Isoform-specific promotion of breast cancer tumorigenicity by TBX3 involves induction of angiogenesis.

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG.

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

MRI surveillance of cancer cell fate in a brain metastasis model after early radiotherapy.

PURPOSE: Incidence of brain metastasis attributed to breast cancer is increasing and prognosis is poor. It is thought that disseminated dormant cancer cells persist in metastatic organs and may evade treatments, thereby facilitating a mechanism for recurrence. Radiotherapy is used to treat brain metastases clinically, but assessment has been limited to macroscopic tumor volumes detectable by clinical imaging. Here, we use cellular MRI to understand the concurrent responses of metastases and nonproliferative or slowly cycling cancer cells to radiotherapy. METHODS: MRI cell tracking was used to investigate the impact of early cranial irradiation on the fate of individual iron-labeled cancer cells and outgrowth of breast cancer brain metastases in the human MDA-MB-231-BR-HER2 cell model. RESULTS: Early whole-brain radiotherapy significantly reduced the outgrowth of metastases from individual disseminated cancer cells in treated animals compared to controls. However, the numbers of nonproliferative iron-retaining cancer cells in the brain were not significantly different. CONCLUSIONS: Radiotherapy, when given early in cancer progression, is effective in preventing the outgrowth of solitary cancer cells to brain metastases. Future studies of the nonproliferative cancer cells' clonogenic potentials are warranted, given that their persistent presence suggests that they may have evaded treatment. Magn Reson Med 78:1506-1512, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

The transcriptional regulator TBX3 promotes progression from non-invasive to invasive breast cancer.

BACKGROUND: TBX3 is a T-box transcription factor repressor that is elevated in metastatic breast cancer and is believed to promote malignancy of tumor cells, possibly by promoting cell survival and epithelial-mesenchymal transition. METHODS: The relative expression of TBX3 was assessed in the 21T cell lines which were derived from an individual patient and represent three distinct stages of breast cancer progression: 21PT, atypical ductal hyperplasia; 21NT, ductal carcinoma in situ; and 21MT-1, invasive mammary carcinoma. Two different isoforms of TBX3 (TBX3iso1 and TBX3iso2) were overexpressed to evaluate cell survival/colony forming ability, growth, and invasion in the ductal carcinoma in situ-like 21NT cell line using an in vitro Matrigel model of cancer progression. In addition, TBX3 expression was knocked down to evaluate the effects of downregulating TBX3 on the invasive mammary carcinoma-like 21MT-1 cell line. Finally, PCR array profiling was used to assess alterations in gene expression due to TBX3 overexpression in the 21NT cells. RESULTS: TBX3 is abundant in the invasive 21MT-1 cell line, while being minimally expressed in the non-invasive 21NT and 21PT cell lines. Overexpression of either TBX3iso1 or TBX3iso2 in 21NT cells resulted in increased cell survival/colony forming ability, growth vs. apoptosis and invasion in Matrigel. In contrast, short hairpin RNA-mediated knockdown of TBX3 in the 21MT-1 cells resulted in smaller colonies, with a more regular, less dispersed (less infiltrative) morphology. Array profiling of the 21NT TBX3 iso1 and iso2 transfectants showed that there are common alterations in expression of several genes involved in signal transduction, cell cycle control/cell survival, epithelial-mesenchymal transition and invasiveness. CONCLUSIONS: Overall, these results indicate that TBX3 (isoform 1 or 2) expression can promote progression in a model of early breast cancer by altering cell properties involved in cell survival/colony formation and invasiveness, as well as key regulatory and EMT/invasiveness-related gene expressions.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential.

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

Cyclin A2, a novel regulator of EMT.

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination.

Assessment of osteopontin in early breast cancer: correlative study in a randomised clinical trial.

