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Tumor Discovery Energy metabolism in bladder cancer

In light of the foregoing, the aims of the current study Specific antigens corresponding to (GLUT1; rabbit
were to feature and compare the cell energy metabolism polyclonal antibody, sc7903, Santa Cruz Biotechnology,
profiles among normal bladder tissue and NMIBC tissues USA), (PFK; mouse monoclonal antibody, LS-C173559,
with different histological grades (low-grade pTa, high- LifeSpan BioSciences, Inc., USA), (GAPDH; rabbit
grade pTa, and high-grade pT1). The present research polyclonal antibody, ab37168, abcam, USA), (LDH;
adjusted the IBEC to these specific tumor types in order mouse monoclonal antibody, sc137243, Santa Cruz
to establish a clinically relevant criterion with prognostic Biotechnology, USA), (PDH; rabbit polyclonal antibody,
significance to classify this neoplasia. GTX104040, GeneTex, USA), (CS; mouse monoclonal
antibody, sc390693, Santa Cruz Biotechnology, USA),
2. Materials and methods (HADHSC; rabbit polyclonal antibody, sc 292196, Santa
2.1. Human samples Cruz Biotechnology, USA), mitochondrial ATP synthase
F1-beta-subunit (β-F1-ATP synthase [ATPase]; rabbit
This retrospective study analyzed 40 urinary bladder polyclonal antibody, sc134962, Santa Cruz Biotechnology,
tissue samples from patients (median age of 62 years) USA), and (hsp60; mouse monoclonal antibody, sc376240,
both with and without a diagnosis of urothelial lesions by Santa Cruz Biotechnology, USA) were identified by reacting
maintaining male-to-female ratio of 2:1. Tissue specimens the tissue specimens with specific primary antibodies,
were obtained at the Urology Service at Amaral Carvalho which were diluted in 1% bovine serum albumin (BSA) and
Hospital, Jaú City, Brazil. Ten bladder tissue samples were stored overnight at 4°C. Bound antibodies were detected
sourced from autopsies of patients without a diagnosis based on using EasyLink One kit (EasyPath EP-12-20504,
of urothelial injury or urological disease. In addition, the Sao Paulo, Brazil). Subsequently, sections were slightly
remaining 30 bladder tissue samples were obtained from counterstained with Harris’ hematoxylin and visualized in
patients who underwent transurethral resection of the Leica DM2500 microscope equipped with DFC295 camera
bladder tumor (TURBT). (Leica, Munich, Germany). 21
Histopathological diagnoses were meticulously The rate of positively stained cells was assessed in
assessed by a senior uropathologist in accordance with the ten fields for each antibody, under high magnification
classification consensus put forth by the International Society (×400), to assess antigen immunoreactivity intensity
of Urological Pathology/World Health Organization. The within urothelial cells in ImageJ software (https://imagej.
20
categorization process lied in splitting the samples into nih.gov/ij/). Quantitative data were analyzed in two
three different groups, with each comprising ten samples, different manners, namely total immunoreactivity and
namely normal (no urothelial lesions) group, low-grade immunoreactivity intensity. Total immunoreactivity was
pTa group, high-grade pTa group, and high-grade pT1 calculated as complement of the negative rate of urothelial
group. Subsequently, these tissue samples were subjected cells observed for a given antibody, subtracted from 100%.
to rigorous analyses using techniques encompassing In other words, it represented the total number of urothelial
immunohistochemistry and Western blotting. cells in the field that presented immunoreactivity to the
All procedures herein were conducted in full compliance assessed antibody. Immunoreactivity intensity was assessed
with the ethical standards set forth by the Ethics and based on categorizing the observed immunoreactivity
Research Committee of Amaral Carvalho Hospital, Jaú within urothelial cells by taking into consideration
City, Brazil (CAAE number: 47475815.9.0000.5434). intensity criteria. These categories were established in
ImageJ software, at scale ranging from 0 to 3 wherein: 0
2.2. Immunohistochemical analysis (no immunoreactivity) means 0% positive urothelial cells,
The same set of bladder samples (n = 10 per group), which 1 (weak immunoreactivity) indicates 1 – 35% positive
had been previously used for histopathological analysis, urothelial cells, 2 (moderate immunoreactivity) represents
was also used for immunolabeling procedures. These 36 – 70% urothelial cells, and 3 (intense immunoreactivity)
tissue samples were cut into 5 μm-thick sections. Antigen features >70% positive urothelial cells. 22
retrieval was achieved by subjecting the aforementioned
sections to three 5-min heating cycles in 10 mM citrate 2.3. Western blotting
buffer at pH 6.0, in standard microwave oven. Subsequently, Urinary bladder samples were collected from all
sections were incubated in peroxidase blocker (EasyPath 40 patients and subjected to immunoblotting analysis
21
EP12-20523, Sao Paulo, Brazil) and this procedure performed according to the established protocols. In
was followed by incubation in 5% goat serum blocking total, 70 μg protein was loaded into each well on the SDS-
solution (EasyPath EP12-20523, Sao Paulo, Brazil) at room polyacrylamide gel for electrophoresis. Subsequently,
temperature for 10 min. proteins were transferred to nitrocellulose membranes,

Volume 3 Issue 1 (2024) 3 https://doi.org/10.36922/td.2290