results. Among identified 428 unique proteins in all CSF samples, we quantified 10 altered proteins with diverse biological functions aer ft induction chemotherapy. Conclusions.The levels of those 10 proteins change during the treatment of CNSL. Some of the proteins are likely to play a vital biological role as biomarkers for the development of ALL. In addition, our results indicated the feasible and reproducible utility of CSF for diagnosis and prognosis of patients with CNSL. 1. Introduction of a wide range of diseases, especially nervous system-relative disorders [8], such as Alzheimer, disseminated sclerosis ALL is the most frequent malignancy in children and the Parkinson, chronic nervous headache, acute brain injury, peak incidence pertains to 1–5 years age cohort [1]. Recently, and mental disorders [9–12]. Increased evidence showed that a study showed that during 2010–2014 in 14 countries, the the proteomic analysis of CSF could also provide candidate lowest rate of 5-year survival of paediatric patients with ALL protein biomarkers for brain tumours [13], especially glioma was of Ecuadorians (49.80%), and the highest was of Finns [14]. Unfortunately, few researches aimed at finding the CSF (95.20%), the range of which was elevated 10% or more proteome change of hematological malignancies [15]. There- compared with that in 1995 [2]. Paediatric ALL has been fore, we undertook a research focusing on the quantitative considered to be potentially curable as a result of the rapidly proteomics of CSF in patients with nasal-type of extranodal improved cytogenetic, molecular, and immunophenotyping natural killer cell/T-cell lymphoma (NKTCL) [16]. In the stratification of leukemic blasts and risk-directed treatment present study, we performed a high-throughput quantitative [3–5]. However, CNS relapse still occurs in 3–8% of the chil- CSF proteomic analysis of patients with CNSL before and dren with ALL and it is a major factor causing death related to aer ft conventional treatment by label-free LC-MS/MS. We cancer of children over 5. Moreover, patients with CNSL are found that the levels of ten proteins with different locations apt to have a relatively poor outcome compared with CNS- and functions significantly changed during the induction negative patients [6]. Therefore, in patients with CNSL, a chemotherapy for ALL. These proteins are associated with more sensitive and noninvasive diagnosis method and new inflammatory processes and tumour development in varying biomarkers reefl cting treatment response and prognosis are degrees. u Th s, this work may provide useful information in urgent need [7]. for molecular mechanisms of ALL development, as well as To date, CSF proteomics has rapidly developed and predicting clinical prognosis of patients with ALL on high become a new method of diagnosis, treatment, and prognosis risk of CNS infiltration. 2 Journal of Oncology 2. Materials and Methods perform separations, and then the trap column was switched with an analytical column of 75𝜇 mx12cm (200A, 5𝜇 m; .. Participants and CSF Collection. Eligible patients, aged 1- Michrom Bioresources). 11 years, had confirmed B-lineage ALL with CNS involvement A 3 min column washing was conducted with mobile when the white blood cells were 1-5/𝜇 L with detectable phase A (2.0% ACN, 0.1% FA). The 60-min liquid-chroma- leukemic blasts in CSF. These patients received additional tography (LC) gradient was used with a rising percentage intrathecal chemotherapy (ITC) involving methotrexate, of bueff r B (95.0% ACN, 0.1% FA) from initially 4% to 90% cytarabine, and dexamethasone weekly during induction for peptide elution purposes (flow rate: 300 nL/min). Data (comprised prednisolone, daunorubicin, vincristine, and were collected by positive ion mode. MS spectra selection pegaspargase according to the protocols of CCCG-ALL- scan range was m/z 375 – 1800 (resolution: 70,000, m/z: 200). 2015). CSF samples were obtained by lumbar puncture from The operation of the LTQ-Orbitrap was n fi ished in data- subjects at Pediatric Hematology of West China Second dependent acquisition mode. The value of automatic gain University Hospital, Sichuan University. The CSF samples control (AGC) was set at 3e6. The top 15 most intense parent collected before the treatment and aeft r achieving a complete ions (number: 20) were selected and the fragmentation was response were divided into two groups: PRE-CSF and POST- performed in HCD collision cell (the set mass resolution: CSF, respectively. After standard checks in laboratory, all CSF 35000). For the purpose of preventing repetitive selection of samples were spun at 3000 rpm under the temperature of peptide, a 30 s’ dynamic exclusion was applied. Collection 4 C for 10 min to remove any cellular debris and were then of the raw files was acquired using Q-Exactive, after which stored in aliquots under the temperature of−80 Caeft r 2h they were analyzed by Maxquant v1.3. The searching was of harvesting. The Medical Ethics Committee belonging to accomplished according to Swiss-Prot human database. To Sichuan University’s West China Hospital confirms the study. ensure the confidence in these listed proteins, identification Also, the informed consents had been signed by all these of peptides was made with at least 1 unique peptide with patients before they hospitalized. a peptide false discovery rate<1% on the basis of Peptide Prophet Algorithm in Elucidator. Five missed trypsin cleav- .. CSF Sample Preparation. Aliquots of CSF were dissolved ages were allowed; cysteine carbamido methylation was set as at room temperature. Also, the dilution of a xe fi d amount a xfi ed modification in this search. of CSF (30𝜇 L) was lysed in RIPA bueff r (150mm NaCl, 50 mm Tris-HCl (pH 7.61), NP-40, 1% deoxycholic acid) with .. Proteomics Enrichment Analysis. After filtering the data phosphatase and protease inhibitors for 20 min on ice. After set, enrichment analysis of the CSF proteome was taken in 10 min’s centrifugation at 13000 x g under the temperature of Gene Ontology (GO). Then, the identified proteins’ Kyoto 4 C, determination of the supernatant protein concentrations Encyclopedia of Genes and Genomes (KEGG) pathways were was finished by BCA assay. The buffer containing 100 mM carried out using David 6.8 (https://david-d.ncifcrf.gov/). NH HCO was added to obtain the 50 mM NH HCO ’s final Signicfi antly altered-protein expression prolfi es were present 4 3 4 3 protein concentration, making an alkaline environment for by heatmap in Mev software. Finally, we took visualized the trypsin digestion. Then the samples were reduced with a known STRING actions (https://string-db.org/) for signifi- n fi al concentration of 5 mM DL-Dithiothreitol (DTT) for 1 h cantly changed CSF proteins [17]. under the temperature of 56 C. For the purpose of alkylating the cysteines, 55 mM iodoacetamide (IAA) was added to .. Western Blot. CSF samples were diluted in 5x SDS- reach the concentration of 15 mM, which then reacted in PAGE sample loading buffer (Beyotime) aer ft determining darkness under room temperature for 30 min. Then 30 mM the protein concentration and heated at 95 Cfor 10 minutes. L-cysteine was added to block redundant IAA, and digestion Fifty micrograms of total proteins from each sample were of protein samples was finished with trypsin (Promega) at loaded into each well, then separated on 12.5% SDS-PAGE gel a ratio of 50:1 (protein to trypsin) under the temperature of (Bio-Rad) and transferred to the polyvinylidene diu fl oride 37 C overnight under pH 8.0. Finally, the samples were heated membrane (Millipore). Aeft r incubation with blocking solu- to the temperature of 95 C for 10 min to stop the reaction. tion (containing 5% milk) for 120 min at room temperature, Before LC−MS/MS analysis, desalination of the peptides was membranes were incubated with the following primary anti- n fi ished by C18 ZipTip (Millopore, ZTC18S096). bodies overnight at 4 C: rabbit-anti-HRG (Abcam), rabbit- anti-SPARC (Abcam). Then, membranes were washed three .. Liquid Chromatography-Tandem Mass Spectrum Analysis. times and incubated with horseradish peroxidase-conjugated During the LC-MS/MS analysis process, the same method secondaryantibody(ThermoFisherScienticfi ) at 37 Cfor 1h. of our previous report was adopted [16]. In brief, all these Chemiluminescent visualization (Clinx Science Instruments) lyophilized peptide digests were resuspended in buffer A was used to visualize the signals. Western blot data were (2.0% ACN, 0.1% FA), after which LC-MS/MS analysis was normalized by Image J software. conducted in triplicate by nanoflow EASY-nLC system, cou- pled online to Q-Exactive quadrupole-orbitrap mass spec- .. Statistical Analysis. Multiple comparisons of PRE-CSF trometer (Thermo Fisher Scientific) with a nanoelectrospray and POST-CSF samples were performed using paired t- ionization source. The determination of run orders for all the test. The analysis of protein proles fi was implemented for samples was made in a random manner. A 100𝜇 mx2cmtrap candidate proteins using univariate analysis. We lfi tered our column (200A, 5𝜇 m; Michrom Bioresources) was used to list of significant altered proteins at p value <0.05. P values Journal of Oncology 3 KEGG PATHWAY Systemic lupus erythematosus Staphylococcus aureus infection Protein digestion and absorption Prion diseases CSF from the cured individuals treatment PPAR signaling pathway −ＦＩＡ (PVale) PI3K-Akt signaling pathway Pertussis Lysosome Centrifugation Supernatants of CSF Glycolysis/Gluconeogenesis Fructose and mannose metabolism CSF from paediatric ALL patients Reductive alkylation Focal adhesion Count ECM-receptor interaction Complement and coagulation cascades Cell adhesion molecules (CAMs) desalting Carbon metabolism Biosynthesis of amino acids Arrhythmogenic right ventricular cardiomyopathy (ARVC) Amoebiasis Bioinformatic analysis LC-MS/MS analysis (triplicate) Trypsin digestion 10 20 Fold Enrichment (a) (b) GO BIOLOGICAL PROCESS GO MOLECULAR FUNCTION Cell adhesion, other, GO CELLULAR COMPLEX 11.14% other 25.30% protein binding other 25% Platelet 36% 27% extracellular Degranulation, exosome 10.84% extracellular 22% receptor matrix binding organization proteolysis 5% , 5.42% serine-type blood , 9.64% endopeptidase microparticle receptor- activity 7% mediated heparin 8% negative endocytosis, binding regulation of 6.33% 5% extracellular endopeptidase calcium ion space complement activity,... binding serine-type 16% activation,. . . endopeptidase inhibitor 8% identical protein plasma extracellular complement activation, activity innate immune binding membrane region classical pathway, 7.83% 5% 8% response, 8.13% 12% 16% (c) (d) (e) Figure 1: The comprehensive proteomics analysis of CSF with CNSL. (a) The experimental work flow. The process of sample preparation, data acquisition, and analysis is shown in the diagram. ALL, leukeamia. (b) In-depth KEGG-pathway analysis of all identified proteins in PRE-CSF samples. (c), (d), and (e) Gene Ontology (GO) analysis of all identified proteins expressed in PRE-CSF samples. All identified proteins were put into the three GO domains (BP, MF, and CC). The names and percentages of term are located next to their position on the charts. were calculated in accordance with Mann–Whitney U test for domain was demonstrated with all expressed proteins. There nonparametric data, or the two-tailed t-test for parametric was no substantial difference among the three subcategories data. The data were shown as means ± standard deviations proportions between pretreated and posttreated groups of (SD). patients with ALL (Supplementary Figure S1). The analysis in BP ontology showed significant protein enrichment in the cell adhesion, platelet degranulation, and proteolysis 3. Results (Figure 1(c)). MF analysis indicated that most of the identified proteins were noted as protein binding and calcium ion .. Global CSF Protein Analysis. Ultimately, six eligible binding as well as serine−type endopeptidase inhibitor activ- patients were included in our study. The demonstration of ity (Figure 1(d)). Analysis of CC ontology mainly pointed our experimental workow fl was presented in Figure 1(a). To investigate the proteomic profile of CSF in patients towards extracellular exosome, extracellular space, and extra- cellular region (Figure 1(e)). with CNSL, we collected PRE- and POST-CSF samples and used the same volume of samples for subsequent laboratory .. Label-Free Quantification of Relevant Protein Abundance. tests and analysis. Within these pooled CSF samples, an average of 428 unique proteins of all PRE-CSF and POST- Aiming at identifying differentially expressed proteins in CSF samples was identified by measuring peptides signal these PRE-CSF and POST-CSF samples, we performed a intensity for protein abundance (Supplementary Table S1). strict t-test difference criteria (Method section) and several During the treatment, the following pathway was revealed statistically significant proteins were identified (Table 1). The activated by depth KEGG-pathway analysis: complement expression of all these proteins was presented in Figure 2(a). and coagulation cascades, related to the usage of antitumor Our label-free quantification demonstrated that the follow- drugs during induction therapy to ALL [18] (Figure 1(b)). ing seven proteins were upregulated: histidine rich gly- Next, we grouped those proteins according to annotations in coprotein (HRG), kallikrein related peptidase 6 (KLK6), Gene Ontology (GO) domains, including ‘Biological Process carnosine dipeptidase 1 (CNDP1), alpha-2-macroglobulin (BP)’, ‘Molecular Function (MF)’, and ‘Cellular Complex (A2M), complement factor H (CFH), complement C4A (CC)’, to determine the constitutions of the CSF proteome. (C4A), and apolipoprotein A1 (APOA1). On the contrary, Then, the percentage representation of each GO subcategory WAP, follistatin/kazal, immunoglobulin, kunitz and netrin 4 Journal of Oncology Table 1: Statistically significant increased or decreased proteins in ALL samples during induction with P <0.05. Gene name Protein ID Change trend P value Protein complement C4A (Rodgers blood group) C4A P0C0L4 ↑ 0.011 HRG P04196 ↑ 0.016 Histidine rich glycoprotein APOA1 P02647 ↑ 0.025 apolipoprotein A1 KLK6 Q92876 ↑ 0.025 kallikrein related peptidase 6 carnosine dipeptidase 1 CNDP1 Q96KN2 ↑ 0.026 CFH P08603 ↑ 0.029 complement factor H A2M P01023 ↑ 0.05 alpha-2-macroglobulin apolipoprotein D APOD P05090 ↓ 0.018 WAP, follistatin/kazal, immunoglobulin, WFIKKN2 Q8TEU8 ↓ 0.024 kunitz and netrin domain containing 2 SPARC P09486 ↓ 0.037 secreted protein acidic and cysteine rich APOD -2.38 SPARC WFIKKN2 -1.18 HRG A2M 0.02 APOA1 CFH 1.22 KLK6 C4A 2.42 CNDP1 (a) CNDP1 SPARC A2M KLK6 WFIKKN2 APOA1 APOD C2A CFH (b) Figure 2: Quantification of changed CSF proteins during the treatment of CNSL. (a) Heatmap analysis of deproteins in PRE-CSF and POST- CSF group: unsupervised clustering analysis showing expression profiles of DE proteins with Euclidean Distance of proteins. (b) eTh altered- protein interactions generated by STRING analysis. Required confidence (score): medium confidence (0.400). Edge width reflects the strength of the STRING action score and the edge length is arbitrary. domain including 2 (WFIKKN2), secreted protein acidic as interaction among these 10 proteins is showed in Figure 2(b). well as cysteine rich (SPARC), apolipoprotein D (APOD) Therefore, these proteins are possibly related to ALL devel- were significantly downregulated. In addition, we used west- opment directly or indirectly, and their changes during the ern blotting technique to validate the LC-MS/MS results, treatment might be used as an indication of the better demonstrated in Supplementary Figure S2. HRG and SPARC therapeutic effects in CNS. were selected for the confirmation of the low intensity and potential applied value as the biomarkers of ALL. Consistent 4. Discussion with our LC-MS/MS results, the content of SPARC was obviously decreased (p=0.044) and HRG had the trend of In this research, the label-free LC−MS/MS was adopted increasing (p=0.072) aer ft treatment of ALL. The protein for constructing overall CSF proteomic proles fi of pediatric PRE-CSF 1 PRE-CSF 2 PRE-CSF 3 PRE-CSF 4 PRE-CSF 5 PRE-CSF 6 POST-CSF 1 POST-CSF 2 POST-CSF 3 POST-CSF 4 POST-CSF 5 POST-CSF 6 Gene Expression Journal of Oncology 5 CNSL samples. Strong concordance could be seen in the of TGF𝛽 family members to the receptor to regulate the bal- proportion of identified proteins assigned to BP, MF, and CC anced relation between the activation of non-Smad and Smad of GO domains between PRE and POST-CSF samples from pathways of TGF𝛽 [34]. As a 33-kDa glycoprotein component of high-density lipoprotein in plasma, Apo D is observably our data, demonstrating that the overall composition of CSF proteome is relatively stable [16]. In addition, the different expressed by glial cells in brain [35]. It is essential for the points in relative levels of CSF proteins were detected in development and repair of nervous system as an important regulator of lipid trafficking and conferring protection from CNSL patients during induction. As subclinical CNS mani- festation is in most patients, CNS-directed therapy is critical cell oxidative stress [36, 37]. Apo D could also act as a for increasing leukemia-free survival rates, even in nonde- nonspecific stress protein leading to cell growth arrest, and tectable CNSL pediatric patients [19]. Intrathecal chemother- it is upregulated under several pathological situations such as apy (ITC) with methotrexate or cytarabine is commonly cancers and neurodegenerative diseases [38, 39]. Increased levels of C4A and CFH were involved in com- used to prevent or treat CNSL. However, various adverse events are likely to occur, including severe gastrointestinal, plement and coagulation cascades pathway [40]. Further- hepatic and neurological toxicities, leukoencephalopathy, more, CFH inhibits endothelial cell migration and angio- genesis by indirectly regulating angiogenic effectors of com- and polyradiculopathy [20, 21]. Therefore, improving therapy efficacy and reducing toxicity are the current focus of ALL plement components, including C3a and C5a, leading to management [22]. CSF is proximate to CNS tissue microen- lower vascular endothelial growth factor (VEGF) expression and transformation of growth factor-𝛽 (TGF-𝛽 )[41–43]. vironment and has relative simplex protein contents; thus, it is preferentially considered as an ideal biomarker source The upregulated expression of CFH might be beneficial to for neurological disorders [23]. By analyzing the changes of confront VEGF produced by ALL cells, as VEGF has been expressed proteins during ITC in patients with CNSL, we proved to promote leukemia cell infiltration into CNS [36]. could find new markers to develop alternative CNSL-related As a 720-kDa glycoprotein, A2M is mainly produced by liver Kupffer cells. A2M is also involved in inflammatory reaction diagnosis, prognosis, and treatment strategies. In this work, we found the levels of three proteins as a proteinase inhibitor and carrier for interleukin-6 (IL- (SPARC, WFIKKN2, andAPOD) decreasedduringCNSL 6) [37]. It has been shown to decrease in some pathological conditions such as hematologic malignancy and advanced treatment. SPARC is a 32 kDa secreted glycoprotein that regulates interaction of cell-extracellular matrix proteins to prostate cancer [44, 45]. It was demonstrated previously promote cell adhesion and migration [24]. It is also reported that the interaction of it with the lipoprotein receptor- to be important for tissue remodelling, angiogenesis, and related protein-1(LRP1) of low-density led to an inhibi- tumourigenesis through activation of growth factors and tion of tumour cell proliferation, migration, and invasion matrix metalloproteinases [25–28]. Increased expression of in astrocytoma malignancy [46]. HRG is an 75 kDa a2- SPARC has been found to play a protumorigenic and glycoprotein produced by liver, which keeps high levels in prometastatic role to many malignant tumours, such as brain, plasma and acts as an acute phase reactant [47]. HRG is involved in numerous biologic processes, such as cell adhe- lung, breast, pancreas, skin, and kidney [29]. Another study showed that the downregulated or absent expression of sion and proliferation, angiogenesis as well as coagulation SPARC is related to high-speed progression of human colon and platelet activation [48]. It is also reported that HRG inhibits tumour growth and vascularization, remodels