Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles

Abstract Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


RT-qPCR analysis of Tspan8 -coding mRNA in human tumours
Supplementary Figure S1 Tspan8 mRNA expression was tested by RT-qPCR in 15 samples: 7 biopsies of breast cancer patients, 7 lung and lymph node metastases and 1 tumour-free sample as a control. The data show a heterogeneous expression of Tspan8 mRNA in primary tumours and metastases with a higher expression range and higher heterogeneity in metastases as compared to the primary tumours.   1000 cells were seeded into sterile agarose cavities and maintained for 1 day in medium to form aggregates.
Cavities were then filled with a desired extracellular matrix (collagen I or BME) and maintained in culture for 7 d.
For imaging, agarose bars were disconnected from the carrier and flipped 90°. Images were taken using bright-field microscopy, then quantitative analysis was performed using ImageJ.
For quantification, images were taken using 4x objective and a resolution of 612 pixel / 1000 µm. In each image, contours of cell aggregate (blue) were marked manually using ImageJ and quantified. For each group at least 8 cell aggregates were evaluated (range: 8 -15). For each cell line the experiment was performed at least twice. Using ImageJ the area of cell aggregates was calculated in µm². Additionally, length of outgrowths was assessed.
Supplementary Figure S4 Radiation resistance test of MTPa-pcDNA3 and MTPa-Tspan8 cells under 3D conditions To assess radiation resistance, anchorage independent colony formation ability of cells upon ionizing irradiation was tested. For that, 10 cells/microwell were seeded in customised agarose inserts with 100 microwells/cm 2 . On day 2, cells were exposed to different radiation doses using 137 Cs at 0.66 Gy/min and maintained for 13 d.
To enhance visibility, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) staining was performed on day 13. Agarose inserts were scanned on a high-resolution flatbed scanner (CanoScan 9000F Mark II, Canon Inc.), using the transmitted light modus at 1200 dpi. The projected areas of individual cell aggregates was measured using ImageJ. Tspan8 strongly enhanced cellular resistance to irradiation. A fraction of MTPa-Tspan8 cells maintained colony formation ability following 6 Gy, whereas the number of colonies built by the parental cells was already reduced by more than 50 % after treatment with 2 Gy. It was noted that multicellular aggregates exhibited a variable resistance, resembling the variations in metastases response observed after radiotherapy of patients in the clinical setting.  Figure S5 Impact of Tspan8 on radiation sensitivity using conventional 2D colony formation assays ** * To test radiation sensitivity of different cell lines, colony forming assays were used as described elsewhere [30,31]. Cells were seeded at 1000-5000 cells / 10 cm plate and irradiated at 2 Gy, established as optimal radiation power to test breast cells radiosensitivity [31]. For siRNA, cells were transfected in 24-well plates and transferred after 1 day into 10 cm dishes. Survival data were corrected for siRNA toxicity as described [31]. After 14 d, the cells were fixed and stained with crystal violet. Images were taken and visible colonies were counted using ImageJ.

(B) Counting of metastases in liver and spleen
Each image was processed to an 8 bit image and the background was subtracted. To determine metastases on liver and spleen surface from healthy tissue, a suitable threshold was set by using Auto Threshold. Set ups for counting metastases were size: 20-2000 pixels and circularity: 0.1-1.00. Brightness and contrast of images were adjusted and a scale bar was included. All image editing was performed with ImageJ.
Supplementary Figure S8 Primary tumour and metastases in liver and spleen of rats after intraperitoneal injection of MTPa and MTPa-Tspan8 cells in Fischer rats Rats were injected with 1 million MTPa or MTPa-Tspan8 cells. 3 wk after injection, the animals were sacrificed and primary tumours and organs with metastases were isolated.