Urine proteome changes in rats subcutaneously inoculated with approximately ten tumor cells

Animal treatment

Male Wistar rats (n = 12, 150 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were maintained with a standard laboratory diet under controlled indoor temperature (21 ± 2 °C), humidity (65–70%) and 12 h/12 h light–dark cycle conditions. The experiment was approved by Peking Union Medical College (Approval ID: ACUC-A02-2014-008).

Walker-256 (W256) carcinosarcoma cells were purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences (Beijing, China). W256 tumor cells were intraperitoneally inoculated into Wistar rats. The W256 ascites tumor cells were harvested from the peritoneal cavity after seven days. After two cell passages, the W256 ascites tumor cells were collected, centrifuged, and resuspended in 0.9% normal saline (NS). Then, W256 tumor cells were serially diluted to a concentration of 10²/mL. The viability of W256 cells was evaluated by the Trypan blue exclusion test using a Neubauer chamber, and only 95% viable tumor cells were used for serially dilution.

The rats were randomly divided into the following two groups: rats subcutaneously inoculated with tumor cells (n = 9) and control rats (n = 3). In the experimental group, rats were inoculated with 10 W256 cells in 100 µL of NS into the right flank of the rats. The control rats were subcutaneously inoculated with an equal volume of NS. All rats were anesthetized with sodium pentobarbital solution (four mg/kg) before inoculation.

Urine collection

Before urine collection, all rats were accommodated in metabolic cages for 2–3 days. Urine samples were collected from rats subcutaneously inoculated with tumor cells (n = 9) on days 0, 13 and 21. All rats were placed in metabolic cages individually for 12 h to collect urine without any treatment. After collection, urine samples were stored immediately at −80 °C.

Extraction and digestion of urinary proteins

Urine samples (n = 27) were centrifuged at 12,000 × g for 30 min at 4 °C. Then, the supernatants were precipitated with three times the volume of ethanol at −20 °C overnight. The pellets were dissolved sufficiently in lysis buffer (8 mol/L urea, 2 mol/L thiourea, 50 mmol/L Tris, and 25 mmol/L DTT). After centrifugation at 4 °C and 12,000 × g for 30 min, the protein samples were measured by using the Bradford assay. A total of 100 µg of each protein sample was digested with trypsin (Trypsin Gold, Mass Spec Grade, Promega, Fitchburg, WI, USA) by using filter-aided sample preparation (FASP) methods (Wisniewski et al., 2009). These digested peptides were desalted using Oasis HLB cartridges (Waters, Milford, MA, USA) and then dried by vacuum evaporation (Thermo Fisher Scientific, Bremen, Germany).

LC-MS/MS analysis

Digested peptides (n = 27) were dissolved in 0.1% formic acid to a concentration of 0.5 µg/µL. For analysis, one µg of peptide from each sample was loaded into a trap column (75 µm ×2 cm, three µm, C18, 100 Å) at a flow rate of 0.25 µL/min and then separated with a reversed-phase analytical column (75 µm ×250 mm, two µm, C18, 100 Å). Peptides were eluted with a gradient extending from 4%–35% buffer B (0.1% formic acid in 80% acetonitrile) for 90 min and then analyzed with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The MS data were acquired using the following parameters: (i) data-dependent MS/MS scans per full scan were auquired at the top-speed mode; (ii) MS scans had a resolution of 120,000, and MS/MS scans had a resolution of 30,000 in Orbitrap; (iii) HCD collision energy was set to 30%; (iv) dynamic exclusion was set to 30 s; (v) the charge-state screening was set to +2 to +7; and (vi) the maximum injection time was 45 ms. Each peptide sample was analyzed twice.

Label-free quantification

Raw data files (n = 54) were searched using Mascot software (version 2.5.1; Matrix Science, London, UK) against the Swiss-Prot rat database (released in February 2017, containing 7,992 sequences). The parent ion tolerance was set to 10 ppm, and the fragment ion mass tolerance was set to 0.02 Da. The carbamidomethylation of cysteine was set as a fixed modification, and the oxidation of methionine was considered a variable modification. Two missed trypsin cleavage sites were allowed, and the specificity of trypsin digestion was set for cleavage after lysine or arginine. Dat files (n = 54) were exported from Mascot software and then processed using Scaffold software (version 4.7.5, Proteome Software Inc., Portland, OR). The parameters were set as follows: both peptide and protein identifications were accepted at a false discovery rate (FDR) of less than 1.0% and proteins were identified with at least two unique peptides. Different samples were compared after normalization with the total spectra. Protein abundances at different time points were compared with spectral counting, according to previously described procedures (Old et al., 2005; Schmidt et al., 2014).