tran- cancer [30]. us, Th SPARC may have complex function in the development of human solid tumours. However, there are sition from epithelial to mesenchymal, and regulates the few studies discussing the role of SPARC in the formation of formation of protumorigenic microenvironment by skewing hematological malignances. Recently, accumulating evidence tumour-associated macrophages polarization from the M2- suggested that SPARC overexpression is associated with poor like towards M1-like phenotype [49–51]. Besides, HRG pro- outcome in acute myeloid leukemia (AML) patients and motes antitumor immune responses and vessel normalization promotes the growth of aggressive leukemia cell in murine to enhance chemotherapy. KLK6, a member of the secreted models. In addition, knockdown of SPARC could inhibit serine protease family of protein-cleaving enzymes, is also defined as an epigenetically regulated tumour suppressor AML cell proliferation through inducing cell cycle arrest at Phase G1/G0 and inhibit active p53-induced apoptosis [52]. KLK6 expression resulted in significant downregulation through raising the levels of p53, caspase-9, caspase-3, and of vimentin, a critical marker of epithelial-to-mesenchymal transition of tumour cells [53]. APOA1, the major protein Fas [24, 31]. As a marker of poor prognosis and a potential therapeutic target of AML, a similar mechanism may be component of high-density lipoproteins (HDL) particles in helpful for inhibiting ALL development, and the decreased plasma, suppressed neutrophil recruitment in inflammation level of SPARC may account for a better prognosis of CNSL, to avoid excessive inflammatory response [54]. Furthermore, since the levels of SPARC significantly declined in subjects it is a potential antiangiogenic factor to suppress tumour when they CR was achieved in our study. WFIKKN2 is a large progression by targeting c-Src/ERK signaling pathways [55, extracellular multidomain protein, containing a WAP, an 56]. Carnosinase 1(CNDP1), primarily synthesized in brain, immunoglobulin, a follistatin domain (binds mature growth secreted into CSF, and finally secreted into the blood, is likely to be important in the maintenance and bioavailabil- factor), an NTR domain, and two Kunitz-type protease inhibitor domains [32]. It is known to positively activate ity of carnosine, a drug for tumours. It could be verified PI3K pathway, which could regulate cancer cell growth and that CNDP1 has antiproliferative properties on different malignant cells for its therapeutic potential against tumours progression [33]. Moreover, it can promote the combination 6 Journal of Oncology including glioblastoma and metastatic prostate cancer. Its MF: Molecular Function deficiency has been related to many kinds of neurologic NKTCL: Natural killer cell/T-cell lymphoma deficits and cancer cachexia [57–59]. Several statistically POST-CSF: Aeft r achieving a complete response altered proteins have been previously proved to play a role PRE-CSF: Before the treatment of regulation in malignant cells or microenvironment of SD: Standard deviations cancers, which might be related to the molecular mechanisms SPARC: Secreted protein acidic and cysteine rich of ALL development and prognosis of patients with CNSL. TGF-𝛽 : Transforming growth factor-𝛽 Collectively, we constructed CSF’s label-free LC−MS/MS VEGF: Vascular endothelial growth factor proteomic profiles in patients suffering from CNSL, which WAP: Whey acidic protein helpsusidentify valuable biomarkers to understand the WFIKKN2: WAP, follistatin/kazal, immunoglobulin, development of ALL with CNS infiltration. It is noteworthy kunitz and netrin domain containing 2. that those altered proteins should be furtherly investigated as predictive markers of ALL patients with CNS infiltration, Data Availability some of which may have a prospect of becoming the new therapeutic targets in CNSL. However, this study has some The data from LC-MS/MS analysis used to support the limitations. For example, the size of samples is small since findings of this study are included within the article. only 6 subjects are covered, and the period of observation is relatively short. Therefore, we will enlarge the sample size and prolong the observation time in our next study, hoping to Conflicts of Interest further verify the roles of those altered proteins in paediatric The authors declare that there are no conflicts of interest. patients of ALL with CNS infiltration. Abbreviations Authors’ Contributions A2M: Alpha-2-macroglobulin Fei Mo and Xuelei Ma contributed equally to this work. ACN: Acetonitrile AGC: Automatic gain control Acknowledgments ALL: Acute lymphocytic leuk(a)emia AML: Acute myeloid leuk(a)emia We appreciate the patients and their families for participating APOA1: Apolipoprotein A1 in the study. APOD: Apolipoprotein D BP: Biological Process C4A: Complement C4A Supplementary Materials CC: Cellular Complex Supplementary Figure S1: Gene Ontology (GO) analysis CFH: Complement factor H CID: Collision induced dissociation of all identified proteins expressed in POST-CSF samples. CNDP1: Carnosine dipeptidase 1 Supplementary Figure S2: validation of SPARC and HRG protein peaks by western blot. (A) Immunoblot of SPARC CNS: Central nervous system CNSL: Central nervous system leuk(a)emia and HRG proteins in PRE-CSF and POST-CSF samples. Fiyft micrograms of PRE-CSF (lanes 1–3) or POST-CSF (lanes 4–6) CSF: Cerebrospinal uid fl CSF were subjected to the lanes. (B) and (C) The relative DTT: DL-Dithiothreitol FA: Formic acid quanticfi ation of SPARC and HRG proteins from panels (A) using Image J sowa ft re, respectively. Supplementary Table S1: GO: Gene Ontology an average of 428 unique proteins of all PRE-CSF and POST- HCD: Higher energy Collision induced Dissociation CSF samples was identified by measuring peptides signal intensity for protein abundance. (Supplementary Materials) HDL: High-density lipoproteins HRG: Histidine rich glycoprotein IAA: Iodoacetamide References IL-6: Interleukin-6 IT: Intrathecal therapy [1] S. Chiaretti, A. Vitale, G. 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Altered CSF Proteomic Profiling of Paediatric Acute Lymphocytic Leukemia Patients with CNS Infiltration

