Here we report on the generation of two clonal 4T1 cell lines (4T1-12B and 4T1-1V), both of which stably express firefly luciferase at a high level in the absence of selective pressure, and one (4T1-1V) which was also modified by addition of an FRT site in its genome. These lines were shown to have metastatic characteristics similar to the parental 4T1 line displaying metastasis to bone, lungs, and liver and brain organs primarily affected in human breast cancer. The ability to image the cells ex vivo with high sensitivity allowed detection and quantitation of metastases in affected organs more effectively than has been possible previously. Luciferase-expressing 4T1 variant cell pools and lines with increased propensity for metastasis to brain, liver, and bone have recently been isolated (to be published elsewhere). These variants will further expand the repertoire of syngeneic models available for the study of late stage breast cancer.
An acquired immune response was found to play an important role in regulating 4T1 tumor growth and metastasis. 4T1 tumors established in normal BALB/c mice displayed a substantial loss of tumor cells beginning 2–3 weeks after introduction. This effect was not apparent in BALB/c nude and BALB/c SCID mice, in which 4T1 cells in tumors proliferated rapidly and continuously. Antibodies directed against several 4T1 antigens were detected in sera from normal tumor-bearing BALB/c mice further supporting the involvement of an acquired immune response to the cells. Myeloid derived suppressor cells (MDSC) which are known to be induced in 4T1 tumor-bearing mice are likely to be involved in establishment and maintenance of 4T1 tumors by attenuating the immune response to allow survival of the tumor in weeks 3–4 and re-emergence of tumor growth in weeks 5–6. Further work will be required to determine the actual role that MDSC and other immune system components play in regulating the growth and survival of 4T1 tumors.
Metastasis of 4T1 tumors is associated with extensive necrosis and inflammation within the primary tumor and hematopoiesis in several mouse organs including spleen and liver. Elevated hematopoiesis has recently been reported for the 4T1 model [17
]. Whether or not a causal relationship exists between these processes and metastasis remains to be demonstrated although two observations suggest that there may be such a connection. First, the extent of necrosis is greater in 4T1 tumors than those derived from less metastatic sibling cell lines (67NR, 168FARN) as indicated by the occurrence of large areas of visible necrosis in the 4T1 tumors. Second, a causal relationship between inflammation and metastasis is supported by the inhibitory effect of the COX-2 inhibitor, SC-236, on metastasis of 4T1 after primary tumor excision [25
]. Inflammation is known to have a positive effect on metastasis in several systems [26
] and is likely to have a pro-metastatic effect in this system as well.
Inflammation in metastatic tumors is generally thought to result from signals produced by dying cells and ECM fragments in areas of insufficient vascularization [29
]. A noteworthy finding of this study is that 4T1 tumor cells, when cultured under optimal growth conditions, produce a wide range of factors capable of inducing production, recruitment and activation of inflammatory cells. These factors include colony stimulating factors GM-CSF (Csf2) and G-CSF (Csf3); cytokines Ccl5, Cxcl1, Cxcl6 and Tslp; angiogenic factors Agpt2 and Vegfc; and acute phase proteins Saa3, C3, and Lcn2. While this does not preclude the involvement of cell death in initiating an inflammatory response in the tumor, it does suggest that the tumor cells themselves may play a more direct and active role in directing pro-metastatic inflammatory processes than previously envisioned.
The methodology used in this study for analysis of gene expression yielded highly significant data characterizing the 4T1 metastatic phenotype. The majority of genes that differed by more than 2-fold in 4T1 relative to the two non-metastatic sibling lines examined displayed an exceptionally high level of significance (p < 0.0001) and genelists obtained at this level of significance displayed very low false positive rates. The statistics argue that the results obtained provide a relatively complete and accurate picture of expression differences associated with the 4T1 phenotype. The high level of accuracy and reproducibility that was achieved is attributed to use of the Illumina BeadChip platform and analysis of cells cultured under carefully controlled growth conditions that minimize differences between biological replicates. The data obtained from this study provide detailed information regarding the genes and pathways involved in breast cancer progression for this model and will be particularly useful for further analysis of the pathological processes responsible for progression to a metastatic phenotype.
Unlike many cell lines used as xenograft models, subclones of the 4T1-12B cell line that had undergone more than 20 doublings were found to be homogeneous with respect to metastatic properties. These cells also display a high plating efficiency and no visibly apparent differentiation in culture or in vivo
. Thus, the cells resemble stem cells found in populations of cell lines such as MCF7 [30
] in that they are self renewing, but differ in that they do not appear to differentiate. While more work is need to determine the basis for this property, the characteristic has utility for studies aimed at determining gene function since clonal lines in which a specific genes have been over-expressed or knocked down can be expected to retain the properties of the parental line from which they were derived. In this regard, the FRT site in the 4T1-1V line will be useful for production of isogenic lines for analysis of gene function.