2.1 Cell lines

Human umbilical vein endothelial cells (HUVECs) and TNBC Hs-578T cells were obtained from the American Type Culture Collection (ATCC). These cells were cultivated at 37 °C in presence of 5% carbon dioxide (CO₂) and humidity in Dulbecco’s modified Eagle’s medium (DMEM, MT10013CV, Gibco, Waltham, MA, USA) supplemented with 3.7 g/L sodium bicarbonate, 10% fetal bovine serum (FBS, FB12999102, Gibco, Waltham, MA, USA), and 50 units/mL of penicillin and streptomycin (15-140-163, Invitrogen, Carlsbad, CA, USA).

2.2 Cell transfection

TSPAN1 expression in Hs-578T cells was inhibited by specific siRNA targeting TSPAN1 (Thermo Fisher Scientific, Waltham, MA, USA). Scrambled siRNA-transfected cells (si-NC, Thermo Fisher Scientific, Waltham, MA, USA) served as controls. The sequences of the TSPAN1 si-1 group were 5′-GGCUCACGACCAAAAAGUAtt-3′ (sense) and 5′-UACUUUUUGGUCGUGAGCCtt-3′ (antisense). The sequences of the TSPAN1 si-2 group were: 5′-GCUUUUGUAUGACAUCCGAtt-3′ (sense), 5′-UCGGAUGUCAUACAAAAGCtg-3′ (antisense). The sequences of the scrambled siRNA group were 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (sense), 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (antisense). Lipofectamine 3000 (L3000008, Thermo Fisher Scientific, Waltham, MA, USA) was used to transfect Hs-578T cells with 50 nM siRNA following manufacturer’s instructions. TSPAN1 expression was detected 48 h after transfection.

2.3 Angiogenesis assays

For this assay, 50 μL Matrigel (8 mg/mL, CB-40234C, Thermo Fisher Scientific, Waltham, MA, USA) was added into the lower chamber of a 24-well Transwell and incubated at 37 °C for 1 h. Following this, 1 × 10⁴ HUVECs and Hs-578T cells were co-cultured at 37 °C for 24 h in the lower and upper chambers of Transwell, respectively. Images of tubular structures were captured using an optical microscope (Inverted Fluorescence Microscope, Olympus Corporation, Tokyo, Japan), and the images were counted using ImageJ software (v2021.8.0).

2.4 Western blot analysis

Total protein was extracted from tissues and cells using Radioimmunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China), and the concentration was measured using a Bicinchoninic Acid Assay (BCA) kit (P0009, Beyotime, Shanghai, China). Isolated proteins were split into various pieces based on their molecular weights using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The samples were then transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA), blocked with 5% skim milk for 1 h at 25 °C, and incubated with the corresponding primary antibody overnight at 4 °C. After that, the membrane was treated for 1 h with a secondary antibody coupled with horseradish peroxidase (P2369, Beyotime, Shanghai, China). Ultimately, an enhanced chemiluminescence detection kit (P10010, NCM Biotech, Shanghai, China) was used to visualize the protein band. The bands were quantified using ImageJ 8.0 software, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference protein. The primary antibodies (1:1000, Abcam, Cambridge, UK) analysed were as follows: TSPAN1 (ab254730), PI3K (ab302958), p-PI3K (ab278545), Akt (ab314110), p-Akt (ab8805), Cluster of Differentiation 31 (CD31, ab281583), Matrix Metalloproteinase (MMP-2, ab92536), MMP-9 (ab76003) and GAPDH (ab32096).

2.5 Tumor samples

Paraffin-embedded specimens from 126 patients with triple-negative breast cancer admitted to Pingshan District People’s Hospital between January 2021 and December 2023 were gathered for retrospective investigation. Patient’s age at diagnosis, their tumor grade, tumor size, clinical stage, and lymph node status were obtained from the medical data record. The Tumor-Node-Metastasis (TNM) method served as the basis for the histological type. Prior to the follow-up deadline, all patients received routine follow-ups by phone or in-person visits. Informed consent was obtained from those patients in their follow-up visit and their tissue specimens were taken for further investigation. All these cancer tissues were tested for the expression of TSPAN1 by immunohistochemistry.

2.6 Inclusion criteria

Confirmed diagnosis: all patients included in the study were diagnosed with TNBC by pathological examination. Specifically, their tumor showed no expression of the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2).

Sample integrity: the patient’s tumor tissue and corresponding clinical data (including age, tumor size, tumor grade, clinical staging, and lymph node status, etc.) were complete and available.

Ethical approval: all patients or their legal representatives signed an informed consent form, agreeing that their anonymized clinical information will be used for research purposes. The hospital’s ethics committee approved the research plan (approval number: 2023013).

2.7 Exclusion criteria

Non-primary tumor: patients diagnosed with metastatic breast cancer or non-primary breast cancer confirmed by pathological evaluation were excluded.

