High Capacity of the Metastatic Breast Tumor Cells in Sphere Formation: Clue for Chemoresistance in Triple- Negative Breast Cancer

Introduction

Methods

Induction of Syngeneic Animal Model of Breast Cancer
Female BALB/c mice weighing 20 to 25 gram obtained from Royan Institute (Iran). The animals were housed in cages at 12-h photoperiod while they had free access to food and water. All animal experiments were in compliance with the relevant laws, and this study was approved by the Ethics Committee of Tehran University of Medical Sciences (registration number: IR.TUMS. REC.1394.1439). 4T1 cells were subcutaneously injected to the flank (or the right hind limb) of the mice (105 cells suspended in 100 µL PBS) using an insulin syringe with a 32G needle. The mice were monitored daily for appearance and behavior characteristics.

Lung Metastatic and Primary Breast Tumor Cell Extraction
TPrimary and metastatic tumor cells were extracted as previously described.[] Briefly, the primary tumor and lung of cancerous mice were excised after 35 days of tumor induction in mice, and surface blood was removed by rinsing it in PBS. After mincing with scissors, fragments were placed to a 50 ml conical tube. For enzymatic digestion, primary tumor and the lungs were digested in 10 mg / ml collagenase type IV at 37°C for 75 min on a platform rocker. All enzymes were purchased from Sigma (St Louis, MO, USA). The digested organ were filtered through 70-um cell strainers and washed with PBS. In the next step, washed cells were resuspended in medium containing 10% FBS, 100 U/ml Penicillin, and 100 ug/ml Streptomycin (all from Gibco, USA). Ultimately, the cells were cultured at 37°C in 5% CO₂.

Sphere Formation Ability
Heterogeneous population of primary and lung metastatic tumor cells was separately cultured in DMEM containing 10% FBS, 100 U/ml Penicillin, and 100 ug/ml Streptomycin (all from Gibco, USA) at 37°C in 5% CO₂. For Sphere formation, cells were seeded at 2.5 × 104 cells in a petri dish in DMEM supplemented with 10% FBS. After 24 h, cell culture media replaced with fresh medium comprised of DMEM supplemented with 2% FBS. Again, after 72 h, cell culture media replaced with fresh medium comprised of DMEM supplemented with 1% FBS. Primary/1° and secondary/2° mammosphere formation was achieved after eight days. The sphere formation index was determined by two independent investigators by counting the sphere formed in 10 high power fields per petri dish.

Statistical Analysis
Results are expressed as the mean ± standard deviation. Data were analyzed with GraphPad Prism statistical software 6.0 (GraphPad Software, La Jolla, CA, USA) using Paired Samples t-test. P<0.05 was considered statistically significant.

Results

Fig. 1. Primary and lung metastatic tumor cells extraction. (a) Metastatic animal model of triple negative breast cancer was generated after 35 days of tumor induction in Balb/c mice. (b) Primary tumor isolation, H&E staining and primary tumor cell extraction were performed on primary tumor tissues. (c) Lung metastatic tumor isolation, H&E staining and metastatic tumor cell extraction were performed on the lung of cancerous mice.

Higher Capacity of Metastatic Tumor Cells in Sphere Formation
After eight days of culturing primary and metastatic tumor cells in sphere forming media, results indicated that metastatic tumor cell have greater ability in sphere formation. As shown in Figure 2, metastatic tumor cell sphere formation ability was higher and greater than primary tumor cell concerning number and size, respectively.

Fig. 2. Higher capacity of lung metastatic breast tumor cells in sphere formation. a and b, Induction of sphere formation in primary and metastatic tumor cells after serum starvation in culturing media. c. Sphere formation capability was significantly higher in lung metastatic tumor cells. All results were expressed as mean ± SD from at least three independent experiments analyzed by unpaired t-test. ****P<0.0001.

Discussion

References