CD147 and Cyclooxygenase Expression in Feline Oral Squamous Cell Carcinoma

Abstract

Feline oral squamous cell carcinoma (OSCC) is a highly invasive form of cancer in cats. In human OSCC, cluster of differentiation 147 (CD147) contributes to inflammation and tumor invasiveness. CD147 is a potential therapeutic target, but the expression of CD147 in feline OSCC has not been examined. Immunohistochemistry was used to determine if cyclooxygenase 2 (COX-2) and CD147 expression in feline OSCC biopsies was coordinated. Tumor cells were more likely to express COX-2 (22/43 cases or 51%) compared to stroma (8/43 or 19%) and adjacent oral epithelium (9/31 cases or 29%) (p < 0.05). CD147 was also more likely to occur in tumor cells compared to stroma and adjacent mucosa, with 21/43 (49%) of cases having >50% tumor cells with mild or moderate CD147 expression, compared to 9/28 (32%) in adjacent epithelium and only 5/43 (12%) in adjacent stroma (p < 0.05). In feline OSCC cell lines (SCCF1, SCCF2, and SCCF3), CD147 gene expression was more consistently expressed compared to COX-2, which was 60-fold higher in SCCF2 cells compared to SCCF1 cells (p < 0.05). CD147 expression did not correlate with COX-2 expression and prostaglandin E2 (PGE2) secretion, indicating that they may be independently regulated. CD147 potentially represents a novel therapeutic target for the treatment of feline OSCC and further study of CD147 is warranted.

Keywords: Basigin; CD147; COX-1; COX-2; EMMPRIN; PGE2; feline; inflammation; invasion; oral squamous cell carcinoma.

Figure 1

COX-2 and CD147 positive and negative controls for immunohistochemistry. (A–D) Photomicrographs of IHC staining using rabbit anti-COX-2 IgG (1:200) and normal rabbit IgG (1:200, negative control). The chromogen is DAB (brown), and the counterstain is hematoxylin (blue). The feline renal macula densa epithelial cells had an intense cytoplasmic signal (A) that was eliminated when the antibody was replaced with normal IgG (B). Scattered oral squamous cell carcinoma (OSCC) cells showed an intense cytoplasmic COX-2 signal (C), which was eliminated when the primary antibody was replaced with normal IgG (D). (E,F) IHC staining using goat anti-CD147 IgG (1:100) and normal goat IgG (1:100, negative control). Feline enterocytes showed moderately intense cytoplasmic and membranous signal (E), which was markedly reduced when the primary antibody was replaced with normal goat IgG (F). There was widespread moderate staining and scattered heavy staining of OSCC cells (cytoplasm and membrane), and light to moderate widespread staining of stromal cells (G). Staining was markedly reduced when the primary antibody was replaced with normal goat IgG (H). Each pair of positive and negative control images are at the same magnification. Scale bars are 50 μM long.

Figure 2

COX-2 and CD147 expression varied between cases of feline OSCC. Photomicrographs of IHC staining using rabbit anti-COX-2 IgG (1:200) and goat anti-CD147 IgG (1:100). The chromogen is DAB (brown), and the counterstain is hematoxylin (blue). Case 1: There are scattered OSCC cells with an intense COX-2 signal (A). In contrast, OSCC cells showed widespread light to moderate CD147 signal (B). Case 2: This sample was COX-2 negative (C), but showed widespread moderate staining and scattered heavy staining for CD147 (D). Case 3: This sample was COX-2 negative (E) and CD147 negative in tumour cells (F), but had scattered moderate CD147 staining in the stroma (F). All images are at the same magnification. Scale bars are 100 μM long.

Figure 3

Expression of COX-2 and CD147 in feline OSCC samples using an IHC grading system. (A) COX-2 staining results were obtained from 43 samples, 31 of which included adjacent oral epithelium. Each bar represents the percentage of cases that were considered positive for COX-2 expression within each compartment (tumor cells, stroma, and adjacent epithelium). Each bar is subdivided to demonstrate the percentage of cases assigned to each grade. COX-2 expression was highest in the tumor cells. (B) CD147 staining results were obtained from 43 samples, 28 of which included adjacent oral epithelium. CD147 expression was highest in the tumor cells. (C) Paired COX-2 and CD147 IHC results were available from 42 OSCC cases. Twenty-two (22) cases had positive COX-2 expression in the tumor cells (grades 1 and 2) and 20 cases were COX-2 negative in the tumor cells (grade 0). Each bar represents the percentage of positive CD147 cases within the COX-2 positive and COX-2 negative groups. There was no significant difference in CD147 expression between COX-2 positive and COX-2 negative cases. There were no cases with grade 5 staining. All of the statistical comparisons were made using Fisher’s exact test (* p value < 0.05).

Figure 4

COX-1, COX-2, and CD147 mRNA expression, and prostaglandin E2 (PGE2) secretion from feline OSCC cell lines in vitro. (A) Each bar represents the mean fold increase in COX-1 mRNA compared to serum-deprived SCCF2 cells. There was no significant effect of serum exposure on COX-1 expression. The expression of COX-1 differed between the three cell lines in serum deprived (*) and stimulated (**) conditions, with SCCF3 expressing the most COX-1, and SCCF1 cells expressing the least COX-1. (B) Each bar represents the mean fold increase in COX-2 mRNA compared to serum-deprived SCCF3 cells. Serum exposure significantly stimulated COX-2 expression in all three cell lines. The expression of COX-2 differed between the three cell lines in serum-deprived (*) and stimulated (**) conditions, with SCCF2 cells expressing the most, and SCCF1 cells expressing the least. (C) Each bar represents the mean fold increase in CD147 mRNA compared to serum-stimulated SCCF3 cells. Serum exposure only had a significant effect on the SCCF3 expression of CD147. The expression of CD147 differed between the three cell lines in serum-deprived (*) and stimulated (**) conditions, with SCCF3 cells expressing the least CD147. (D) Each bar represents the mean PGE2 concentration relative to conditioned medium from SCCF1 cells. SCCF1 cells secreted the least PGE2. The average concentration of PGE2 in SCCF1-conditioned medium was 22.2 pg/mL per 100,000 cells. Each graph represents combined data from three independent experiments. All of the statistical comparisons of RT-qPCR data were made using the Kruskal–Wallis test (* serum-deprived, p value < 0.05; ** serum-stimulated, p value < 0.05; # serum-deprived compared to serum-stimulated, p value < 0.05). PGE2 concentrations were compared using the Kruskal–Wallis test (* p value < 0.05).