Concurrent malignancies: patients diagnosed with other malignant tumors before or at the time as TNBC diagnosis were excluded.

Patients receiving neoadjuvant therapy: patients who had received chemotherapy, radiation therapy, or other systemic treatments before surgery were excluded, as these treatments could affect the biological characteristics of the tumor.

Incomplete clinical data: patients with missing or incomplete clinical data were excluded to ensure the accuracy and reproducibility of research results.

2.8 Statistical analyses

The statistical analysis was carried out using IBM SPSS software (version 25, Armonk, NY, USA). The relationship between TSPAN1 expression levels and clinicopathological characteristics was investigated using the chi-square test or Fisher’s exact probability method. Differences between the two groups were examined using two-tailed Student’s t-test. Tukey’s post hoc test was employed to determine significance in the group differences. A p value of less than 0.05 was considered statistically significant.

3.1 TSPAN1/PI3K/Akt signaling is upregulated in triple-negative breast cancer

To study the significance of TSPAN1 in breast cancer, we first reviewed data from a public database, The Cancer Genome Atlas (TCGA), and showed that TSPAN1 expression was much greater in breast cancer tissues than in normal breast tissues (Fig. 1A) (p < 0.05). We then tested cancer and adjacent tissues from 20 TNBC patients and found that the expression of TSPAN1 and CD31 was higher in TNBC tissues, accompanied by PI3K/Akt signaling pathway activation (Fig. 1B) (p < 0.001). These results suggested that high expression of TSPAN1, activation of PI3K/Akt signaling pathway, and angiogenesis are closely related to the development of TNBC.

Fig. 1.TSPAN1/PI3K/Akt signaling is upregulated in triple-negative breast cancer. (A) GEPIA website analysis of the expression difference of TSPAN1 in breast cancer tissues and normal tissues. (B) Protein expression of TSPAN1, PI3K, p-PI3K, Akt, p-Akt and CD31 in TNBC cancer tissue and adjacent tissue. *: p < 0.05; ***: p < 0.001. n = 20. PI3K: phosphoinositide 3-kinase; Akt: protein kinase B; TSPAN1: tetraspanin 1; TPM: Transcripts Per Million; BRCA: Breast invasive carcinoma; CD: Cluster of Differentiation; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

We further examined the association between TSPAN1 expression in TNBC tumor samples and clinical pathological features of breast cancer, such as age, tumor size, T classification, N classification, and TNM stage. Table 1 illustrates the approximate correlation between TSPAN1 expression in TNBC and lymph node metastases, N classification, and T classification. These findings showed that TSPAN1 might play a major role in TNBC metastasis.

3.2 Knockdown of TSPAN1 inhibits angiogenesis in breast cancer cells

To elucidate the role and mechanism of TSPAN1 in the development of TNBC, we performed siRNA-mediated knockdown of TSPAN1 in the TNBC cell line Hs-578T (Fig. 2A) (p < 0.001). The results showed that knockdown of TSPAN1 could inhibit the expression of matrix metalloproteinase (MMP)-2 and MMP-9 angiogenic factors (Fig. 2A) (p < 0.001). Additionally, HUVECs angiogenesis assay showed that knockdown of TSPAN1 could reduce the number of blood vessels (Fig. 2B) (p < 0.001). The above results showed that knockdown of TSPAN1 inhibits angiogenesis by reducing MMP-2 and MMP-9.

Fig. 2.Knockdown of TSPAN1 inhibits angiogenesis in breast cancer cells. (A) Western blotting to detect the expression of TSPAN1, MMP-2, MMP-9. (B) Detection of angiogenesis ability of HUVECs. Values are presented as mean ± SD. **p < 0.01 vs. si-NC group; ***p < 0.001 vs. si-NC group. n = 3. si-NC: siRNA-transfected cells; TSPAN: tetraspanin; MMP: Matrix Metalloproteinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

3.3 Knockdown of TSPAN1 inhibits the PI3K/Akt pathway

Because previous results showed elevation of TSPAN1 and increased activation of PI3K/Akt signaling pathway in TNBC tissues, we next investigated whether TSPAN1 regulates the PI3K/Akt pathway in TNBC cells. Knocking down TSPAN1 affects the PI3K/Akt signaling pathway (Fig. 3) (p < 0.001), leading to decreased angiogenesis in TNBC. These findings imply that TSPAN1 plays an essential role in TNBC angiogenesis via the PI3K/Akt signaling pathway.

Fig. 3.Knockdown of TSPAN1 inhibits the PI3K/Akt pathway. Western blotting to detect the expression of TSPAN1, PI3K, p-PI3K, Akt, p-Akt in TNBC Hs-578T cells. Values are presented as mean ± SD. ***p < 0.001 vs. si-NC group. n = 3. TSPAN: tetraspanin; PI3K: phosphoinositide 3-kinase; Akt: protein kinase B; si-NC: siRNA-transfected cells; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.