Expression of bladder cancer-associated glycans in murine tumor cell lines

Abstract

The characterization of murine cell lines is of great importance in order to identify preclinical models that could resemble human diseases. Aberrant glycosylation includes the loss, excessive or novel expression of glycans and the appearance of truncated structures. MB49 and MB49-I are currently the only two murine cell lines available for the development of preclinical bladder cancer models. The glycans Lewis X (LeX), Sialyl lewis X (SLeX) and Sialyl Tn (STn) have previously been associated with aggressiveness, dissemination and poor prognosis in human bladder cancer, additionally N-glycolyl GM3 (NGcGM3) is a neo-antigen expressed in many types of tumors; however, to the best of our knowledge, its expression has not previously been assessed in this type of cancer. Taking into account the relevance of glycans in tumor biology and considering that they can act as targets of therapies and biomarkers, the present study evaluated the expression of LeX, SLeX, STn and NGcGM3 in MB49 and MB49-I cells, in different growth conditions such as monolayer cultures, three-dimensional multicellular spheroids and mouse heterotopic and orthotopic tumors. The expression of LeX was not detected in either cell line, whereas SLeX was expressed in monolayers, spheroids and orthotopic tumors of both cell lines. STn was only identified in MB49 monolayers and spheroids. There are no reports concerning the expression of NGcGM3 in human or murine bladder cancer. In our hands, MB49 and MB49-I expressed this ganglioside in all the growth conditions evaluated. The assessment of its expression in cancer cell lines and patient tumors is of great importance, considering the relevance of this ganglioside in tumor biology. The data obtained by the present study demonstrates that glycan expression may be substantially altered depending on the growth conditions, highlighting the importance of the characterization of murine cancer models. To the best of our knowledge, the present study is the first to examine the expression of cancer-associated glycans, in the two murine cell lines available for the development of preclinical studies in bladder cancer.

Keywords: MB49; MB49-I; bladder cancer; glycans; murine models.

Figure 1.

Monosaccharide structure of the glycans analyzed in the present study. LeX, Lewis X; SLeX, Sialyl Lewis X; STn, Sialyl Tn; NGcGM3, N-glycolyl GM3. The letter R represents any structure of a protein or lipid.

Figure 2.

Evaluation of lectins (A) Mal II and (B) SNA binding to MB49 and MB49-I cell lines by FACS. Results correspond to median fluorescence intensity relative to negative control (PBS) of five independent experiments. Data are expressed as median with interquartile range; Mann Whitney test *P<0.05. FACS, fluorescence activated cell sorting; MFI, Median fluorescence intensity values.

Figure 3.

Evaluation of glycan expression in (A) MB49 and (B) MB49-I cell lines by FACS. The percentage of positive cells for each glycan is indicated in the graphs. Representative dot plots of at least three independent experiments. FSC-H, forward side scatter.

Figure 4.

Evaluation of glycan expression in (A) MB49 and (B) MB49-I three-dimensional tumor spheroids by IHC. Photos are representative of at least three experiments. Original magnification, ×400.

Figure 5.

Evaluation of glycan expression in tumors by IHC. (A) MB49 heterotopic tumors; (B) MB49-I heterotopic tumors; (C) MB49 orthotopic tumors; (D) MB49-I orthotopic tumors. Photos are representative of at least three experiments. Original magnification, ×400.

Figure 6.

Evaluation of NGcGM3 expression in (A) T24 cell line by FACS; Representative dot plots of at least three independent experiments. (B) T24 three-dimensional tumor spheroids by IHC. Photos are representative of at least three experiments. Original magnification, ×400. The percentage of positive cells for each glycan is indicated in the graphs. FSC-H, forward side scatter.