Identification of genes involved in the formation of lymph node metastasis from human tumour xenograft models of breast cancer

Background

Lymph node metastasis is associated with considerable morbidity and is linked to poor prognosis in breast cancer. We have developed experimental models of lymphatic metastasis from the human breast carcinoma cell lines GI 101a and MDA-MB-435. Several sublines of cells derived from lymph node metastases in vivo have been developed. When injected into mammary fat pads (MFP) of athymic mice, all cell lines produced spontaneous lymph node metastases. These cell lines also generated lymph node metastases (in addition to the expected lung metastases) when injected intravenously. In the latter, the tumour cells need to traverse the pulmonary capillary bed and either show tropism for, or adaptation to, the lymph node environment. These distinct patterns of spread – due respectively to direct (intralymphatic) and indirect (haematogenous) colonisation of nodes – will enable us to explore determinants of both putative passive and active (nodal tropism) mechanisms independently.

Methods

RNA was extracted from frozen primary tumours and lymph node metastases derived from the different cell lines, after MFP or intravenous injection, and was used to generate gene expression profiles. A supervised learning method from the BRB ArrayTools 3.5.0 software was used to identify the genes that were differentially expressed between the lymph node metastases obtained from the two routes of dissemination, as well as between matched primary tumours and their lymph node metastases.

Results

Microarray results indicate that it is possible to distinguish between the lymph node metastases and matched primary tumours. Additionally, the nodal metastases derived from the MFP primary site segregate from those derived from the peripheral circulation. These samples cluster together irrespective of the cell line of origin. We have now identified genes upregulated and downregulated in each cluster, and are validating their expression at the protein level.

Conclusion

The presented results will provide more information about the molecules involved in the generation of lymph node metastases. Furthermore, the identification of genes differentially expressed between metastases originating from MFP and intravenously suggests that at some level distinct molecular mechanisms may be in operation in active and passive modes of dissemination.