Figure 1.
Tumor formation in the mammary fat pad upon injection of total bone marrow aspirates isolated from femurs containing metastatic tumor cells.
A. Experimental design to determine the tumorigenicity of disseminated human cancer cells in the bone marrow of mice previously injected with tumorspheres into the mammary fat pad. Bone marrow, aspirated from femurs of mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice to determine the tumor-forming ability of dormant cancer cells in the bone marrow. B. Injection of 12.5×106 cells/pad aspirated from the femurs of mice injected with tumorspheres resulted in large tumor formation in the mammary fat pad two months post-injection. C. Injection of 12.5×106 cells/pad aspirated from the femurs of non-injected mice resulted in no tumor formation in the mammary fat pad three months post injection. D. Summary of tumor formation and metastasis for sample 5–9 BM, including ER/PR/Her2 status of patients samples from which the parental tumorspheres were first derived. E. Representative positive HNA staining of 5 µm paraffin-embedded sections of sample 5 BM tumors demonstrated the presence of human cells. F. No positive nuclear HNA staining of 5 µm paraffin-embedded sections of mouse kidney from non-tumor bearing mouse (negative control). G. Positive HNA staining of 5 µm paraffin-embedded sections of a human MCF-7 xenograft (positive control). 200× magnification in all panels.
Figure 2.
Expression of markers for epithelial and mesenchymal lineages in tumor samples.
A–E. H+E staining of tumors formed in the mammary fat pad upon injection of bone marrow aspirated from the femurs of mice injected with tumorspheres isolated from samples 5–9 BM, respectively. F–T. Representative IHC demonstrating patterns of expression of E-cadherin (F–J), β-catenin (K–O), and fibronectin (P–T) in sample 5–9 BM tumors. 200× magnification in all panels.
Figure 3.
Metastatic lesions in the lungs of mice bearing mammary fat pad tumors that were derived from transplantation of bone marrow aspirate containing metastatic tumor cells.
A–D. H+E staining performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM illustrates metastatic lesions. Metastatic lesions indicated by £; normal mouse tissue indicated by §. 100× magnification. E–H. IHC performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM using a monoclonal antibody to E-cadherin. I–L. IHC performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM using a polyclonal antibody to β-catenin.
Figure 4.
Metastatic lesions in the livers of mice bearing mammary fat pad tumors that were derived from transplantation of bone marrow aspirate containing metastatic tumor cells.
A–D. H+E staining performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM illustrates metastatic lesions. Lesion indicated by £; normal tissue indicated by §. 100× magnification. E–H. IHC performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM using a monoclonal antibody to E-cadherin. I–L. IHC performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM using a polyclonal antibody to β-catenin.
Figure 5.
Metastatic profile of bone marrow transplantation experiments.
A. Graphical representation of the metastatic burden determined for the lungs, kidneys, and livers analyzed by H+E staining from samples 5–9 BM. The metastatic burden in each organ was calculated by dividing the pixels present in the metastatic lesion/s (x pixels) by the total pixels comprising the field of view (y pixels) then multiplying by 100 [(x pixels/y pixels)*100] resulting in a percent value. Values are reported as mean +/− SD. B. Graphical representation of the percent metastatic burden, previously calculated as above, compared to days post-injection of total bone marrow into the mammary fat pad for sample 5–9 BM. Values are reported as mean +/− SD.