The Cancer Genome Atlas-Breast Cancer (TCGA-BRCA)-related data were downloaded from the Gene Expression Datasets Associated with BC using the Genomic Data Commons (GDC) data portal (https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). The analysis of the differential expression of CD47 in breast cancer and adjacent non-cancerous tissues was conducted using Gene Expression Profiling Interactive Analysis (GEPHIA2; http://gepia2.cancer-pku.cn/).

In the TCGA-BRCA, GSE15852, and GSE42568 datasets, using the “DESeq2” software package (dds - DESeqDataSetFromMatrix (countData, colData, design = ~ condition); alpha = 0.05; lfcThreshold $>$1; Version 3.0; Michael Love and Wolfgang Huber; London, USA), we screened out the DEGs in the samples with high and low expression of CD47 in tumor tissues. Venn diagrams showed co-expressed genes, and functional enrichment analysis utilizing Gene Ontology (GO) alongside Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was conducted through the cluster-Profiler R package 4.3.2 (Michael Love and Wolfgang Huber; NY, USA) [12]. This analysis aimed to categorize a range of biological processes, molecular functions, cellular components, and pathways associated with the identified marker genes within the target clusters [13, 14] (Supplementary Fig. 1A,B).

This research conducted a retrospective analysis of breast cancer cases stored in the Pathology Department at Shandong Third Hospital from 2020 to 2022. The analysis included 108 patients who had been diagnosed with invasive ductal carcinoma of the breast, all of whom did not receive preoperative chemotherapy, radiotherapy, or endocrine treatment. This study was conducted in accordance with the Declaration of Helsinki and was approved by the relevant ethics committee. The Ethics Committee and Institutional Review Board of the Third Hospital of Shandong Province approved the study, and the necessity for informed consent was signed (No. KYLL-2021066). An immunohistochemical staining assay was used to detect 108 paired BC tissue samples. Rabbit anti-human CD47 monoclonal antibody was purchased (ab300124, Cell Signaling Technology, Inc. Boston, USA; Dilution concentration: 1:200). Staining results were analyzed using ImageJ software (Version 1.8.0.345; National Institutes of Health; NY, USA) [15] and interpreted according to the semi-quantitative method utilized to assess the expression of CD47 in BC and paraneoplastic samples.

Human BC cells (MDA-MB-231 cell line; ZQ0118) and mouse BC 4T-1 cells (ZQ0201; Shanghai Zhongqiao Xinzhou Biological Company, Shanghai, China) were inoculated in Dulbecco’s Modified Eagle Medium (Gibcp 11885084, Suzhou, Jiangsu, China) with 10% fetal bovine serum (Suolaibao technology company, Beijing, China) and 100 IU/mL penicillin/streptomycin at 37 °C with 5% CO₂. All cell lines were validated by STR profiling and tested negative for Mycoplasma.

Cells were placed in six-well plates at a density of 2 $\times$ 10⁵ cells per well and allowed to incubate overnight to achieve 90% confluency. For each well, plasmid DNA [2 µg CD47-short hairpin RNA (shRNA); D010003, GenePharma company; Shanghai, China] was diluted in 250 µL of DMEM (ZQ-100; Shanghai Zhong Qiao Xin Zhou Biotechnology Co, Shanghai, China) and mixed gently. Separately, 10 µL of transfection reagent (e.g., Lipofectamine 3000, ThermoFisher, Shanghai, China) incubated for 5 min at 24 °C. The diluted DNA and transfection reagent were then combined, incubated for 20 min, and were added dropwise to the cells, which were then incubated at 37 °C in a CO₂ incubator. After 6 hours, the medium was replaced with a fresh fetal medium. Transfection efficiency was assessed 24–48 h post-transfection using quantitative real-time (qPCR) and Western blot (WB) assays (Supplementary Tables 1,2).

Total RNA was extracted from cultured cells by Easy Pure RNA Isolation Kit (R0018S; Beyotime Biotechnology, Shanghai, China) and a reverse-transcription (RT) Kit (D7153; BeyoRT M-MuLV, Beyotime Biotechnlogy Company, Shanghai, China). The cDNA was subjected to qRT-PCR with gene-specific primers in the presence of qPCR Master Mix (BeyoRT II cDNA, Beyotime Biotechnlogy Company, Shanghai, China). Relative mRNA expression levels were calculated using the 2-ΔΔCt method, where ΔCt represents the difference in threshold cycle (Ct) values between the target gene and a reference gene (e.g., GAPDH (Supplementary Table 3). All samples were analyzed in triplicate.

The total protein was extracted from the cells using the Radio Immunoprecipitation Assay (RIPA) extraction reagent (Beyotime Biotechnology Company, Shanghai, China), and the protein concentration was determined using a bicinchoninic acid protein assay (Beyotime Biotechnology Company, Shanghai, China). Subsequently, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The primary antibody (anti-CD47:1:2000, 20305–1-AP; anti-$\beta$-actin: 1:2000, 10494-1-AP, Proteintech, Rosemont, IL, Beijing, China) and the secondary antibody (IgG-HRP, 1:3000, AS014; Wolburn, Boston, MA, USA) were incubated, respectively. Protein bands were visualized using chemiluminescence and analyzed using the ImageJ software (Version 2.0; available from the National Institutes of Health, USA) (Supplementary Fig. 2).