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Copyright © 2019 Fei Mo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2019, Article ID 3283629, 8 pages https://doi.org/10.1155/2019/3283629 Research Article Altered CSF Proteomic Profiling of Paediatric Acute Lymphocytic Leukemia Patients with CNS Infiltration Fei Mo, Xuelei Ma , Xiaobei Liu, Ruofan Zhou, Yunuo Zhao, and Hui Zhou Department of Biotherapy, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, China Correspondence should be addressed to Xuelei Ma; drmaxuelei@gmail.com Received 5 January 2019; Accepted 9 April 2019; Published 2 May 2019 Academic Editor: om Th as E. Adrian Copyright © 2019 Fei Mo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. For childhood acute lymphocytic leukemia (ALL), central nervous system leukemia (CNSL) is still the main reason of treatment failure. Changes of cerebrospinal uid fl (CSF) proteome are deemed to occur aer ft intrathecal chemotherapy. Objective. To find critical CSF biomarkers, which could be utilized to increase diagnostic and prognostic accuracy of CNSL. Methods.We performed proteomic profiling of CSF before and after the treatment of six sporadic paediatric patients diagnosed as ALL with central nervous system (CNS) involvement. CSF samples were properly processed and analyzed through the use of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results. Among identified 428 unique proteins in all CSF samples, we quantified 10 altered proteins with diverse biological functions aer ft induction chemotherapy. Conclusions.The levels of those 10 proteins change during the treatment of CNSL. Some of the proteins are likely to play a vital biological role as biomarkers for the development of ALL. In addition, our results indicated the feasible and reproducible utility of CSF for diagnosis and prognosis of patients with CNSL. 1. Introduction of a wide range of diseases, especially nervous system-relative disorders [8], such as Alzheimer, disseminated sclerosis ALL is the most frequent malignancy in children and the Parkinson, chronic nervous headache, acute brain injury, peak incidence pertains to 1–5 years age cohort [1]. Recently, and mental disorders [9–12]. Increased evidence showed that a study showed that during 2010–2014 in 14 countries, the the proteomic analysis of CSF could also provide candidate lowest rate of 5-year survival of paediatric patients with ALL protein biomarkers for brain tumours [13], especially glioma was of Ecuadorians (49.80%), and the highest was of Finns [14]. Unfortunately, few researches aimed at finding the CSF (95.20%), the range of which was elevated 10% or more proteome change of hematological malignancies [15]. There- compared with that in 1995 [2]. Paediatric ALL has been fore, we undertook a research focusing on the quantitative considered to be potentially curable as a result of the rapidly proteomics of CSF in patients with nasal-type of extranodal improved cytogenetic, molecular, and immunophenotyping natural killer cell/T-cell lymphoma (NKTCL) [16]. In the stratification of leukemic blasts and risk-directed treatment present study, we performed a high-throughput quantitative [3–5]. However, CNS relapse still occurs in 3–8% of the chil- CSF proteomic analysis of patients with CNSL before and dren with ALL and it is a major factor causing death related to aer ft conventional treatment by label-free LC-MS/MS. We cancer of children over 5. Moreover, patients with CNSL are found that the levels of ten proteins with different locations apt to have a relatively poor outcome compared with CNS- and functions significantly changed during the induction negative patients [6]. Therefore, in patients with CNSL, a chemotherapy for ALL. These proteins are associated with more sensitive and noninvasive diagnosis method and new inflammatory processes and tumour development in varying biomarkers reefl cting treatment response and prognosis are degrees. u Th s, this work may provide useful information in urgent need [7]. for molecular mechanisms of ALL development, as well as To date, CSF proteomics has rapidly developed and predicting clinical prognosis of patients with ALL on high become a new method of diagnosis, treatment, and prognosis risk of CNS infiltration. 2 Journal of Oncology 2. Materials and Methods perform separations, and then the trap column was switched with an analytical column of 75𝜇 mx12cm (200A, 5𝜇 m; .. Participants and CSF Collection. Eligible patients, aged 1- Michrom Bioresources). 11 years, had confirmed B-lineage ALL with CNS involvement A 3 min column washing was conducted with mobile when the white blood cells were 1-5/𝜇 L with detectable phase A (2.0% ACN, 0.1% FA). The 60-min liquid-chroma- leukemic blasts in CSF. These patients received additional tography (LC) gradient was used with a rising percentage intrathecal chemotherapy (ITC) involving methotrexate, of bueff r B (95.0% ACN, 0.1% FA) from initially 4% to 90% cytarabine, and dexamethasone weekly during induction for peptide elution purposes (flow rate: 300 nL/min). Data (comprised prednisolone, daunorubicin, vincristine, and were collected by positive ion mode. MS spectra selection pegaspargase according to the protocols of CCCG-ALL- scan range was m/z 375 – 1800 (resolution: 70,000, m/z: 200). 2015). CSF samples were obtained by lumbar puncture from The operation of the LTQ-Orbitrap was n fi ished in data- subjects at Pediatric Hematology of West China Second dependent acquisition mode. The value of automatic gain University Hospital, Sichuan University. The CSF samples control (AGC) was set at 3e6. The top 15 most intense parent collected before the treatment and aeft r achieving a complete ions (number: 20) were selected and the fragmentation was response were divided into two groups: PRE-CSF and POST- performed in HCD collision cell (the set mass resolution: CSF, respectively. After standard checks in laboratory, all CSF 35000). For the purpose of preventing repetitive selection of samples were spun at 3000 rpm under the temperature of peptide, a 30 s’ dynamic exclusion was applied. Collection 4 C for 10 min to remove any cellular debris and were then of the raw files was acquired using Q-Exactive, after which stored in aliquots under the temperature of−80 Caeft r 2h they were analyzed by Maxquant v1.3. The searching was of harvesting. The Medical Ethics Committee belonging to accomplished according to Swiss-Prot human database. To Sichuan University’s West China Hospital confirms the study. ensure the confidence in these listed proteins, identification Also, the informed consents had been signed by all these of peptides was made with at least 1 unique peptide with patients before they hospitalized. a peptide false discovery rate<1% on the basis of Peptide Prophet Algorithm in Elucidator. Five missed trypsin cleav- .. CSF Sample Preparation. Aliquots of CSF were dissolved ages were allowed; cysteine carbamido methylation was set as at room temperature. Also, the dilution of a xe fi d amount a xfi ed modification in this search. of CSF (30𝜇 L) was lysed in RIPA bueff r (150mm NaCl, 50 mm Tris-HCl (pH 7.61), NP-40, 1% deoxycholic acid) with .. Proteomics Enrichment Analysis. After filtering the data phosphatase and protease inhibitors for 20 min on ice. After set, enrichment analysis of the CSF proteome was taken in 10 min’s centrifugation at 13000 x g under the temperature of Gene Ontology (GO). Then, the identified proteins’ Kyoto 4 C, determination of the supernatant