Differential ability among SCPs to metastasize to the adrenal gland. (A) After intracardiac injection of individual SCPs, bioluminescence images were acquired and analyzed for signals originating from regions consistent with adrenal metastasis (arrows). Shown are representative mice at 7 weeks after injection with SCPs that show varying abilities to give rise to adrenal metastasis. (B) At necropsy, left and right adrenal glands (with the kidneys) were removed and were imaged ex vivo for bioluminescence. Arrows show the locations of the left and right adrenal glands, respectively, from a representative mouse with adrenal metastasis.

Segregation of primary breast carcinomas using the bone metastasis gene signature. The microarray data for primary breast tumors from patients that developed distant metastasis were used in hierarchical clustering using the 50 bone metastasis genes described in Figure 7C. Both the patient samples (columns) and the genes (rows) were clustered. The patient samples were classified into two major clusters with an overall R index of 0.90 (a robustness index; see Methods). The site(s) of distant recurrence for each patient is (are) listed along the bottom, with the site of first recurrence listed first. The genes were clustered into six groups (labeled along the left), with the gene symbol of each gene shown (on the right). The asterisks indicate genes without symbols (from top to bottom, Affymetrix probe set identifiers 211429_s_at and 211796_s_at). Orbit, orbit of the eye.

Verification of macroscopic and microscopic metastases by fluorescence histology. (A–I) A pathological fracture involving the proximal tibia (A–E) or vertebrae (F–I) is demonstrated by skeletal x-ray (A and B) and an overlay of this x-ray with BLI (B and G) from the same mouse as that described in Figure 2. To confirm metastases, we performed whole-mount frozen sectioning. Regions corresponding to the fractured tibia and vertebra were analyzed by H&E staining (C, D, and H) or unstained sections were analyzed for GFP fluorescence (E and I). (J–M) A lateral projection of a bioluminescence image from day 45 (J) corresponding to the same image as that in Figure 2L reveals a signal originating from the adrenal gland (green arrow), as shown by H&E staining (K). Magnification of the boxed region in K (L) and GFP fluorescence (M) of the left adrenal gland are shown. (N–Q) Inspection of organs in the left upper abdominal quadrant with areas of bioluminescence signal (N) reveals a focus of tumor growth in the pancreas (O). Magnification of the boxed region in O (P) and GFP fluorescence (Q) are shown.

SCPs exhibit different abilities to metastasize to bone. (A and B) Each of the SCPs was labeled with the TGL reporter, and 1 × 10⁵ cells were injected into the left cardiac ventricle. At the indicated days after xenografting, bioluminescence images were acquired. (A) Representative mice injected with a representative set of SCPs are shown in the supine position. The intensity of the signal from days 1, 4, and 8 are on equivalent scales, while day 24 and day 48 are each on separate scales due to increasing signal strength and to avoid signal saturation. (B) The normalized photon flux from the dominant signal originating from the hindlimbs, forelimbs, or pelvis of all the SCPs studied was measured over the indicated time course. SCPs were ranked according to their growth kinetics in either bone or lung. SCPs with a higher rank order for bone are shown in red, and those with a higher rank order for lung are shown in green. The bottom three SCPs for both bone and lung are classified as being the least metastatic and are shown in blue.

SCPs demonstrate different abilities to metastasize to the lung. (A–C) Each of the SCPs was labeled with the TGL reporter, and 2 × 10⁵ cells were injected into the tail vein. At the indicated day after xenografting, bioluminescence images were acquired. (A) Representative mice injected with a representative set of SCPs are shown in the supine position. The intensity of the signal from day 0 is displayed on one scale, while that of days 14 and 49 (Day ≥14) are on a different scale due to increasing signal strength and to avoid signal saturation. (B) The normalized photon flux from the lung of all the SCPs studied was measured over the indicated time course. SCPs are color-coded as described in Figure 4B. (C) The lungs of SCPs that show growth in lung were analyzed histologically. A lung section from a representative SCP is shown stained for CD31, a marker for vascular endothelial cells, and counterstained with eosin. Asterisks mark regions of parenchymal tumor growth. The red arrow shows a CD31-positive blood vessel with an associated perivascular tumor growth pattern.