Experimental cells were plated in a 96-well plate with a density of 5000 cells per well. The proliferation rates of 4T-1 and MB-231 cells from both the short hairpin (sh)-CD47 and control groups were assessed. Cell viability measurements occurred every 12 hours. Daily, 10 µL of CCK-8 reagent (MCE, Shanghai, China) was added to each well of the 96-well plates and incubated at 37 °C for one hour. Optical density values were then determined using a microplate reader (Infinite® M1000PRO, TECAN, Männedorf, Switzerland) at a 450-nm wavelength.

In summary, 400 cells were placed in a six-well plate and allowed to incubate in culture medium containing 10% fetal bovine serum at a temperature of 37 °C with 5% CO₂ for a duration of 2 weeks. The culture medium was replaced every three days. Prior to staining with 0.1% crystal violet, the cells were fixed using 4% paraformaldehyde for 30 minutes. The clones were then quantified using ImageJ software.

Cells from each group were inoculated in six-well plates (2 $\times$ 10⁵ cells/well). When the cell growth reached 85% confluence, the bottom of the six-well plate was scratched vertically and cultured in Dulbecco’s Modified Eagle Medium with DMEM. The scratches were observed under a microscope (Olympus, IX-71, Tokyo, Japan,) and photographed at 0, 24, and 48 h. The healing rate was determined using the formula ((initial scratch area - scratch area at time t)/(initial scratch area)) $\times$ 100%.

The capability of cell migration was assessed using 24-well transwell chambers (with an 8-µm pore size; Sigma-Aldrich, Corning, NY, USA). In the upper compartment, 100 µL of serum-free medium was combined with 2 $\times$ 10⁴ cells, while the lower compartment contained 600 µL of medium supplemented with 10% fetal bovine serum. Following 48 hours of culture in a 37 °C incubator, the cells were fixed using 4% paraformaldehyde and subsequently treated with 0.4% crystal violet solution (Sigma-Aldrich, V5265, Shanghai, China). The stained cells were then photographed (Olympus, Tokyo, Japan) and quantified.

Culture the cells until they reach 70–80% confluence, then detach and re-suspend them in fresh medium. Use diluted Matrigel (3 mg/mL; M8370, Suolaibao company, Beijing, China) coated on the upper surface of the transwell inserts and add a predetermined number of cells to the upper chamber, while adding complete medium to the lower chamber. This process should be performed on ice, allowing the Matrigel to solidify at 37 °C for 45 minutes. Incubate the setup at 37 °C with 5% CO₂ for 24–48 hours. After incubation, fix the migrated cells on the lower surface of the insert using a fixative solution, followed by staining with a crystal violet. Once stained, rinse to remove excess dye, allow the inserts to dry, and then examine them under a microscope to count the number of invaded cells.

Female BALB/C nude mice (5-weeks-old; 17.54 $\pm$ 0.867 g; 45 in all; Beijing Viewsolid Biotech Co., Ltd., Beijing, China) were utilized to construct the xenograft model. Knocked-down 4T-1/sh-NC (Negative Control) and 4T-1/sh-CD47 cells were suspended in 100 µL of PBS and injected subcutaneously. Tumors were measured every 2–3 d. After 33 days, mice were euthanized with a 20% concentration of isoflurane (1217828, Shanghai Yuyan Scientific Instrument Co., LTD, Shanghai, China) in airtight equipment for three minutes according to the ethical guidelines [16], and xenograft tumors were dissected for subsequent experiments. All animal experimental processes were approved by the Laboratory Animal Center of Shandong Third Hospital (No. DWKYLL-2021023).

Statistical analyses were conducted utilizing IBM SPSS Statistics software (IBM Corp., Armonk, NY, USA) version 29.0, along with GraphPad Prism 10.0 (GraphPad Software, La Jolla, CA, USA), and R version 3.6.1 (R Foundation for Statistical Computing, Vienna, Austria) [12]. The results of the IHC were evaluated employing the chi-square test. A p-value of less than 0.05 was deemed statistically significant. To compare the two sample groups, an independent samples t-test was used, whereas a one-way ANOVA was applied for comparisons involving multiple groups. Following one-way ANOVA, post-hoc multiple comparisons were performed using the Tukey’s HSD (Honestly Significant Difference) test to evaluate differences between group means. This method was chosen for its ability to control the Type I error rate while comparing all pairs of means. All statistical tests were two-sided, and each experiment was conducted three times for every group.

