Reagents were from Sigma Chemical Co (St. Louis, MO, USA), tissue culture flasks, dishes, and multi-well plates were from Falcon Orange Scientific (Graignette Business Park, Belgium), culture media for cell lines was from Gibco BRL (Carlsbad, CA, USA), culture media for tissues, VEGF and bovine fetal serum were from Sigma Aldrich (St. Luis, MO, USA).

Patients with suspected kidney cancer or healthy kidney donors were enrolled. After signing the informed consent, subjects were interviewed using a standard questionnaire that requested information about socio-demographic, medical, and lifestyle factors. Human adipose tissue explants from cancerous kidneys were obtained from patients to whom a partial or total nephrectomy was performed, who had not received previous chemotherapy or radiotherapy treatment. Human adipose tissue explants from normal kidneys were obtained from live kidney donors. Perirenal adipose tissue biopsies were taken as follows: (1) in the case of living kidney donors, the adipose tissue fragment was taken 1 cm away from the kidney from the middle zone (middle pole); (2) in the case of patients with kidney cancer, the fragment of adipose tissue was taken 1 cm from the kidney, also considering the location of the tumor. Trying, in all cases, to take the sample 1 cm from the location of the tumor, getting as close as possible to the middle zone. In all cases, biopsies were taken distant from the adrenal gland (sources of norepinephrine).

The median body mass index (BMI) of patients was: 26.8 kg/m² for patients with renal tumor (hRAT), and 24.9 kg/m² for living kidney donors (hRAN). BMI (kg/m²) was calculated as weight (kg) divided by height (m) squared. Of the living kidney donors, 57% are female and 43% are male, with a mean age of 44 years (+/− 7). Of the ccRCC patients, 27% are female and 73% are male, with a mean age of 56 years (+/− 14). All patients are from the population of the Province of Mendoza, Argentina.

Samples were transported in PBS and processed immediately; a fragment of each one was stored at −80 °C, another fragment was processed under a sterile laminar flow hood. The project was approved by the Medical School’s ethics committee (Universidad Nacional de Cuyo, Argentina, EXP-CUY: 11722–2016) according to the Declaration of Helsinki of experimentation with human subjects. All patients gave their informed consent to undergo tissue harvesting for this research (14, 15).

Total RNA was extracted from 100 mg of tissue (hRAN n = 8; hRAT n = 8) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified according to its absorbance at 260/280 nm. Two micrograms of total RNA were reverse transcribed at 37 °C using random hexamer primers and Moloney murine leukemia virus retrotranscriptase in a 20 μL reaction mixture. The RNA was first denatured at 70 °C for 5 min. in the presence of 2.5 μg of random hexamer primers (Thermo Fisher Scientific, Carlsbad, CA, USA). For the subsequent RT reaction, the following mixture was added: RT buffer [50 mM Tris–HCl (pH8.4), 75 mM KCl, 3 mM MgCl2], 0.5 mM dNTPs, 5 mM DTT, 200 units M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, CA, USA). The reaction was incubated at 37 °C for 50 min., next, the reaction was inactivated by heating at 70 °C for 15 min. The cDNA was stored at −20 °C. The mRNA content of each gene was estimated by RT Real-Time PCR using specific primers described in Table 1. The PCR reactions were performed using a Corbett Rotor Gene 6000 Real-Time Thermocycler (Corbett Research Pty Ltd. Sydney, Australia) and the HOT FIREPol® DNA Polimerase (Solis BioDine, Tartú, Estonia). The PCR reactions were initiated with 15 min incubation at 95 °C, followed by 40 cycles of 95 °C for 30 s, 30 s at the annealing temperatures shown in Table 1 and 72 °C for 30 s. Melt curve analysis was used to check that a single specific amplified product was generated. Real time quantification was monitored by measuring the increase in fluorescence at the end of each amplification cycle, according to the manufacturer’s protocol. Relative expression was determined using the Comparative Quantitation method of normalized samples in relation to the expression of a calibrator sample (control sample). Each PCR run included a no-template control and a sample without reverse transcriptase. All measurements were performed in duplicate by sample of two independent experiments. The reaction conditions and quantities of cDNA added were calibrated so that the assay response was linear with respect to the amount of input cDNA for each pair of primers. Relative levels of mRNA were normalized to the GAPDH reference gene (14, 15).