protein concentrations Encyclopedia of Genes and Genomes (KEGG) pathways were was finished by BCA assay. The buffer containing 100 mM carried out using David 6.8 (https://david-d.ncifcrf.gov/). NH HCO was added to obtain the 50 mM NH HCO ’s final Signicfi antly altered-protein expression prolfi es were present 4 3 4 3 protein concentration, making an alkaline environment for by heatmap in Mev software. Finally, we took visualized the trypsin digestion. Then the samples were reduced with a known STRING actions (https://string-db.org/) for signifi- n fi al concentration of 5 mM DL-Dithiothreitol (DTT) for 1 h cantly changed CSF proteins [17]. under the temperature of 56 C. For the purpose of alkylating the cysteines, 55 mM iodoacetamide (IAA) was added to .. Western Blot. CSF samples were diluted in 5x SDS- reach the concentration of 15 mM, which then reacted in PAGE sample loading buffer (Beyotime) aer ft determining darkness under room temperature for 30 min. Then 30 mM the protein concentration and heated at 95 Cfor 10 minutes. L-cysteine was added to block redundant IAA, and digestion Fifty micrograms of total proteins from each sample were of protein samples was finished with trypsin (Promega) at loaded into each well, then separated on 12.5% SDS-PAGE gel a ratio of 50:1 (protein to trypsin) under the temperature of (Bio-Rad) and transferred to the polyvinylidene diu fl oride 37 C overnight under pH 8.0. Finally, the samples were heated membrane (Millipore). Aeft r incubation with blocking solu- to the temperature of 95 C for 10 min to stop the reaction. tion (containing 5% milk) for 120 min at room temperature, Before LC−MS/MS analysis, desalination of the peptides was membranes were incubated with the following primary anti- n fi ished by C18 ZipTip (Millopore, ZTC18S096). bodies overnight at 4 C: rabbit-anti-HRG (Abcam), rabbit- anti-SPARC (Abcam). Then, membranes were washed three .. Liquid Chromatography-Tandem Mass Spectrum Analysis. times and incubated with horseradish peroxidase-conjugated During the LC-MS/MS analysis process, the same method secondaryantibody(ThermoFisherScienticfi ) at 37 Cfor 1h. of our previous report was adopted [16]. In brief, all these Chemiluminescent visualization (Clinx Science Instruments) lyophilized peptide digests were resuspended in buffer A was used to visualize the signals. Western blot data were (2.0% ACN, 0.1% FA), after which LC-MS/MS analysis was normalized by Image J software. conducted in triplicate by nanoflow EASY-nLC system, cou- pled online to Q-Exactive quadrupole-orbitrap mass spec- .. Statistical Analysis. Multiple comparisons of PRE-CSF trometer (Thermo Fisher Scientific) with a nanoelectrospray and POST-CSF samples were performed using paired t- ionization source. The determination of run orders for all the test. The analysis of protein proles fi was implemented for samples was made in a random manner. A 100𝜇 mx2cmtrap candidate proteins using univariate analysis. We lfi tered our column (200A, 5𝜇 m; Michrom Bioresources) was used to list of significant altered proteins at p value <0.05. P values Journal of Oncology 3 KEGG PATHWAY Systemic lupus erythematosus Staphylococcus aureus infection Protein digestion and absorption Prion diseases CSF from the cured individuals treatment PPAR signaling pathway −ＦＩＡ (PVale) PI3K-Akt signaling pathway Pertussis Lysosome Centrifugation Supernatants of CSF Glycolysis/Gluconeogenesis Fructose and mannose metabolism CSF from paediatric ALL patients Reductive alkylation Focal adhesion Count ECM-receptor interaction Complement and coagulation cascades Cell adhesion molecules (CAMs) desalting Carbon metabolism Biosynthesis of amino acids Arrhythmogenic right ventricular cardiomyopathy (ARVC) Amoebiasis Bioinformatic analysis LC-MS/MS analysis (triplicate) Trypsin digestion 10 20 Fold Enrichment (a) (b) GO BIOLOGICAL PROCESS GO MOLECULAR FUNCTION Cell adhesion, other, GO CELLULAR COMPLEX 11.14% other 25.30% protein binding other 25% Platelet 36% 27% extracellular Degranulation, exosome 10.84% extracellular 22% receptor matrix binding organization proteolysis 5% , 5.42% serine-type blood , 9.64% endopeptidase microparticle receptor- activity 7% mediated heparin 8% negative endocytosis, binding regulation of 6.33% 5% extracellular endopeptidase calcium ion space complement activity,... binding serine-type 16% activation,. . . endopeptidase inhibitor 8% identical protein plasma extracellular complement activation, activity innate immune binding membrane region classical pathway, 7.83% 5% 8% response, 8.13% 12% 16% (c) (d) (e) Figure 1: The comprehensive proteomics analysis of CSF with CNSL. (a) The experimental work flow. The process of sample preparation, data acquisition, and analysis is shown in the diagram. ALL, leukeamia. (b) In-depth KEGG-pathway analysis of all identified proteins in PRE-CSF samples. (c), (d), and (e) Gene Ontology (GO) analysis of all identified proteins expressed in PRE-CSF samples. All identified proteins were put into the three GO domains (BP, MF, and CC). The names and percentages of term are located next to their position on the charts. were calculated in accordance with Mann–Whitney U test for domain was demonstrated with all expressed proteins. There nonparametric data, or the two-tailed t-test for parametric was no substantial difference among the three subcategories data. The data were shown as means ± standard deviations proportions between pretreated and posttreated groups of (SD). patients with ALL (Supplementary Figure S1). The analysis in BP ontology showed significant protein enrichment in the cell adhesion, platelet degranulation, and proteolysis 3. Results (Figure 1(c)). MF analysis indicated that most of the identified proteins were noted as protein binding and calcium ion .. Global CSF Protein Analysis. Ultimately, six eligible binding as well as serine−type endopeptidase inhibitor activ- patients were included in our study. The demonstration of ity (Figure 1(d)). Analysis of CC ontology mainly pointed our experimental workow fl was presented in Figure 1(a). To investigate the proteomic profile of CSF in patients towards extracellular exosome, extracellular space, and extra- cellular region (Figure 1(e)). with CNSL, we collected PRE- and POST-CSF samples and used the same volume of samples for subsequent laboratory .. Label-Free Quantification of Relevant Protein Abundance. tests and analysis. Within these pooled CSF samples, an average of 428 unique proteins of all PRE-CSF and POST- Aiming at identifying differentially expressed proteins in CSF samples was identified by measuring peptides signal these PRE-CSF and POST-CSF samples, we performed a intensity for protein abundance (Supplementary Table S1). strict t-test difference criteria (Method section) and several During the treatment, the following pathway was revealed statistically