INTRODUCTION: Osteopontin (OPN) is a malignancy-associated glycoprotein that contributes functionally to tumor aggressiveness. In metastatic breast cancer, we previously demonstrated that elevated OPN in primary tumor and blood was associated with poor prognosis. METHODS: We measured OPN in plasma by ELISA, and in tumors by immunohistochemistry, in 624 (94%) and 462 (69%), respectively, of 667 postmenopausal women with hormone responsive early breast cancer treated by surgery followed by adjuvant treatment with tamoxifen +/- octreotide in a randomized trial (NCIC CTG MA.14; National Cancer Institute of Canada Clinical Trials Group Mammary.14). RESULTS: Plasma OPN was measured in 2,540 samples; 688 at baseline and 1,852 collected during follow-up. Mean baseline plasma OPN was 46 ng/ml (range 22.6 to 290) which did not differ from normal levels. Mean percentage OPN tumor cell positivity was 33.9 (95% CI: 30.2 to 37.9). There was no correlation between plasma and tumor OPN values. In multivariate analysis, neither was associated with event-free survival (EFS), relapse-free survival (RFS), overall survival (OS), bone RFS or non-bone RFS. An exploratory analysis in patients with recurrence showed higher mean OPN plasma levels 60.7 ng/ml (23.9 to 543) in the recurrence period compared with baseline levels. CONCLUSIONS: The hypothesis that OPN tumor expression would have independent prognostic value in early breast cancer was not supported by multivariate analysis of this study population. Plasma OPN levels in women with hormone responsive early breast cancer in the MA.14 trial were not elevated and there was no evidence for prognostic value of plasma OPN in this defined group of patients. However, our finding of elevated mean OPN plasma level around the time of recurrence warrants further study. TRIAL REGISTRATION: NCT00002864, http://clinicaltrials.gov/show/NCT00002864.

Dissemination and growth of cancer cells in metastatic sites.

Metastases, rather than primary tumours, are responsible for most cancer deaths. To prevent these deaths, improved ways to treat metastatic disease are needed. Blood flow and other mechanical factors influence the delivery of cancer cells to specific organs, whereas molecular interactions between the cancer cells and the new organ influence the probability that the cells will grow there. Inhibition of the growth of metastases in secondary sites offers a promising approach for cancer therapy.

Neuropeptide Y stimulates proliferation and migration in the 4T1 breast cancer cell line.

Stress has long been thought of to be associated with increased risk of cancer. Chronic stress is associated with elevated levels of sympathetic neurotransmitter (norepinephrine and neuropeptide Y: NPY) release and immunosuppression. The expression of NPY receptors has been reported in human breast carcinomas. Recently, activation of the NPY Y5 receptor was shown to stimulate cell growth and increase migration in human breast cancer cells; however the effects of NPY have yet to be investigated in a murine model of breast cancer. Thus, the specific aims of the current study were to: (i) characterize NPY receptor expression in 4T1 breast cancer cells and orthotopic tumors grown in BALB/c mice and (ii) investigate the impact of NPY receptor activation on 4T1 cell proliferation and migration in vitro. Positive expression of NPY receptors (Y1R, Y2R and Y5R) was observed in cells and tumor tissue. As well, NPY treatment of 4T1 cells promoted a concentration-dependent increase in proliferation, through increased phosphorylation of ERK 1/2. Using NPY receptor antagonists (Y1R:BIBP3226, Y2R:BIIE0246 and Y5R:L-152,804), we found the proliferative response to be Y5R mediated. Additionally, NPY increased chemotaxis through Y2R and Y5R activation. These data are in congruence with those from human cell lines and highlight the 4T1 cell line as a translatable model of breast cancer in which the effects of NPY can be studied in an immunocompetent system.

An emerging role for the nuclear localization of maspin in the suppression of tumor progression and metastasis.

Maspin, a member of the serpin family of serine protease inhibitors, was originally identified as a tumor suppressor that is expressed in normal mammary epithelial cells but is reduced or absent in breast carcinomas. Early enthusiasm for maspin as a biomarker for disease progression has been tempered by clinical data that associates maspin with favourable outcomes in some studies and poor prognosis in others. Here, we review all of the published clinical studies for maspin in breast and ovarian cancers and propose that the apparent discordance between clinical reports is a consequence of differential cellular distribution of maspin. Indeed, it was thought that an extracellular pool of maspin possessed tumor suppressor activity, acting by inhibiting migration and increasing cell adhesion. Recent evidence from our group and others indicates, however, that the nuclear localization of maspin in cancer cells is necessary for its tumor suppressor activity. We provide additional data here to demonstrate that nuclear-localized maspin binds to chromatin and is required to effectively prevent cells from metastasizing. Our knowledge of other serpins that localize to the nucleus should help to inform future studies of nuclear maspin. Elucidation of the molecular mechanisms regulating the localization and activities of maspin should pave the way for the development of improved diagnostics and therapies for cancer.