Adipose tissues were washed with cold PBS 1× (Gibco, USA) and weighed. hRAN or hRAT were plated in culture flasks with M199 culture medium (Invitrogen™; 1 g tissue/10 mL M199) and incubated for 1 h at 37 °C in 5% CO₂. After that, the medium was removed and replaced with fresh medium and the tissues were incubated for 24 h. Afterwards, the supernatant was collected and filtered with sterile 0.22 μm membranes. Then, supernatants were aliquoted into 1 mL fractions and immediately stored at –80 °C. The control-CMs were obtained from the collection of the serum-free M199 medium after 24 h of incubation in a culture flask at 37 °C in 5% CO₂ (14, 15).

Levels of VEGF in hRAN- (n = 10) or hRAT-CMs (n = 19) were determined using enzyme-linked immunoassay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). In brief, high-binding 96-well microplates (Costar; Corning) were coated with capture Ab (2 μg/mL purified rabbit anti-VEGF polyclonal IgG) in 0.1 M sodium carbonate, pH 9.5. After incubation for 18 h at 4 °C, wells were rinsed three times with wash buffer (0.05% Tween-20 in PBS) and incubated for 1 h at room temperature with blocking solution [2% Bovine Serum Albumin (BSA) in Phosphate buffer saline (PBS)] Samples and standards (100 μL) were diluted in 1% BSA–Tween-20 and incubated for 18 h at 4 °C. Plates were then washed and incubated with 100 ng/mL biotinylated detection Ab (purified rabbit anti-VEGF polyclonal IgG) for 1 h. Plates were rinsed three times before adding 0.33 μg/mL HRP-labeled streptavidin (Sigma-Aldrich) for 30 min. After washing, 100 μL 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (0.1 mg/mL tetramethylbenzidine and 0.06% H₂O₂ in citrate–phosphate buffer, pH 5.0) was added to plates. The reaction was stopped by adding 2 N H₂SO₄. Optical densities were determined at 450 nm in a Multiskan MS Microplate Reader (Thermo Fisher Scientific). A standard curve ranging from 2.5 to 160 ng/mL VEGF was run in parallel (27).

Isolation of HUVECs from umbilical cords was performed essentially as described in Bannoud et al. (27), following approval by the Ethics Committee from the Faculty of Medicine, National University of Cuyo (Mendoza, Argentina) (ref. EXP-CUY: 4693–2018 and updated in 2019); and by the Research Ethics Committee from the Central Hospital of Mendoza (Mendoza, Argentina) (Approval record: 10/2022).

The 96-well plates were coated with Geltrex® and maintained at 37 °C for 30 min. After that, excess gelatin was recovered, and cells were seeded (2.5×10⁴ cells per well). HUVEC cells were grown on complete medium RPMI 15% FBS with 10 ng/mL VEGF. Confluent cell monolayers were wounded with a pipette tip, washed twice with PBS and treated with hRAN-CM (n = 6), hRAT-CM (n = 6), or M199 growth medium as negative control, all diluted with RPMI 2% FBS and positive control (RPMI 15% BFS with 20 ng/mL VEGF). Images at time zero (0 h) were captured to record the initial width of the wounds. The recovery of the wounded monolayers due to cell migration toward the denuded area was evaluated after 6 and 12 h. The images were acquired by an inverted phase-contrast microscope (Olympus CKX-41) using a 4× objective. Quantification was performed using ImageJ analysis software (NIH, Bethesda, MD, USA) by a polygon selection mode and determining the percentage of the wounded area at 6 or 12 h respect to control (0 h) (14, 15).

