Patients and study approval
The study included newly diagnosed RCC (n=43) and non-RCC (n=7) patients that underwent radical nephrectomy (Supplemental Table S1). The primary tumor and the adjacent healthy renal tissue samples were obtained from the patients during the surgical procedures within a four-year time frame.
Non-RCC cases such as rectal carcinoma (n=1), urothelial carcinoma (n=1) and benign RCC angiomyolipoma (n=1) were excluded from all analyses. The study was approved by the Helsinki University Hospital ethical committee (Dnro 115/13/03/02/15) and was conducted in accordance with the Declaration of Helsinki. All patient samples were taken after a signed informed consent.
Sample preparation and processing
A prospective sample collection of freshly excised tumor and matching adjacent healthy tissue samples were collected (2016-2020) and stored in MACS® tissue storage solution (Miltenyi Biotec 130-100-008) at 4°C upon harvest. All samples were processed directly upon arrival at our facilities within minimal transportation time. Each sample was independently dissociated using Miltenyi’s Tumor Dissociation kit protocol (Miltenyi Biotec 130-095-929). A portion of the freshly dissociated cells were viably frozen in 10% FBS-DMSO solution and preserved at -150°C for the cytokine assay.
An assessment of 18 clinical parameters (tumor size and weight, TNM staging, WHO/ISUP 2016 tumor grading simplified into two classes: low (G1-G2) and high (G3-G4), presence of necrosis, peri-renal and peri-pelvic fat infiltration, rhabdoid histology, age at relapse) and other medical histories were included (Supplemental Table S1).
Conditioned media were obtained from each dissociated tumor (n=50) and adjacent healthy renal tissue (n=24) sample. First, 100 000 cells in 150mL/well were seeded in 96-U well culture plates and cultured in tumor cell media (RPMI-1640, 10% FBS, 1% Penicillin/Streptomycin, 2mM L-Glutamine, 10mM sodium pyruvate, hydrocortisone sodium succinate (Solu-Cortef) 0.0004mg/mL) for 24h at 37°C, 5% CO2. The next day, cells were washed twice with PBS to remove any remaining serum, replaced with unsupplemented RPMI-1640, and were incubated for 24h at 37°C, 5% CO2. The conditioned media were collected and filtered through a 0.2mm syringe before use. Details of the conditioned media protocol have previously been described in detail14,15.
Multiplex cytokine assay
The presence of various cytokines such as chemokines, growth factors, and interleukins (pg/mL) from the serum-free conditioned media were analyzed using the Bio-Plex ProTM human cytokine screening panel (Bio-Rad) according to the manufacturer’s instructions.
I. Tumor-Healthy comparisons
Non-parametric Mann-Whitney U-test (unpaired, two-tailed) with a 95% confidence level was used to compare two groups. All scatter dot plots show error bars with the median and range as horizontal lines. The statistical analyses were performed using Prism 9 Version 9.2.0 (GraphPad Software Inc.). For all graphs: ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
II. Heatmap and correlation analyses
Unsupervised heatmap clustering was carried out by using the Euclidean distance and ward.D2 linkage method after log2 transforming and Z-score scaling the data. The Z-score scaling was performed for each cytokine (row). Spearman’s correlation and hierarchical clustering with the complete linkage method were used for the correlation plot (Fig. 2), which was created with the R package, corrplot16. R version 220.127.116.11, RStudio version 1.3.1056 and the Python package CytoMod18 were used for the analyses. Benjamini and Hochberg-corrected p-values with a false discovery rate (FDR) < 0.05 with were considered significant.