Gene signatures of breast cancer progression and metastasis.

Breast cancer is a heterogeneous disease. Patient outcome varies significantly, depending on prognostic features of patients and their tumors, including patient age, menopausal status, tumor size and histology, nodal status, and so on. Response to treatment also depends on a series of predictive factors, such as hormone receptor and HER2 status. Current treatment guidelines use these features to determine treatment. However, these guidelines are imperfect, and do not always predict response to treatment or survival. Evolving technologies are permitting increasingly large amounts of molecular data to be obtained from tumors, which may enable more personalized treatment decisions to be made. The challenge is to learn what information leads to improved prognostic accuracy and treatment outcome for individual patients.

Nuclear localization of maspin is essential for its inhibition of tumor growth and metastasis.

Maspin (mammary serine protease inhibitor or SerpinB5) acts as a tumor suppressor when overexpressed in aggressive cancer cell lines. However, its role in human cancer is controversial. Maspin expression has been associated with a poor prognosis in some studies, whereas in others, with favorable outcome. The clinical data suggest, however, that nuclear-localized maspin is associated with improved survival. We hypothesized that the tumor suppressor activity of maspin may require nuclear localization, and that the discordance between clinical and experimental reports is a consequence of the variable subcellular distribution of maspin. Furthermore, we surmized that nuclear maspin could function as a tumor suppressor through the regulation of genes involved in tumor growth and invasion. Maspin or maspin fused to a nuclear export signal were expressed in metastatic human breast and epidermoid carcinoma cell lines. We found that pan-cellular localized maspin inhibited in vivo tumor growth and metastasis when assessed in xenograft chicken embryo and murine mammary fat pad injection models. However, when maspin was excluded from the nucleus via a nuclear exclusion signal, it no longer functioned as a metastasis suppressor. Using chromatin immunoprecipitation, we show that nuclear maspin was enriched at the promoter of colony-stimulating factor-1 (CSF-1) and associated with diminished levels of CSF-1 mRNA. Our findings demonstrate that the nuclear localization of maspin is required for its tumor and metastasis suppressor functions in vivo, and suggest that its mechanism of action involves, in part, direct association of maspin with target genes.

In vivo single scan detection of both iron-labeled cells and breast cancer metastases in the mouse brain using balanced steady-state free precession imaging at 1.5 T.

PURPOSE: To simultaneously detect iron-labeled cancer cells and brain tumors in vivo in one scan, the balanced steady-state free precession (b-SSFP) imaging sequence was optimized at 1.5 T on mice developing brain metastases subsequent to the injection of micron-sized iron oxide particle-labeled human breast cancer cells. MATERIALS AND METHODS: b-SSFP sequence parameters (repetition time, flip angle, and receiver bandwidth) were varied and the signal-to-noise ratio, contrast between the brain and tumors, and the number of detected iron-labeled cells were evaluated. RESULTS: Optimal b-SSFP images were acquired with a 26 msec repetition time, 35° flip angle, and bandwidth of ±21 kHz. b-SSFP images were compared with T(2) -weighted 2D fast spin echo (FSE) and 3D spoiled gradient recalled echo (SPGR) images. The mean tumor-brain contrast-to-noise ratio and the ability to detect iron-labeled cells were the highest in the b-SSFP images. CONCLUSION: A single b-SSFP scan can be used to visualize both iron-labeled cells and brain metastases.

Role of the metastasis-promoting protein osteopontin in the tumour microenvironment.

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post-translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients' tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross-talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN-mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns.

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

Changes over time of extracellular domain of HER2 (ECD/HER2) serum levels have prognostic value in metastatic breast cancer.

