Telocytes and ezrin expression in normal-appearing tissues adjacent to urothelial bladder carcinoma as predictors of invasiveness and recurrence

Abstract

Recurrence and progression rates vary widely among different histological subtypes of bladder cancer (BC). Normal-appearing mucosa in non-muscle invasive bladder cancer and muscle-invasive bladder cancer in cystoscopy and histopathology is a factor in staging and treatment. Telocytes (TCs) are spindle-shaped cells that connect with other cell types allowing communication though cytoskeletal signaling. They are involved in tissue regeneration and pathogenesis of diseases and cancer. In this study, 12 normal-appearing tissues from urinary bladder with BC, both invasive and non-invasive were evaluated in patients who had either trans-urethral resection of bladder tumor or cystectomy. In each case, cystoscopy, intraoperative inspection, and histopathology all confirmed the absence of cancerous elements. Five patients with neurogenic bladder were used as a control group. Immunohistochemistry revealed that c-Kit + cells were intensively distributed in bladder layers from BC samples, while they were seldom detected in the control group. Ultrastructural examination of reprocessed tissue showed an intense distribution of TCs and telopodes in the submucosa and between smooth muscle cells in BC. Telopodes were numerous, arborizing, and intercommunicating. Whereas TCs and telopodes were scarce in the neurogenic bladder. Also, cancerous tissue had the highest expression level of ezrin protein, and this level gradually decreased as we moved away from the tumor. Our finding of TCs number in normal-appearing tissues in conjunction with ezrin expression may compete invasiveness and possibly a trail to reduce recurrence rates.

Figure 1

Light microscopy with H&E and cKit IHC findings in normal-appearing tissue of urinary bladder with UBC: (A,B) showed no evidence of cancer in mucosa, submucosa and muscularis propria layers. (C,D) cKit IHC shows intense distribution of cKit + ve cells in the muscle layer (long arrow). Neurogenic bladder: (A,B) showing increased collagen within the muscle layer. (C,D) cKit IHC shows of cKit + ve cells mainly close to blood vessels (short arrow). (A,B,C: ×100, scale bar 100 µm, D: 200×, scale bar 50 µm). (×200, scale bar 50 µm).

Figure 2

cKit IHC shows distribution of c-Kit positive cells in different samples of normal-appearing tissue of urinary bladder with UBC. (A,B,C): MIBC. (D,E): NMIBC, high grade and (F): NMIBC low grade. (× 200, scale bar 50 µm).

Figure 3

TEM microgram of submucosa and muscularis propria of normal-appearing tissue of urinary bladder with MIBC. (A,A′) (A: original findings), (A’: digitalized image) showing TC (TC colored light blue), TC nucleus (colored violet) with distinctive cytoplasm containing numerous vesicles (yellow V), and Tps (Tp red Asterix). TCs and Tps are infiltrating between smooth muscle cells (SMC) and dense collagen (green C). (B,B′) (B: original findings), (B’: digitalized image) showing Tp (colored light blue, pointed with short blue arrows, they have many vesicles (yellow V), and three Tp are in close contact with blood vessels. (C,C′) (C: original findings), (C′: digitalized image) showing two TC cells (colored light blue) with a distinctive nucleus in one of them (N), network of Tps appearing numerous, branching, and arborizing (small blue arrows and red Asterix), Tps cytoplasm are rich in vesicles (yellow V), TCs and Tps are infiltrating dense collection of collagens (green C). (Scale bar 2 μm).

Figure 4

TEM microgram of submucosa and muscularis propria of normal-appearing tissue of urinary bladder with NMIBC. (A,A′) (A: original findings), (A’: digitalized image) showing TC with its distinct nucleus, and Tp (red Asterix) appearing thin, tortuous, and having numerous vesicles (V). TC and Tp are insinuated between two smooth muscle cells (SMC) and abundant collagen (green C). (Scale bar 2 μm). (B,B′) (B: original findings), (B′: digitalized image) showing Tp (blue short arrows and red Asterix) that has numerous vesicles (yellow V) and is attached to the smooth muscle cell (SMC) and collagen (green C). (Scale bar 2 μm). (C,D) showing smooth muscle cells (SMC) with condensation of collagen (c) between smooth muscle cells (SMC), and fibroblast cell (Fb) in close contact. No TCs or Tps were found. (Scale bar C:500 nm, D:2 μm).

Figure 5

Findings in urinary bladder in cases of neurogenic bladder dysfunction. (A) Transmission electron microgram shows TC with its nucleus (n) and short thin cytoplasmic extension of Tp (red triangle). (B) Digitalized image of ultrastructural findings in neurogenic bladder dysfunction shows TC with its nucleus (n), Tp (red arrows) having many vesicles. Telopodes appeared in close contact to smooth muscle cells (SMC) and excess collagen. (Scale bar 2 μm).

Figure 6

Relative quantitative distribution of TCs/TEM fields. Results are expressed as mean ± SD, ****p < 0.001, **p < 0.01, *p < 0.05.

Figure 7

(A) Relative expression levels of ezrin protein. (B) Western blot images for ezrin expression. Results are expressed as mean ± SD, ****p < 0.001, **p < 0.01.