Genome-wide “unsupervised” classification of the SCPs correlates with metastatic phenotype. (A) A multidimensional scaling plot illustrates the relationship between the various SCPs and their primary metastatic tropism based on genes that are differentially expressed across the SCPs starting from the more than 22,000 present on the Affymetrix U133A GeneChip. SCPs are color-coded according to their primary metastatic tropism (green for lung, red for bone, and blue for weakly metastatic). The plot demonstrates that SCPs with the same primary metastatic tropism group together in 3-dimensional space. Each group is each enclosed in a circle. MCF10A is shown by itself (gold dot). (B) Hierarchical clustering of the SCPs based on genes differentially expressed reveals similar relationships and a similar association with metastatic tropism, as summarized in the table below the dendrogram. (C) A Venn diagram demonstrates the relationship between the genes differentially expressed across the SCPs and a previously described bone metastasis gene set. Of 1,267 differentially expressed genes, 50 of the 127 bone metastasis genes (102 are unique) overlap. (D) A Northern blot showing the expression levels of 4 of the bone metastasis genes among the SCPs used in this study (boxed and labeled by SCP, with the color of the label corresponding to tissue tropism). GAPDH, loading control.

Noninvasive BLI to monitor the development of osteolytic metastases from the same mouse. (A–D) SCP2, a highly metastatic clone from MDA-MB-231, was transduced with the luciferase-containing TGL reporter gene and was injected into the left cardiac ventricle of an immunodeficient mouse. At the indicated times after xenografting, the bioluminescence signal was captured. The intensity of the signal, measured as photon flux, is shown as a color scale. Images for days 0, 1, and 8 are displayed on the same scale, while the day-35 image is shown on a different scale due to the exponential growth of the metastases. A metastasis to the right hindlimb is circled in red. (E) The growth kinetics of the right hindlimb metastasis outlined by the red circle shown in B–D was quantified by measurement of photon flux. (F–H) A bioluminescence image (F) and a skeletal x-ray image (G) were obtained on day 16 after xenografting. Images were superimposed (H) to demonstrate registration of the bioluminescence signals with skeletal anatomy. (I–N) A superimposed image from day 45 (I and L) reveals extensive areas of osteolytic destruction that correspond to bioluminescence signals. Magnification of regions outlined in red shows involvement of the femur/tibia, iliac creast of the pelvis, and the sacrum (J and K), in addition to the vertebrae (M and N). The bioluminescence signal from the region outlined in yellow on the left lateral projection (L) does not overlap with skeletal structures and originates from the adrenal gland (Figure 3, J–M).

SCPs from MDA-MB-231 cells have a poor-prognosis gene expression signature. (A) Microarray expression data of 46 of the 70 poor-prognosis genes (7) that are present on the Affymetrix U133A GeneChip for the MCF10A normal breast epithelial cell line, parental MDA-MB-231 cell line, and various SCPs from MDA-MB-231. Each column represents a gene (denoted along the bottom) and each row represents a cell line (denoted along the right). Genes of the poor-prognosis signature that are expressed at higher levels in poor-prognosis tumors are above the red line, and those that are underexpressed are above the green line. Genes with low trust values due to low or absent expression are shaded in darker colors (Trust; wedge). (B) Microarray expression data of primary human breast carcinoma from 63 patients treated at our institution who had at least 5 years of clinical follow-up and/or developed metastatic disease. Hierarchical clustering of the patients’ data was performed with the 46 poor-prognosis genes. Each column represents a patient and each row, a gene. The MDA-MB-231 cell line was included and is denoted by a blue dot in the dendrogram. Those patients in the good-prognosis versus the poor-prognosis cluster are separated by the yellow line. (C) Five-year metastasis-free survival data for the 63 patients classified according to the hierarchical clustering described in B. The P value shown in the graph was calculated by the χ² test. (D) Dendrogram showing hierarchical clustering of the SCPs and MCF10A using the poor-prognosis genes. A scale of the distance metric used is shown on the left.