significant proteins were identified (Table 1). The activated by depth KEGG-pathway analysis: complement expression of all these proteins was presented in Figure 2(a). and coagulation cascades, related to the usage of antitumor Our label-free quantification demonstrated that the follow- drugs during induction therapy to ALL [18] (Figure 1(b)). ing seven proteins were upregulated: histidine rich gly- Next, we grouped those proteins according to annotations in coprotein (HRG), kallikrein related peptidase 6 (KLK6), Gene Ontology (GO) domains, including ‘Biological Process carnosine dipeptidase 1 (CNDP1), alpha-2-macroglobulin (BP)’, ‘Molecular Function (MF)’, and ‘Cellular Complex (A2M), complement factor H (CFH), complement C4A (CC)’, to determine the constitutions of the CSF proteome. (C4A), and apolipoprotein A1 (APOA1). On the contrary, Then, the percentage representation of each GO subcategory WAP, follistatin/kazal, immunoglobulin, kunitz and netrin 4 Journal of Oncology Table 1: Statistically significant increased or decreased proteins in ALL samples during induction with P <0.05. Gene name Protein ID Change trend P value Protein complement C4A (Rodgers blood group) C4A P0C0L4 ↑ 0.011 HRG P04196 ↑ 0.016 Histidine rich glycoprotein APOA1 P02647 ↑ 0.025 apolipoprotein A1 KLK6 Q92876 ↑ 0.025 kallikrein related peptidase 6 carnosine dipeptidase 1 CNDP1 Q96KN2 ↑ 0.026 CFH P08603 ↑ 0.029 complement factor H A2M P01023 ↑ 0.05 alpha-2-macroglobulin apolipoprotein D APOD P05090 ↓ 0.018 WAP, follistatin/kazal, immunoglobulin, WFIKKN2 Q8TEU8 ↓ 0.024 kunitz and netrin domain containing 2 SPARC P09486 ↓ 0.037 secreted protein acidic and cysteine rich APOD -2.38 SPARC WFIKKN2 -1.18 HRG A2M 0.02 APOA1 CFH 1.22 KLK6 C4A 2.42 CNDP1 (a) CNDP1 SPARC A2M KLK6 WFIKKN2 APOA1 APOD C2A CFH (b) Figure 2: Quantification of changed CSF proteins during the treatment of CNSL. (a) Heatmap analysis of deproteins in PRE-CSF and POST- CSF group: unsupervised clustering analysis showing expression profiles of DE proteins with Euclidean Distance of proteins. (b) eTh altered- protein interactions generated by STRING analysis. Required confidence (score): medium confidence (0.400). Edge width reflects the strength of the STRING action score and the edge length is arbitrary. domain including 2 (WFIKKN2), secreted protein acidic as interaction among these 10 proteins is showed in Figure 2(b). well as cysteine rich (SPARC), apolipoprotein D (APOD) Therefore, these proteins are possibly related to ALL devel- were significantly downregulated. In addition, we used west- opment directly or indirectly, and their changes during the ern blotting technique to validate the LC-MS/MS results, treatment might be used as an indication of the better demonstrated in Supplementary Figure S2. HRG and SPARC therapeutic effects in CNS. were selected for the confirmation of the low intensity and potential applied value as the biomarkers of ALL. Consistent 4. Discussion with our LC-MS/MS results, the content of SPARC was obviously decreased (p=0.044) and HRG had the trend of In this research, the label-free LC−MS/MS was adopted increasing (p=0.072) aer ft treatment of ALL. The protein for constructing overall CSF proteomic proles fi of pediatric PRE-CSF 1 PRE-CSF 2 PRE-CSF 3 PRE-CSF 4 PRE-CSF 5 PRE-CSF 6 POST-CSF 1 POST-CSF 2 POST-CSF 3 POST-CSF 4 POST-CSF 5 POST-CSF 6 Gene Expression Journal of Oncology 5 CNSL samples. Strong concordance could be seen in the of TGF𝛽 family members to the receptor to regulate the bal- proportion of identified proteins assigned to BP, MF, and CC anced relation between the activation of non-Smad and Smad of GO domains between PRE and POST-CSF samples from pathways of TGF𝛽 [34]. As a 33-kDa glycoprotein component of high-density lipoprotein in plasma, Apo D is observably our data, demonstrating that the overall composition of CSF proteome is relatively stable [16]. In addition, the different expressed by glial cells in brain [35]. It is essential for the points in relative levels of CSF proteins were detected in development and repair of nervous system as an important regulator of lipid trafficking and conferring protection from CNSL patients during induction. As subclinical CNS mani- festation is in most patients, CNS-directed therapy is critical cell oxidative stress [36, 37]. Apo D could also act as a for increasing leukemia-free survival rates, even in nonde- nonspecific stress protein leading to cell growth arrest, and tectable CNSL pediatric patients [19]. Intrathecal chemother- it is upregulated under several pathological situations such as apy (ITC) with methotrexate or cytarabine is commonly cancers and neurodegenerative diseases [38, 39]. Increased levels of C4A and CFH were involved in com- used to prevent or treat CNSL. However, various adverse events are likely to occur, including severe gastrointestinal, plement and coagulation cascades pathway [40]. Further- hepatic and neurological toxicities, leukoencephalopathy, more, CFH inhibits endothelial cell migration and angio- genesis by indirectly regulating angiogenic effectors of com- and polyradiculopathy [20, 21]. Therefore, improving therapy efficacy and reducing toxicity are the current focus of ALL plement components, including C3a and C5a, leading to management [22]. CSF is proximate to CNS tissue microen- lower vascular endothelial growth factor (VEGF) expression and transformation of growth factor-𝛽 (TGF-𝛽 )[41–43]. vironment and has relative simplex protein contents; thus, it is preferentially considered as an ideal biomarker source The upregulated expression of CFH might be beneficial to for neurological disorders [23]. By analyzing the changes of confront VEGF produced by ALL cells, as VEGF has been expressed proteins during ITC in patients with CNSL, we proved to promote leukemia cell infiltration into CNS [36]. could find new markers to develop alternative CNSL-related As a 720-kDa glycoprotein, A2M is mainly produced by liver Kupffer cells. A2M is also involved in inflammatory reaction diagnosis, prognosis, and treatment strategies. In this work, we found the levels of three proteins as a proteinase inhibitor and carrier for interleukin-6 (IL- (SPARC, WFIKKN2, andAPOD) decreasedduringCNSL 6) [37]. It has been shown to decrease in some pathological conditions such as hematologic malignancy and advanced treatment. SPARC is a 32 kDa secreted glycoprotein that regulates interaction of cell-extracellular matrix proteins to prostate cancer [44, 45]. It was demonstrated previously promote cell adhesion and migration [24]. It is also reported that the interaction of it with the lipoprotein receptor- to be important for tissue remodelling, angiogenesis, and related protein-1(LRP1) of low-density led to an inhibi- tumourigenesis through activation of growth factors and tion of tumour cell proliferation, migration, and