An Immunocompetent Murine Model of Metastatic Mammary Cancer Accessible to Bioluminescence Imaging

Materials and Methods

Vectors. luc2, an improved firefly luciferase gene showing strong expression in mammalian cells, was excised from a pGL4.10 vector (Promega, Madison, WI, USA) by digestion with NheI/XbaI. The luc2 gene fragment was ligated into a NheI/XbaI- digested pTracer-CMV/Bsd plasmid vector containing a gene conferring resistance to blasticidin (Invitrogen, Inc., Carlsbad, CA, USA). The plasmid vector was extracted from Escherichia coli (DH5α strain) and initially purified using a modified alkaline lysis procedure and a Qiagen Plasmid Maxi Kit (Qiagen Inc., Valencia, CA, USA), with further purification using centrifugal filters (Ultrafree-MC, Millipore Co., Bedford, MA, USA). The combined vector is referred to as pTracer-Luc2.

Parent mammary tumor cell line. Mouse mammary tumor virus (MMTV), purified from medium in which Jyg-MC cells (established from mammary tumors of the Chinese wild mouse) were grown, was inoculated into the inguinal mammary glands of female BALB/c mice, resulting in the development of mammary carcinomas (4). The BJMC3879 mammary adenocarcinoma cell line was subsequently derived from a metastatic focus within a lymph node from one of the inoculated mice and the cell line continues to show a high metastatic propensity, especially to lymph nodes and lungs (5-7). The BJMC3879 cell line was maintained in either RPMI-1640 medium or Dulbecco's modified Eagle's medium containing 10% fetal bovine serum supplemented with streptomycin/penicillin in an incubator at 37°C and under a 5% CO₂ atmosphere.

Stable transfection of luciferase gene. BJMC3879 mammary carcinoma cells were subjected to electroporation with the pTracer-Luc2 vector. Transfection was conducted using an amaxa Nucleofector device (amaxa GmbH, Köln, Germany). In the transfection process, BJMC3879 cells (1×10⁷) were suspended in 100 μl of Nucleofector V solution (amaxa GmbH) to which 2.5 μg of the pTracer-Luc2 vector were added and gene electrotransfer then conducted using the amaxa Nucleofector system with program number T03. The transfected cells were then plated in T-75 flasks. Three days later, the cells were exposed to 5 μg/ml blasticidin. Medium containing 5 μg/ml blasticidin was changed twice a week for 2 weeks, once a week for 1 week, and changed once a month thereafter. Concentration of the blasticidin in the medium was increased to 10 μg/ml in a stepwise manner at each medium change.

D-Luciferin potassium salts (Wako Pure Chemical Industries, Osaka, Japan) were added to the medium of the transfected BJMC3879-Luc2 cells or to the non-transfected parental BJMC3879 cells to a final concentration of at 150 μg/ml. Expression of luciferase was confirmed in the transfected cultures by bioluminescent imaging using a Photon Imager (Biospace Lab, Paris, France).

Animals. A total of 10 female BALB/c mice at 6 weeks of age were used in this study (Japan SLC, Inc., Hamamatsu, Japan). The mice were housed no more than 5 per plastic cage on wood chip bedding with free access to water and food and maintained under conditions of controlled temperature (21±2°C), humidity (50±10%), and lighting (12 h-12 h light-dark cycle). All were held for a 1-week acclimatization period prior to test initiation. All manipulations and treatments of the mice were performed in accordance with procedures outlined in the Guide for the Care and Use of Laboratory Animals at Osaka Medical College. At the termination of the study, all mice were euthanized under isoflurane anesthesia.

Bioluminescence imaging in vivo. While under isoflurane inhalation using an SBH Scientific anesthesia system (SBH Designs Inc., Ontario, Canada), 5×10⁶ BJMC3879-Luc2 cells in 0.3 ml phosphate-buffered saline were inoculated subcutaneously into the right inguinal region of the 10 female BALB/c mice. The mice were then injected intraperitoneally with D-luciferin potassium salts (Wako Pure Chemical Industries) at 3 mg/mouse. Bioluminescence imaging with a Photon Imager (Biospace Lab) was performed weekly from weeks 2 to 10 post-inoculation. The bioluminescent signals received during the 6 min acquisition time were quantified using Photovision software (Biospace Lab). Each developing mammary tumor was measured weekly using digital calipers, and tumor volumes were calculated using the following formula: maximum diameter × (minimum diameter)² ×0.4 (8).

Histopathology. At necropsy, tumors and lymph nodes — routinely those from the axillary and femoral regions, as well as those appearing abnormal — were removed, fixed in 10% formaldehyde solution in phosphate buffer, and processed through to paraffin embedding. Lungs were inflated with formaldehyde solution prior to excision and immersion in fixative; the individual lobes were subsequently removed from the bronchial tree and examined for metastatic foci and similarly processed to paraffin embedding. All paraffin-embedded tissues were cut into sequential 4-μm thick sections and stained with hematoxylin and eosin for histopathological examination.

To identify lymphatic vessels in the primary mammary carcinomas, the avidin-biotin immunohistochemical complex method (LSAB kit; DakoCytomation, Carpinteria, CA, USA) was used. A hamster anti-podoplanin monoclonal antibody (AngioBio Co., Del Mar, CA, USA) against a lymphatic endothelium marker was used.

Statistical analysis. Significant differences in sequential changes in the quantitative data (tumor volumes and photon counts) were analyzed using Student's t-test. The quantitative data between tumor volumes and photon counts was analyzed using Pearson's correlation.