HUVEC cells (3×10⁴ cells/0.2 mL) were placed into the top transwell with 8 μm pore membranes (COSTAR® cat. #03123015). They were then incubated with hRAN-CM (n = 6), hRAT-CM (n = 6), negative control (M199 growth medium n = 6), all diluted with RPMI 2% FBS, and positive control (RPMI 15% FBS with 20 ng/mL VEGF n = 3) and allowed to transmigrate across the porous membrane for 18 h. At the end of the assay, inserts were removed, and the cells were stained with a 0.1% crystal-violet in 20% methanol (Sintorgan®) solution for 10 min. The inserts were washed in distilled water and the cells on the upper membrane surface were removed with a cotton swab. The air-dried membranes were viewed under 20× magnification and migrated cells were counted in five randomly chosen fields per membrane (14, 27).

The formation of capillary-like tubular structures was assessed in Geltrex-coated plates as described (26). In brief, HUVECs (1.5 × 10⁴ cells) were mixed with different CMs, and then plated on Geltrex-coated 96-well plates at 37 °C for 12 h or, alternatively, on Geltrex-coated μ-slide angiogenesis (Ibidi®) for 6–8 h. Capillary-like tubular structures were visualized by phase-contrast microscopy and recorded by counting the number of tubules (closed areas) per well in a phase-contrast microscope (Nikon E-100) (27).

GraphPad Prism version 8 (GraphPad Software Inc.) was used for statistical analysis. For comparisons between two datasets we performed t-test with Mann–Whitney or Welch’s corrections as appropriate. For multiple comparisons, one-way ANOVA followed by Tukey’s or Dunnett’s posttest (parametric analysis) or the Kruskal–Wallis test followed by Dunn’s posttest (nonparametric analysis), were used. p-values < of 0.05 or less were considered significant.

We evaluated gene expression profiles of angiogenesis regulatory factors in different fragments of adipose tissue. To do so, we measured mRNA levels of HIF-1α, HIF-2α, and VEGF in AT from normal and tumor kidney samples. We found higher gene expression of HIF1α and HIF2α in hRAT vs. hRAN explants (Figures 1a,b, p < 0.01). No statistically significant differences were found in VEGF mRNA expression among groups (Figure 1c).

In order to evaluate the secretion of VEGF protein in hRAN- and hRAT-CMs, we conducted an ELISA assay. We observed a close to significance trend toward higher VEGF secretion in hRAT-CMs compared to hRAN-CMs (Figure 2, p = 0.052).

To determine the migration capacity of HUVEC cells after incubation with different CMs, we performed wound healing assays. HUVEC cells were grown, wounded, and treated with hRAN-, hRAT-, VEGF control- (positive control), and M199 control- (negative control) CMs. After 6 and 12 h of incubation, hRAT-CMs significantly enhanced HUVEC migration vs. hRAN-CMs and M199 control-CMs (p < 0.05 and p < 0.0001 respectively, Figures 3a,b). However, the positive control enhanced HUVEC migration, vs. hRAN-CMs and M199 control CMs, only after 12 h (p-value < 0.001).

To evaluate the transmigration capacity of HUVEC cells, due to chemoattractant factors secreted by AT, we performed a Transwell assay. HUVEC cells were grown with complete growth medium and treated with hRAN-, hRAT-, VEGF- (positive control) and M199- (negative control) CMs. After 18 h of incubation, hRAT-CMs significantly enhanced HUVEC transmigration, through the insert, vs. hRAN- and M199-CMs (p < 0.0001, Figure 4).

To evaluate the angiogenic activity of hRAT- and hRAN-CMs, we performed a tubulogenesis assay to model the formation of three-dimensional vessels. A VEGF-CM group for tubulogenesis was performed as a positive control of the assay, in order to evaluate whether HUVEC cells were indeed able to form new tubuli. This group showed a very significant increase of tubulogenesis compared to the hRAN-CM (p < 0.0001, data not shown). In addition, we observed that hRAT-CMs significantly increased the formation of ECs tubular networks compared to hRAN-CMs (Figure 5, p < 0.05).