Background: The 4T1 mouse mammary tumor cell line is one of only a few breast cancer models with the capacity to metastasize efficiently to sites affected in human breast cancer. Here we describe two 4T1 cell lines modified to facilitate analysis of tumor growth and metastasis and evaluation of gene function in vivo. New information regarding the involvement of innate and acquired immunity in metastasis and other characteristics of the model relevant to its use in the study of late stage breast cancer are reported.

It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar protontranslocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in ϳ18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.The leading cause of mortality from cancer is metastasis, making inhibition of metastasis an important strategy in controlling cancer progression. The metastatic cascade involves a series of steps that include escape of cells from the site of the primary tumor into the circulation or lymphatic system and the extravasation of cells from the circulation or lymphatic system into secondary sites (1, 2). Both of these processes require the tumor cells to display an invasive phenotype in which they degrade extracellular matrix of the surrounding tissue. The vacuolar H ϩ -ATPases (or V-ATPases) 2 are a family of ATP-dependent proton pumps that have been implicated in tumor cell invasion (3). V-ATPases are up-regulated in tissue samples from highly invasive pancreatic carcinomas (4), and treatment of a human cancer cell line with antisense oligonucleotides to the V-ATPase c subunit decreased invasion in vitro (5). Inhibition of V-ATPase expression in hepatocellular carcinoma cells using siRNAs reduces invasiveness of these cells in vitro and metastasis in vivo (6). In addition, treatment of hig...

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