Breast cancer cell competition and invasion in the tumor microenvironment

Abstract

Advances in microarray profiling in patients with breast cancer have identified a large number of genes correlated with prognosis whose functional relevance is unknown. The purpose of this study was to develop an in vivo assay for functionally screening potential regulators of tumor growth and metastasis. Lentiviral vectors were used to drive expression of shRNAs to suppress the expression of a gene in individual transductant cell lines derived from the breast cancer line MDA-MB-231. A pooled population of transductants was injected into the mammary fat pads of mice to evaluate regulators of tumor growth, and tail vein injection was done to evaluate potential regulators of metastasis. The relative proportions of individual transductants were evaluated using a barcode sequence specific to each shRNA transductant. We were able to characterize the homeobox 2, HOXB2, transcription factor as a negative regulator, decreasing cell proliferation in the tumor microenvronment.;To study the functional contributions of a gene such as ErbB2 (Her2/Neu), we used the Neu transgenic mouse model with add-back tyrosine sites in a Neu receptor with C-terminal tyrosines mutated to phenylalanine. We found that the Neu-YB had increased while the Neu-YD strain was defective in invasion, intravasation, and metastasis. The Neu-YB tumors have previously been shown to secrete increased CXCL12 compared to the other Neu add-back strains. We showed that the YB in vivo invasion is dependent on this CXCR4/CXCL12 signaling loop. The in vivo invasion was also inhibited by CSF-1R blocking antibody, indicating macrophages are still needed for invasion. The CXCL12 signaling may be part of a paracrine loop involving tumor associated macrophages in the tumor microenvironment. In MTLn3 cells CXCL12 increased in vivo invasion as well as macrophage recruitment. We hypothesized that increased CXCL12 in the tumor microenvironment brings in more CXCR4 positive macrophages which may act to increase invasion of the tumor cells and set up a more aggressive tumor microenvironment.;These studies highlight the importance of the tumor microenvironment and in vivo assays to study tumor progression and the steps leading to metastasis for functionally characterizing the contributions of genes found to correlate with breast cancer patient prognosis.