Inhibition of Anti-Inflammatory Macrophage Phenotype Reduces Tumour Growth in Mouse Models of Brain Metastasis.

Breast cancer brain metastasis is a significant clinical problem and carries a poor prognosis. Although it is well-established that macrophages are a primary component of the brain metastasis microenvironment, the role of blood-derived macrophages (BDM) and brain-resident microglia in the progression of brain metastases remains uncertain. The aim of this study, therefore, was to determine the role, specifically, of pro- and anti-inflammatory BDM and microglial phenotypes on metastasis progression. Initial in vitro studies demonstrated decreased migration of EO771 metastatic breast cancer cells in the presence of pro-inflammatory, but not anti-inflammatory, stimulated RAW 264.7 macrophages. In vivo, suppression of the anti-inflammatory BDM phenotype, specifically, via myeloid knock out of Krüppel-like Factor 4 (KLF4) significantly reduced EO771 tumour growth in the brains of C57BL/6 mice. Further, pharmacological inhibition of the anti-inflammatory BDM and/or microglial phenotypes, via either Colony Stimulating Factor 1 Receptor (CSF-1R) or STAT6 pathways, significantly decreased tumour burden in two different syngeneic mouse models of breast cancer brain metastasis. These findings suggest that switching BDM and microglia towards a more pro-inflammatory phenotype may be an effective therapeutic strategy in brain metastasis.

Automated Co-in Situ Hybridization and Immunofluorescence Using Archival Tumor Tissue.

In situ hybridization (ISH) and immunohistochemistry (IHC) are valuable tools for molecular pathology and cancer research. Recent advances in multiplex technology, assay automation, and digital image analysis have enabled the development of co-ISH IHC or immunofluorescence (IF) methods, which allow researchers to simultaneously view and quantify expression of mRNA and protein within the preserved tissue spatial context. These data are vital to the study of the control of gene expression in the complex tumor microenvironment.

Anti-inflammatory Microglia/Macrophages As a Potential Therapeutic Target in Brain Metastasis.

Brain metastasis is a common complication of cancer patients and is associated with poor survival. Histological data from patients with brain metastases suggest that microglia are the major immune population activated around the metastatic foci. Microglia and macrophages have the ability to polarize to different phenotypes and to exert both tumorigenic and cytotoxic effects. However, the role of microglia/macrophages during the early stages of metastatic growth in the brain has not yet been determined. The aim of this study was to profile microglial/macrophage activation in a mouse model of breast cancer brain metastasis during the early stages of tumor growth, and to assess the role of the anti-inflammatory microglial/macrophage population, specifically, during this phase. Following intracerebral injection of 5 × 103 4T1-GFP mammary carcinoma cells into female BALB/c mice, robust microglial/macrophage activation around the 4T1 metastatic foci was evident throughout the time-course studied (28 days) and correlated positively with tumor volume (R2 = 0.67). Populations of classically (proinflammatory) and alternatively (anti-inflammatory) activated microglia/macrophages were identified immunohistochemically by expression of either induced nitric oxide synthase/cyclooxygenase 2 or mannose receptor 1/arginase 1, respectively. Temporally, levels of both pro- and anti-inflammatory cells were broadly stable across the time-course. Subsequently, selective depletion of the anti-inflammatory microglia/macrophage population by intracerebral injection of mannosylated clodronate liposomes significantly reduced metastatic tumor burden (p < 0.01). Moreover, increased levels of apoptosis were associated with tumors in clodronate liposome treated animals compared to controls (p < 0.05). These findings suggest that microglia/macrophages are important effectors of the inflammatory response in the early stages of brain metastasis, and that targeting the anti-inflammatory microglial/macrophage population may offer an effective new therapeutic avenue for patients with brain metastases.

Early Diagnosis of Brain Metastases Using a Biofluids-Metabolomics Approach in Mice.

