Project protocol — Contents
Steps Procedure performed Age Data collected 1 Transgenic FVB/N-TgN(MMTVPyMT) males are bred to females from each of the 28 inbred strains to create transgenic F1 females for the strain survey 6-7 wks - 2 Transgenic F1 females are screened by manual palpation 3 times per week for the presence of primary mammary tumor ~35-90 days Location of tumor, BW 3 An additional wk is used to confirm and to establish diagnosis ~42-97 days - 4 Tumor positive mice are aged for 40 days following diagnosis to allow the development and progression of metastases ~82-137 days - 5 Mice are sacrificed 40 days post-diagnosis, necropsied, and their lungs harvested and then processed for histopathology ~82-137 days BW, lung volume 6 Histology of the lungs stained with H&E are examined for metastasis - tumor count, size and density
- Dissecting tools: scissors, forceps, scalpels, and ruler.
- Weight scale: for obtaining body weight measurements.
- Dissecting microscope: Leica M420 Macroviewer with an Apozoom lens under 103 magnification with the objective 10 cm above the stage, equipped with Leica Q500MC Image Analysis System
- Microtome: for sectioning paraffin embedded tissues
- Tissue fixative: 10% paraformaldehyde
- Tissue stain: hematoxylin-eosin (H&E)
- Microscope slides: for mounting tissue samples
- Anesthetic: Avertin or liquid carbon dioxide and cull jar for euthanasia
I. Creation of F1 transgenic females
a. FVB/N-TgN(MMTVPyMT) transgenic males are bred with females from 28 different inbred strains of mice.
b. F1 virgin female progenies from the above intercross are screened for genetic transmission of the transgene by PCR amplification (see Lifsted et, al., 1998) of tail DNA samples.
II. Initial phenotyping for primary mammary tumor
a. Transgenic positive F1 virgin females are manually palpated (see Figure 1 below) 3 times per week beginning at birth for the presence of primary mammary tumor.
b. Upon detection of primary mammary tumor, the exact location of the lesion, body weight, and age of the mouse are recorded.
c. The diagnosis of primary mammary tumor is confirmed within 1 wk from initial detection.
d. To allow the development and progression of metastasis, the tumor positive mice are aged for an additional 40 days.
Figure 1. Illustrations of mammary tissues and their locations in the female mouse.
III. Gross phenotyping for metastasis
a. Aged tumor positive females are sacrificed by carbon dioxide inhalation using cull-jars or by injection of Avertin and cervical dislocation.
b.Full necropsy is performed, which include visual and manual inspection for any lumps and bumps throughout the body.
c.The number of tumors and their locations, sizes, and weights are recorded along with carcass weight following dissection.
d.The entire lung (see Figure 2 below) is harvested and visually and manually inspected for metastasis.
e. The lung is inflated gently with an injection of fixative into the trachea before being immersed in 10% paraformaldehyde for histology. Fixation is accomplished for at least 24 h.
2. Schematic drawing of the different lung lobes of the mouse and examples of areas examined for histopathology.
IV. Histopathological examination of the lungs for metastasis
a. Following 24 h of fixation, the lungs are imbedded in paraffin in the proper orientation for coronal and serial (consecutive) sectioning.
b. At least 3 coronal noncontiguous sections of the lung lobes (see Figure 2 above) and separated by a distance of at least 100 µm, are sampled for macroscopic examination.
c. Slide samples of the lungs stained with H&E are first examined using a Leica M420 Macroviewer with an Apozoom lens under 103 magnification, and with the objective 10 cm above the stage.
d. Three fields are scored per slide for a total of 9 fields examined per mouse.
e. A Leica Q500MC Image Analysis System is used to determine pulmonary metastatic density.
f. To minimize operator bias, a single inspector, blind to the identity of the samples, is allowed to do the analysis.
Data collected by investigator
Data collected included latency of primary mammary tumor diagnosis, tumor burden, lung histopathology (number and area of lung metastasis), and total lung area.
Definitions & formulas
Pulmonary metastatic density: accounting of lung metastasis using Leica Q500MC Image Analysis System.
Metastasis index: the number of multicellular metastatic lesions counted per square micron of lung tissue examined.
Average metastasis size: total area of metastatic tissue surveyed on a slide divided by the number of metastatic lesions observed and counted.