BACKGROUND: Blood levels of the extracellular domain of HER-2/neu (ECD/HER2) have been suggested to have potential as a tumor marker in breast cancer. Our aim was to assess the prognostic value of baseline levels of ECD/HER2, but more importantly changes in levels over time, in women with metastatic breast cancer. METHODS: Baseline and serial levels of ECD/HER2 were measured in 158 women with newly-diagnosed metastatic breast cancer, in whom we previously performed serial measurement of plasma osteopontin. ECD/HER2 was measured in 1,282 serum samples using a validated ELISA at baseline and every 3-12 weeks during and after therapy until death (median, n=8 samples per patient). Multivariate time-dependent survival analyses were conducted using models that right-censored patient outcomes 3, 6 and 12 months after last known ECD/HER2 measurement. RESULTS: Thirty-four patients (22%) had elevated baseline ECD/HER2 (median 10.2 ng/ml: range 4.1-40.4 ng/ml). In univariate analysis, elevated baseline ECD/HER2 was associated with short survival (P=0.001). In a multivariate model incorporating standard clinical prognostic factors, baseline ECD/HER2 was significantly associated with survival duration (RR 1.029; P=0.020). Presence of visceral metastases and ECOG status 2-4 also retained significance. In a multivariate model incorporating standard prognostic factors and changes in sequential ECD/HER2 levels, an ECD/HER2 increase of >12 ng/ml at any time was the variable with most prognostic value for poor survival (RR 6.10; P=0.0003); poor ECOG status also retained significance. CONCLUSION: Increases over time of ECD/HER2 levels were strongly associated with poor survival in this cohort of women with metastatic breast cancer.

New dual monoclonal ELISA for measuring plasma osteopontin as a biomarker associated with survival in prostate cancer: clinical validation and comparison of multiple ELISAs.

BACKGROUND: A previously developed monoclonal/polyclonal ELISA (Mono/Poly) to detect plasma concentrations of osteopontin (OPN) was shown to provide prognostic information in breast, prostate, and other cancers. Here we describe the clinical validation of a new dual monoclonal (Dual Mono) assay. We compared both assays with 4 assays that recognize defined regions of OPN protein (dual polyclonal systems 5-1, 4-1, 4-3 and polyclonal-monoclonal system 1-3). METHODS: OPN sequences recognized by the monoclonal antibodies that make up the Dual Mono ELISA were identified by Pepscan CLIPS analysis. Using the 6 ELISAs, we measured OPN in plasma from 66 patients with castration-resistant prostate cancer, and we assessed the ability of each assay to predict patient survival. RESULTS: The assays varied in measured plasma OPN concentrations, with median values ranging from 112 to 1740 mug/L, and ability to predict patient survival. By Cox univariable regression of survival by tertiles of OPN, the Mono/Poly and Dual Mono ELISAs had the highest log-rank chi(2) values. After adjustment for risk factors independently associated with survival in our samples, OPN remained associated with survival only for the Mono/Poly and Dual Mono systems. CONCLUSIONS: OPN plasma values varied significantly depending on the assay used. Only the Mono/Poly and Dual Mono systems were independently associated with survival in a population of men with castration-resistant prostate cancer. The availability of a clinically validated, dual monoclonal-based ELISA will provide consistent reagents for studies of OPN plasma concentrations in cancer and other pathologies.

Lymphatic metastasis of breast cancer cells is associated with differential gene expression profiles that predict cancer stem cell-like properties and the ability to survive, establish and grow in a foreign environment.

Although lymphatic dissemination is a major route for breast cancer metastasis, there has been little work to determine what factors control the ability of tumor cells to survive, establish and show progressive growth in a lymph node environment. This information is of particular relevance now, in the era of sentinel lymph node biopsy, where smaller intranodal tumor deposits are being detected earlier in the course of disease, the clinical relevance of which is uncertain. In this study, we compared differentially expressed genes in cell lines of high (468LN) vs. low (468GFP) lymphatic metastatic ability, and related these to clinical literature on genes associated with lymphatic metastatic ability and prognosis, to identify genes of potential clinical relevance. This approach revealed differential expression of a set of genes associated with 'cancer stem cell-like' properties, as well as networks of genes potentially associated with survival and autonomous growth. We explored these differences functionally and found that 468LN cells have a higher proportion of cells with a cancer stem cell-like (CD44+/CD24-) phenotype, have a higher clonogenic potential and a greater ability to survive, establish and grow in a foreign (lymph node and 3D Matrigel) microenvironment, relative to 468GFP cells. Differentially expressed genes which reflect these functions provide candidates for investigation as potential targets for therapy directed against early lymphatic metastasis.