Statistical analysis

Average normalized spectral counts of each sample were used for the following statistical analysis. The levels of proteins identified on days 13 and 21 were compared with their levels on day 0. Differential proteins were selected with the following criteria: unique peptides ≥2; fold change ≥1.5 or ≤0.67; average spectral count in the high-abundance group ≥3; comparison between two groups were conducted using two-sided, unpaired t-test; and P-values of group differences were adjusted by the Benjamini and Hochberg method (Benjamini & Hochberg, 1995). Group differences resulting in adjusted P-values <0.05 were considered statistically significant. All results are expressed as the mean ± standard deviation.

Functional enrichment analysis

Differential proteins on days 13 and 21 were analyzed by Gene Ontology (GO) based on the biological process, cellular component and molecular function categories using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang da, Sherman & Lempicki, 2009). The biological pathway enrichment at the two time points was analyzed with IPA software (Ingenuity Systems, Mountain View, CA, USA).

Characterization of rats subcutaneously inoculated with tumor cells

A total of 12 male Wistar rats (150 ± 20 g) were randomly divided into the following two groups: a control group (n = 3) and a group of rats subcutaneously inoculated with W256 tumor cells (n = 9). The body weight of these 12 rats was recorded every 3–5 days, and the daily behavior changes of the two groups were observed. The body weight of the group of rats subcutaneously inoculated with W256 tumor cells was slightly lower than that of the rats in the control group, but there were no significant differences until day 41 (Fig. 2). In addition, we did not observe any detectable tumor mass in the whole period. The rats in the control group performed normal daily activities and had shiny hair. There were no significant differences in daily behavior between these two groups.

Urine proteome changes

Twenty-seven urine samples at three time points (days 0, 13, and 21) were used for label-free LC-MS/MS quantitation. A total of 824 urinary proteins with at least 2 unique peptides were identified with <1% FDR at the protein level (Table S1). A hierarchical clustering was performed by using the complete linkage method. As shown in Fig. 3A, all technical replications within one sample were clustered together, indicating that the technical variation was smaller than the interindividual variation. In addition, all 824 proteins were clustered into three clusters, which almost corresponded to the urine proteome samples from the same group on day 0, day 13 and day 21 (except rat4-D0 and rat6-D21), indicating that intragroup technical variation was smaller than the intergroup biological variation and showed good consistency among these nine rats when inoculated with the same limited number of tumor cells. Using screening criteria, 34 and 59 differential proteins were identified on days 13 and 21, respectively. The overlap of these differential proteins is shown by a Venn diagram in Fig. 3B. Details are presented in Table 1.

Functional analysis

Functional enrichment analysis of differential proteins was performed by DAVID (Huang da, Sherman & Lempicki, 2009). Differential proteins were classified into biological processes, cellular components and molecular functions. The major biological pathways of differential proteins were enriched by IPA software. A significance threshold of P < 0.05 was used in all these representative lists.

Lists of fourteen representative biological processes on days 13 and 21 are presented in Fig. 4A. Cell adhesion, negative regulation of endopeptidase activity and organ regeneration were overrepresented both on days 13 and 21. Blood coagulation, acute-phase response, autophagic cell death, positive regulation of cell proliferation, extracellular matrix organization, and response to glucose were independently overrepresented on day 13. On day 21, heterophilic cell–cell adhesion via plasma membrane cell adhesion molecules, proteolysis, positive regulation of phagocytosis and angiogenesis were independently enriched.

To identify the biological pathways involved with the differential urine proteins, IPA software was used for canonical pathway enrichment analysis. A total of 18 and 13 significant pathways were enriched on days 13 and 21, respectively (Fig. 4B). Among these pathways, enriched intrinsic prothrombin activation pathway, coagulation system, acute phase response signaling, extrinsic prothrombin activation pathway and NAD phosphorylation and dephosphorylation were overrepresented on days 13 and 21. In addition, some representative pathways, such as autophagy, phagosome maturation and the role of tissue factor in cancer, were independently enriched on day 13, and the complement system was enriched only on day 21.

The enriched cellular components and molecular functions are presented in Fig. S1. The majority of differential proteins were derived from the extracellular exosomes, extracellular space and extracellular matrix (Fig. S1A). In the molecular function category, serine-type endopeptidase inhibitor activity and calcium ion binding were overrepresented on both on days 13 and 21 (Fig. S1B).

Random allocation statistical analysis

To confirm that these differential proteins on days 13 and 21 were indeed due to the ten subcutaneously inoculated W256 tumor cells, we randomly allocated the data of these 27 samples (Number 1 to Number 27) into three groups. We tried three random allocations, and the numbers in these three groups are shown in Table 2. In each iteration, we used the data of group 1 as the control group. When we used the previous criteria to screen differential urinary proteins, it was found that the adjusted P-values value on days 13 and 21 were all >0.05. No differential proteins were selected in these three randomly allocated trials. Details are shown in Tables S2, S3 and S4.