To elucidate the divergence in CD47 expression in BC, we analyzed two datasets derived from TCGA-BRCA databases: GSE42568 and GSE15852. Our findings indicated that CD47 expression was significantly high in BC samples comparing to normal breast samples (Fig. 1A,B). Subsequently, we used a Venn gram to identify approximately 11,194 genes co-expressed across the three datasets (Fig. 1C,D). To conduct a comprehensive analysis of the signaling pathways correlated to the regulation of activated CD47 from KEGG database [13], we established that elevated CD47 levels in BC were linked to several critical pathways, including cytokine-cytokine receptor interactions, the JAK/STAT signaling pathway, chemokine signaling pathway, and cell adhesion molecule pathways. These pathways are pivotal in driving processes such as cell proliferation, apoptosis, metabolism, and invasiveness (Fig. 1E; Supplementary Fig. 3). Additionally, the GO analysis indicated that differentially expressed genes (DEGs) associated with elevated CD47 expression were connected to the enhancement of cytokine production, activity of receptor ligands, and the activation of signaling receptors (Fig. 1F).

Fig. 1.

Bioinformatic analysis of CD47 in two databases. (A) Column diagrams of CD47 expression in tumors and normal tissues based on The Cancer Genome Atlas and Gene Expression Omnibus databases. (B) Volcano plots of differentially expressed genes according to three databases. (C) Venn diagram of three databases showing co-expressed genes. (D) Gene numbers in the three databases. (E,F) The Gene Ontology/Kyoto Encyclopedia of Genes and Genomes clustering analysis of high CD4 expression-associated signaling pathways and functions. BP, Biological Process; CC, Cellular Component; MF, Molecular Function.

To investigate the relationship between CD47 level and BC, we collected data from 108 clinical cases between January 2020 and January 2022. IHC staining demonstrated that CD47 was primarily localized on the membrane of BC cells, and these levels were significantly higher in cancer tissues than in para-cancerous tissues (Fig. 2A,B; Table 1; p 0.0001). Furthermore, statistical analyses uncovered that increased CD47 expression was associated with several factors, including high Ki-67 levels, age $>$60 years, and TNM stage. Nevertheless, there were no statistically significant differences in CD47 levels between the estrogen receptor-, progesterone receptor, or Human Epidermal Growth Factor Receptor-positive and negative groups (Table 2).

Fig. 2.

The clinical significance of CD47 expression in breast cancer. (A,B) CD47 expression in cancer and paired para-cancerous tissues by immunohistochemical staining. The scale bar = 100 or 200 µm. All experiments had three biological replicates with similar results. Data are presented as mean $\pm$ standard deviation. ****p $\le$ 0.0001.

We performed in vitro and in vivo experiments to verify the function of CD47 in BC. Given the elevated levels of CD47, we transfected three shRNA plasmids into 4T1 and MB-231 cells to downregulate CD47 expression (Supplementary Fig. 1). Notably, cell viability was overtly lower in the sh-CD47 group than that in the control group, which was further corroborated by monoclonal proliferation results using CCK-8 staining (Fig. 3). The wound healing assay results for 4T1 and MB-231 cells demonstrated that the migratory ratios at 24 and 48 hours were reduced in the sh-CD47 group compared with the control groups (p 0.0001; Fig. 4A–D). Combined with the outcomes of the cell migration assay, the invasive ability of sh-CD47 cancer cells decreased compared to that of normal cancer cells (p 0.05 in 4T1 cells; p 0.001 in MB-231 cells; Fig. 4E,F). Furthermore, we established subcutaneous tumors in nude mice using sh-CD47 4T1, control 4T1, and 4T1 cells with empty plasmids. The results indicated that the tumor volume was distinctly lower in the sh-CD47 group than that in the other two groups (Fig. 5A–E). In terms of the proliferative markers, Cyclin dependent kinase 4 (CDK4) and cyclin dependent kinase 6 (CDK6) expression in BC tumors in the sh-CD47 group had lower levels than that in the control groups (Fig. 5F–I).

Fig. 3.

The functional experiment of CD47 in breast cancer cells. (A,B) Cell viability of 4T1 and MB-231 cells transfected withshort hairpin-CD47, negative control (NC) plasmids were detected by Cell Counting Kit-8 (CCK-8) staining assay. (C–F) Cell monoclonal proliferation of 4T1 and MB-231 cells. The scale bar = 1 cm. *p 0.05, **p $\le$ 0.01, ***p 0.001, ****p $\le$ 0.0001.

Fig. 4.

CD47 knockdown attenuated the invasion and migration behavior of breast cancer cells. (A–D) Wound healing of 4T1 and MB-231 cells transfected with sh-CD47 and sh-NC plasmids was measured at 24 h and 48 h. The scale bar = 500 µm. (E,F) The transwell detection of 4T1 and MB-231 cells transfected with sh-CD47 and sh-NC plasmids is shown. The scale bar = 50 or 100 µm. *p 0.05, ***p 0.001, ****p $\le$ 0.0001.

Fig. 5.

CD47 knockdown decreased xenograft tumor volume and weight in nude mice. (A) Gross images of nude mice with BC tumors in the sh-CD47 and control groups. (B,C) Distinct tumor volumes and weights of nude mice are shown. (D,E) The gross species of BC tumors in the sh-CD47 group and the control group are shown; tumor weights were analyzed. (F,G) The cyclin dependent kinase 4/6 expression of BC tumors by immunohistochemistry staining assay. The scale bar = 100 or 500 µm. (H,I) Relative expression values of cyclin dependent kinase 4/6 (n=5). (***p 0.001; ****p 0.0001).