In Vivo Selection of Intermediately- and Highly-Malignant Variants of Triple-negative Breast Cancer in Orthotopic Nude Mouse Models

Materials and Methods

Cell culture. Parental and metastatic variants of the TNBC MDA-MB-231P-RFP cell line were maintained and cultured in Dulbecco's modified Eagle's medium (DMEM) on plastic with 10% fetal bovine serum and 5% penicillin/streptomycin.

Mice. Athymic nude mice (AntiCancer Inc., San Diego, CA, USA) were kept in a barrier facility under HEPA filtration. Mice were fed with autoclaved laboratory rodent diet (Tecklad LM-485, Envigo, Indianapolis, IN, USA). All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals under assurance A3873– 01.

Establishment of intermediately- and highly-metastatic variants by serial orthotopic implantation. Initially, MDA-MB-231-RFP cells (1×10⁷ cells/site) were injected subcutaneously into the flank of 5 nude mice. For orthotopic transplantation, 7-mm³ fragments of the harvested subcutaneous tumor were grafted into a mammary gland of 5 nude mice. After growth, the tumor was resected. Residual cancer cells grew into a tumor which was harvested and divided into fragments and re-implanted into a mammary gland. After the tumor metastasized to lymph nodes, it was harvested and divided into fragments and re-implanted into mammary gland. After growth, the orthotopic tumor was resected. The residual cancer cells formed primary and metastatic tumors. The metastasis was harvested and divided into fragments re-implanted into the mammary gland. Highly-metastatic MDA-MB-231-RFP tumors were developed after seven orthotopic transplantations described above. Intermediately-metastatic variants of MDA-MB-231-RFP were isolated by serial orthotopic transplantation three times (3-6).

In vivo whole-body/whole-tumor imaging. The OV100 small animal imaging system (Olympus Corp., Tokyo, Japan), was used. The OV100 contains an MT-20 light source (Olympus Biosystems, Planegg, Germany) and DP70 CCD camera (Olympus), for whole body, as well as subcellular imaging in live mice (7-9). The optics of the OV100 have been specially developed for macroimaging as well as microimaging with high light-gathering capacity. Four individually optimized objective lenses, parcentered and parfocal, provide a 105-fold magnification range. High-resolution images were captured directly on a PC (Fujitsu Siemens, Munich, Germany). Images were processed for contrast and brightness and analyzed with the use of Paint Shop Pro 8 and CellR (10-15).

Confocal laser scanning microscopy (CLSM) was performed using the FV1000 (Olympus Corp.) with 2-laser diodes (473 nm and 559 nm). A 4x (0.20 numerical aperture immersion) objective lens and 20 x (0.95 numerical aperture immersion) objective lens (Olympus) were used. Scanning and image acquisition were controlled by Fluoview software (Olympus) (16).

Statistical analysis. Data for fluorescence intensity (Figure 1C, 2C) are shown as means±standard deviation (SD). For comparison between two groups, significant differences were determined using the Student's t-test. P-values of less than 0.05 were considered significant.