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. 2023 Jan 21;24(3):2152.
doi: 10.3390/ijms24032152.

Expression of CD22 in Triple-Negative Breast Cancer: A Novel Prognostic Biomarker and Potential Target for CAR Therapy

Tahir Zaib  1   2   3 , Ke Cheng  1   2   3 , Tingdang Liu  1   2   3 , Ruyi Mei  1   2   3 , Qin Liu  1   2   3 , Xiaoling Zhou  1   2   3 , Lifang He  4   5   6 , Hibba Rashid  7 , Qingdong Xie  1   2   3 , Hanif Khan  8 , Yien Xu  3   6 , Pingnan Sun  1   2   3 , Jundong Wu  4   5   6
Affiliations

Expression of CD22 in Triple-Negative Breast Cancer: A Novel Prognostic Biomarker and Potential Target for CAR Therapy

Tahir Zaib et al. Int J Mol Sci. .

Abstract

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Due to the lack of expression of well-known molecular targets [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)], there is a need for more alternative treatment approaches in TNBC. Chimeric antigen receptor (CAR)-T cell-based immunotherapy treatment is one of the latest treatment technologies with outstanding therapeutic advances in the past decade, especially in the treatment of hematologic malignancies, but the therapeutic effects of CAR-T cells against solid tumors have not yet shown significant clinical benefits. Identification of highly specific CAR-T targets in solid tumors is also crucial for its successful treatment. CD22 is reported to be a multifunctional receptor that is mainly expressed on the surface of mature B-cells (lymphocytes) and is also highly expressed in most B-cell malignancies. This study aimed to investigate the expression of CD22 in TNBC. Bioinformatic analysis was performed to evaluate the expression of CD22 in breast carcinoma and normal tissues. RNA-seq data of normal and breast carcinoma patients were downloaded from The Cancer Genome Atlas (TCGA), and differential gene expression was performed using R language. Additionally, online bioinformatics web tools (GEPIA and TNM plot) were used to evaluate the expression of CD22 in breast carcinoma and normal tissues. Western blot (WB) analysis and immunofluorescence (IF) were performed to characterize the expression of CD22 in TNBC cell lines. Immunohistochemical (IHC) staining was performed on tumor specimens from 97 TNBC patients for CD22 expression. Moreover, statistical analysis was performed to analyze the association of clinical pathological parameters with CD22 expression. Correlation analysis between overall survival data of TNBC patients and CD22 expression was also performed. Differential gene expression analysis of TCGA data revealed that CD22 is among the upregulated differentially expressed genes (DEGs) with high expression in breast cancer, as compared to normal breast tissues. WB and IF analysis revealed high expression of CD22 in TNBC cell lines. IHC results also showed that approximately 62.89% (61/97) of TNBC specimens were stained positive for CD22. Cell membrane expression of CD22 was evident in 23.71% (23/97) of TNBC specimens, and 39.18% (38/97) of TNBC specimens showed cytoplasmic/membrane expression, while 37.11% (36/97) specimens were negative for CD22. Furthermore, significant associations were found between the size of tumors in TNBC patients and CD22 expression, which unveils its potential as a prognostic biomarker. No significant correlation was found between the overall survival of TNBC patients and CD22 expression. In conclusion, we demonstrated for the first time that CD22 is highly expressed in TNBC. Based on our findings, we anticipated that CD22 could be used as a prognostic biomarker in TNBC, and it might be a potential CAR-T target in TNBC for whom few therapeutic options exist. However, more large-scale studies and clinical trials will ensure its potential usefulness as a CAR-T target in TNBC.

Keywords: CAR-T; CD22; biomarkers; immunohistochemistry; triple negative breast cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bioinformatics analysis of CD22 expression in breast cancer and normal tissues. (A) Heatmap of the expression values for CD22 from RNA-seq data in TCGA. (B) BOX plot for CD22 expression from GEPIA shows that CD22 is relatively highly expressed in Breast tumor as compared to normal tissues. (C) TNMplot for CD22 expression also shows high expression of CD22 in breast tumor as compared to normal tissues.
Figure 2
Figure 2
Expression of CD22 in breast cancer cell lines. (A) Western blot results for CD22 expression. BT549, MCF7, and MDA-MB-231 were positive for CD22 expression. Raji was used as the positive control, and K562 and MSCs were used as negative controls for CD22. (B) Graphical representation of relative CD22 protein expression values from the western blot analysis. (C) Representative images of CD22 expression by immunofluorescence of MSCs and breast cancer cell lines: BT549, MDA-MB-231, and MCF7. MSCs (negative control) showed no fluorescence. Membrane expression of CD22 stained in green can be seen in the BT549 and MDA-MB-231cell lines. Cytoplasmic expression of CD22 can be seen in the MCF7. Nuclei were stained with DAPI (4′, 6-diamidino-2-phenylindole) (blue). (Scale Bar: 100 µm).
Figure 3
Figure 3
Representative images of immunohistochemical staining of CD22 expression in TNBC specimens. (A) Tonsillar carcinoma incubated with PBS showed no signal; (B) Tonsillar carcinoma incubated with anti-CD22 antibody showed positive expression for CD22; (C) TNBC sample negative for CD22. Relative expression of CD22 in TNBC samples (D) weak-positive, (E) moderate-positive, (F) strong-positive. (G,H) Membrane expression of CD22 in TNBC cells. The red arrows marked membrane expression of CD22 in TNBC cells. Magnifications: 20×, Scale bar 100 µm.
Figure 4
Figure 4
Correlation analysis of CD22 expression and overall Survival of TNBC patients (A) Correlation analysis of overall survival of TNBC patients and CD22 expression. (B) Survival plots for CD22 expression and breast cancer patients from GEPIA showed no significant association between both. The solid lines represent survival curve, and the dashed lines represent 95% confidence intervals.
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