Over 20% of cancer patients will develop brain metastases. Prognosis is currently extremely poor, largely owing to late-stage diagnosis. We hypothesized that biofluid metabolomics could detect tumours at the micrometastatic stage, prior to the current clinical gold-standard of blood-brain barrier breakdown. Metastatic mammary carcinoma cells (4T1-GFP) were injected into BALB/c mice via intracerebral, intracardiac or intravenous routes to induce differing cerebral and systemic tumour burdens. B16F10 melanoma and MDA231BR-GFP human breast carcinoma cells were used for additional modelling. Urine metabolite composition was analysed by 1H NMR spectroscopy. Statistical pattern recognition and modelling was applied to identify differences or commonalities indicative of brain metastasis burden. Significant metabolic profile separations were found between control cohorts and animals with tumour burdens at all time-points for the intracerebral 4T1-GFP time-course. Models became stronger, with higher sensitivity and specificity, as the time-course progressed indicating a more severe tumour burden. Sensitivity and specificity for predicting a blinded testing set were 0.89 and 0.82, respectively, at day 5, both rising to 1.00 at day 35. Significant separations were also found between control and all 4T1-GFP injected mice irrespective of route. Likewise, significant separations were observed in B16F10 and MDA231BR-GFP cell line models. Metabolites underpinning each separation were identified. These findings demonstrate that brain metastases can be diagnosed in an animal model based on urinary metabolomics from micrometastatic stages. Furthermore, it is possible to separate differing systemic and CNS tumour burdens, suggesting a metabolite fingerprint specific to brain metastasis. This method has strong potential for clinical translation.

Disruption of tumour-host communication by downregulation of LFA-1 reduces COX-2 and e-NOS expression and inhibits brain metastasis growth.

Over 20% of cancer patients will suffer metastatic spread to the brain, and prognosis remains poor. Communication between tumour cells and host tissue is essential during metastasis, yet little is known of the processes underlying such interactions in the brain.Here we test the hypothesis that cross-talk between tumour cells and host brain cells, through tumour cell leukocyte function associated protein-1 (LFA-1), is critical in metastasis development. Temporal expression of LFA-1 and its major ligand intercellular adhesion molecule-1 (ICAM-1) was determined in two different mouse models of brain metastasis. Marked upregulation of both proteins was found, co-localising with astrocytes, microglia and tumour cells themselves. Silencing of LFA-1 expression in MDA231Br-GFP cells prior to intracerebral injection resulted in > 70% reduction in tumour burden compared to control MDA231Br-GFP cells (p < 0.005, n = 5). Subsequent qRT-PCR analysis of brain tissue revealed significant reductions in COX-2, VEGF and eNOS from host brain tissue, but not tumour cells, in mice injected with LFA-1 knockdown cells (p < 0.0001, n = 5). Finally, expression of both LFA-1 and ICAM-1 was demonstrated in human brain metastasis samples.The results of this study suggest LFA-1 as a new target in brain metastasis therapy and highlight the potential synergy with current anti-COX-2 and anti-NOS therapies.

Regulation of CXCR4 gene expression in breast cancer cells under diverse stress conditions.

Chronic inflammation is a critical component in breast cancer progression. Pro-inflammatory mediators along with growth/survival factors within the tumor microenvironment potentiate the expression of pro-inflammatory cytokines (IL-1, IL-6, TNF-α), chemotactic cytokines and their receptors (CXCR4, CXCL12, CXCL8) and angiogenic factors (VEGF) that often overcome the effect of anti-inflammatory molecules (IL-4, IL-10) thus evading the host's antitumor immunity. Detailed knowledge, therefore, of the regulatory mechanisms determining cytokine levels is essential to understand the pathogenesis of breast cancer. HIF-1α and NF-κB transcription factors are important players for the establishment of a pro-inflammatory and potentially oncogenic environment. HIF-1α is the key mediator of the cellular response to oxygen deprivation and induces the expression of genes involved in survival and angiogenesis within solid hypoxic tumors. The expression of these genes is often modulated by the p53 tumor suppressor protein that induces apoptosis or cell cycle arrest in neoplastic cells. Functional crosstalk between HIF-1α and p53 pathways mediated by modulators shared between the two transcription factors such as SRC-1 and SIRT-1 differentially regulate the expression of distinct subsets of their target genes under variable stress conditions. In an attempt to shed light on the complex regulatory mechanisms involved in cancer-related inflammation, we investigated the role of the two common p53 and HIF-1α co-regulators SRC-1 and SIRT-1, in the expression of the highly potent metastatic chemokine receptor CXCR4. Both SRC-1 and SIRT-1 overexpression in DSFX-treated MCF-7 cells reduced CXCR4 cellular levels implying that both co-regulators are crucial factors in the determination of the metastatic potential of breast cancer cells.