SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

Abstract

BackgroundThis laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.MethodsReal time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.ResultsIt was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.ConclusionsTumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

Enrichment of genes associated with squamous differentiation in cancer initiating cells isolated from urothelial cells transformed by the environmental toxicant arsenite

“…The study also determined the expression of nine basal and 16 luminal markers as defined by Choi and coworkers (2014) in the transformed cell lines and in urospheres (spheroids) isolated from the cell lines. Previous studies from this laboratory have shown the urospheres generated from the As 3+ and Cd 2+transformed cell lines to form tumors in immune-compromised mice with a histology indistinguishable from the originating cell lines, including focal areas of squamous differentiation (Slusser-Nore et al, 2016;Sandquist et al 2016). The results from the Hoggarth et al, 2018 study showed the cell lines to display a gene signature with identity to the basal subtype, and this signature was further enriched in urospheres isolated from the cell lines (Hoggarth et al, 2018).…”

Correlation between secreted protein acidic and rich in cysteine protein expression and the prognosis of postoperative patients exhibiting esophageal squamous cell carcinoma

“…Studies have identified that the high expression level of SPARC protein in melanoma and prostate cancer promotes tumor growth and metastasis (32). However, the SPARC protein may act as an antitumor factor in pancreatic and colorectal cancer, resulting in anti-angiogenesis, apoptosis, inhibition of cell proliferation and cell cycle arrest, thus inhibiting tumor growth (33). In the present study, SPARC protein expression in patients with ESCC was associated with the survival prognosis, and the clinical features of the tumor were significantly associated with survival, differentiation and staging.…”

Activation of PPARγ and inhibition of cell proliferation reduces key proteins associated with the basal subtype of bladder cancer in As3+-transformed UROtsa cells

“…The cells were sub-cultured at a 1:4 ratio using trypsin-EDTA and the cultures were fed fresh growth medium every three days. The UROtsa parent cell line has been authenticated using short tandem repeat (STR) analysis [ 31 ]. The As 3+ -transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar, form spheroids when grown in ultra-low attachment flasks and form tumors when injected subcutaneously in immune-compromised mice [ 17 , 31 – 34 ].…”

“…The cells were sub-cultured at a 1:4 ratio using trypsin-EDTA and the cultures were fed fresh growth medium every three days. The UROtsa parent cell line has been authenticated using short tandem repeat (STR) analysis [31]. The As 3…”

“…-transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar, form spheroids when grown in ultra-low attachment flasks and form tumors when injected subcutaneously in immune-compromised mice [17,[31][32][33][34]. The As#3 can also form tumors upon intraperitoneal injection [33].…”