invasion matrix metalloproteinases [25–28]. Increased expression of in astrocytoma malignancy [46]. HRG is an 75 kDa a2- SPARC has been found to play a protumorigenic and glycoprotein produced by liver, which keeps high levels in prometastatic role to many malignant tumours, such as brain, plasma and acts as an acute phase reactant [47]. HRG is involved in numerous biologic processes, such as cell adhe- lung, breast, pancreas, skin, and kidney [29]. Another study showed that the downregulated or absent expression of sion and proliferation, angiogenesis as well as coagulation SPARC is related to high-speed progression of human colon and platelet activation [48]. It is also reported that HRG inhibits tumour growth and vascularization, remodels tran- cancer [30]. us, Th SPARC may have complex function in the development of human solid tumours. However, there are sition from epithelial to mesenchymal, and regulates the few studies discussing the role of SPARC in the formation of formation of protumorigenic microenvironment by skewing hematological malignances. Recently, accumulating evidence tumour-associated macrophages polarization from the M2- suggested that SPARC overexpression is associated with poor like towards M1-like phenotype [49–51]. Besides, HRG pro- outcome in acute myeloid leukemia (AML) patients and motes antitumor immune responses and vessel normalization promotes the growth of aggressive leukemia cell in murine to enhance chemotherapy. KLK6, a member of the secreted models. In addition, knockdown of SPARC could inhibit serine protease family of protein-cleaving enzymes, is also defined as an epigenetically regulated tumour suppressor AML cell proliferation through inducing cell cycle arrest at Phase G1/G0 and inhibit active p53-induced apoptosis [52]. KLK6 expression resulted in significant downregulation through raising the levels of p53, caspase-9, caspase-3, and of vimentin, a critical marker of epithelial-to-mesenchymal transition of tumour cells [53]. APOA1, the major protein Fas [24, 31]. As a marker of poor prognosis and a potential therapeutic target of AML, a similar mechanism may be component of high-density lipoproteins (HDL) particles in helpful for inhibiting ALL development, and the decreased plasma, suppressed neutrophil recruitment in inflammation level of SPARC may account for a better prognosis of CNSL, to avoid excessive inflammatory response [54]. Furthermore, since the levels of SPARC significantly declined in subjects it is a potential antiangiogenic factor to suppress tumour when they CR was achieved in our study. WFIKKN2 is a large progression by targeting c-Src/ERK signaling pathways [55, extracellular multidomain protein, containing a WAP, an 56]. Carnosinase 1(CNDP1), primarily synthesized in brain, immunoglobulin, a follistatin domain (binds mature growth secreted into CSF, and finally secreted into the blood, is likely to be important in the maintenance and bioavailabil- factor), an NTR domain, and two Kunitz-type protease inhibitor domains [32]. It is known to positively activate ity of carnosine, a drug for tumours. It could be verified PI3K pathway, which could regulate cancer cell growth and that CNDP1 has antiproliferative properties on different malignant cells for its therapeutic potential against tumours progression [33]. Moreover, it can promote the combination 6 Journal of Oncology including glioblastoma and metastatic prostate cancer. Its MF: Molecular Function deficiency has been related to many kinds of neurologic NKTCL: Natural killer cell/T-cell lymphoma deficits and cancer cachexia [57–59]. Several statistically POST-CSF: Aeft r achieving a complete response altered proteins have been previously proved to play a role PRE-CSF: Before the treatment of regulation in malignant cells or microenvironment of SD: Standard deviations cancers, which might be related to the molecular mechanisms SPARC: Secreted protein acidic and cysteine rich of ALL development and prognosis of patients with CNSL. TGF-𝛽 : Transforming growth factor-𝛽 Collectively, we constructed CSF’s label-free LC−MS/MS VEGF: Vascular endothelial growth factor proteomic profiles in patients suffering from CNSL, which WAP: Whey acidic protein helpsusidentify valuable biomarkers to understand the WFIKKN2: WAP, follistatin/kazal, immunoglobulin, development of ALL with CNS infiltration. It is noteworthy kunitz and netrin domain containing 2. that those altered proteins should be furtherly investigated as predictive markers of ALL patients with CNS infiltration, Data Availability some of which may have a prospect of becoming the new therapeutic targets in CNSL. However, this study has some The data from LC-MS/MS analysis used to support the limitations. For example, the size of samples is small since findings of this study are included within the article. only 6 subjects are covered, and the period of observation is relatively short. Therefore, we will enlarge the sample size and prolong the observation time in our next study, hoping to Conflicts of Interest further verify the roles of those altered proteins in paediatric The authors declare that there are no conflicts of interest. patients of ALL with CNS infiltration. Abbreviations Authors’ Contributions A2M: Alpha-2-macroglobulin Fei Mo and Xuelei Ma contributed equally to this work. ACN: Acetonitrile AGC: Automatic gain control Acknowledgments ALL: Acute lymphocytic leuk(a)emia AML: Acute myeloid leuk(a)emia We appreciate the patients and their families for participating APOA1: Apolipoprotein A1 in the study. APOD: Apolipoprotein D BP: Biological Process C4A: Complement C4A Supplementary Materials CC: Cellular Complex Supplementary Figure S1: Gene Ontology (GO) analysis CFH: Complement factor H CID: Collision induced dissociation of all identified proteins expressed in POST-CSF samples. CNDP1: Carnosine dipeptidase 1 Supplementary Figure S2: validation of SPARC and HRG protein peaks by western blot. (A) Immunoblot of SPARC CNS: Central nervous system CNSL: Central nervous system leuk(a)emia and HRG proteins in PRE-CSF and POST-CSF samples. Fiyft micrograms of PRE-CSF (lanes 1–3) or POST-CSF (lanes 4–6) CSF: Cerebrospinal uid fl CSF were subjected to the lanes. (B) and (C) The relative DTT: DL-Dithiothreitol FA: Formic acid quanticfi ation of SPARC and HRG proteins from panels (A) using Image J sowa ft re, respectively. Supplementary Table S1: GO: Gene Ontology an average of 428 unique proteins of all PRE-CSF and POST- HCD: Higher energy Collision induced Dissociation CSF samples was identified by measuring peptides signal intensity for protein abundance. (Supplementary Materials) HDL: High-density lipoproteins HRG: Histidine rich glycoprotein IAA: Iodoacetamide References IL-6: Interleukin-6 IT: Intrathecal therapy [1] S. Chiaretti, A. Vitale, G. 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