Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

MicroRNA‐127 is Downregulated by Tudor‐SN Protein and Contributes to Metastasis and Proliferation in Breast Cancer Cell Line MDA‐MB‐231

Tudor‐SN is a multifunctional protein that is highly expressed in multiple cancers including breast cancer. Tudor‐SN, as a component in RNA‐induced splicing complex, was recently reported to regulate gene expression in a microRNA (miRNA)‐dependent manner, such as let‐7, miR‐34a and miR‐221. However, how Tudor‐SN is associated with cancer development still remains largely elusive. In the present study, we explored the role of Tudor‐SN in breast cancer. Stable knockdown of endogenous Tudor‐SN, performed on the breast cancer cell line MDA‐MB‐231 by small hairpin RNA expression vectors, suppressed the in vitro migration and invasion ability of the metastatic breast cancer cell line. Interestingly, we found Tudor‐SN as a miRNA regulator according to microarray analysis, and further identified that Tudor‐SN negatively regulated the expression of miR‐127, and consequently increased the expression of the proto‐oncogene BCL6 which was a convincing target of miR‐127. Moreover, overexpression of miR‐127 reduced the in vitro migration and proliferation ability of breast cancer cell MDA‐MB‐231. Collectively, our results suggested a novel mechanism that Tudor‐SN promoted metastasis and proliferation of breast cancer cells via downregulating the miR‐127 expression. Anat Rec, 296:1842–1849, 2013. © 2013 Wiley Periodicals, Inc.     

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Characterization of choline kinase in human endothelial cells

High choline kinase‐α (Chk‐α) expression is frequently observed in cancer cells, making it a novel target for pharmacological and molecular inhibition. As inhibiting agents are delivered systemically, it is important to determine Chk‐α expression levels in endothelial cells that line both normal and tumor vasculature, and the effect of Chk‐α downregulation on these cells. Here, we characterized Chk‐α expression and the effect of its downregulation in human umbilical vein endothelial cells (HUVECs) relative to MDA‐MB‐231 human breast cancer cells. We used small interfering RNA (siRNA) to downregulate Chk‐α expression. Basal mRNA levels of Chk‐α were approximately three‐fold lower in HUVECs relative to MDA‐MB‐231 breast cancer cells. Consistent with the differences in Chk‐α protein levels, phosphocholine levels were approximately 10‐fold lower in HUVECs relative to MDA‐MB‐231 cells. Transient transfection with siRNA‐Chk resulted in comparable levels of mRNA and protein in MDA‐MB‐231 breast cancer cells and HUVECs. However, there was a significant reduction in proliferation in MDA‐MB‐231 cells, but not in HUVECs. No significant difference in CD31 immunostaining was observed in tumor sections obtained from mice injected with control luciferase‐short hairpin (sh)RNA or Chk‐shRNA lentivirus. These data suggest that systemically delivered agents that downregulate Chk‐α in tumors will not affect endothelial cell proliferation during delivery, and further support the development of Chk‐α downregulation as a cancer‐specific treatment. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel bisphosphonate minodronate (YM529) specifically inhibits osteolytic bone metastasis produced by human small-cell lung cancer cells in NK-cell depleted SCID mice

In the present study, we examined the effects of a newly developed bisphosphonate, minodronate (YM529), on osteolytic bone metastasis caused by lung cancer. Human small-cell lung cancer (SBC-5) cells, injected intravenously into natural killer cell-depleted SCID mice, produced radiologically detectable bone metastasis by day 18 and macroscopically visible visceral metastases (lung, liver, kidney, systemic lymph node) by day 35. Prophylactic treatment with YM529 on day 1 significantly inhibited the formation of osteolytic bone metastasis evaluated on X-ray photographs in a dose-dependent manner. In addition, treatment with YM529 after establishment of bone metastasis (on day 21) also inhibited bone metastasis, although the treatment was more effective when started earlier. Single administration was as effective as repeated treatment, suggesting a sustained inhibitory effect of YM529 on bone metastasis. YM529 reduced the number of osteoclasts in the bone metastatic lesions in vivo, but had no effect on the proliferation or cytokine production of SBC-5 cells in vitro. These results suggest that YM529 is a potent inhibitor of bone metastasis of human lung cancer, probably by suppressing osteoclastic bone resorption. In contrast, treatment with YM529 had no effect on visceral metastasis, even if started on day 1, and did not prolong the survival of the mice. Therefore, development of a combined modality is necessary for prolonging the survival of small-cell lung cancer patients with multiple-organ metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment and characterization of highly osteolytic luminal breast cancer cell lines by intracaudal arterial injection

Bone is one of the most common metastatic sites of breast cancer, and bone metastasis profoundly affects the quality of life of breast cancer patients. Bone metastasis is commonly observed among all the subtypes of breast cancer; however, its molecular mechanism has been analyzed only in triple‐negative subtype of breast cancer (TNBC). To characterize the molecular mechanisms of bone metastasis of luminal breast cancer, we established a bone‐metastatic model of the MCF7, luminal breast cancer cell line, with enhanced osteolytic activity by intracaudal arterial injection (CAI). Pathological analysis of the established cell lines revealed that they exhibited fierce osteolytic ability by promoting osteoclast differentiation and activity. The signature genes extracted from highly osteolytic MCF7 cell lines were differed from those of bone‐metastatic TNBC cell lines. Our results suggest that unique mechanisms of osteolysis in bone‐metastatic lesions of luminal breast cancer. In addition, several up‐regulated genes in MCF7‐BM (Bone Metastasis) 02 cell lines correlated with poor prognosis with luminal breast cancer patients. Our findings support further study on the bone‐metastatic mechanisms of luminal breast cancer.     

Effect of truncated neurokinin‐1 receptor expression changes on the interaction between human breast cancer and bone marrow‐derived mesenchymal stem cells

Previous studies in breast cancer cell lines showed that truncated neurokinin receptor‐1 (NK1R‐Tr) was able to promote malignant transformation of breast cells, and NK1R‐Tr may contribute to tumor progression and promote distant metastasis in human breast cancer. A co‐culture model of breast cancer and bone marrow‐derived human mesenchymal stem (HMSC‐bm) cells showed that HMSC‐bm inhibited the growth of breast cancer cells and entered the bone marrow at early stages. Down‐regulation of NK1R‐Tr may be a key factor in maintaining the quiescent phenotype of breast cancer cells among bone marrow stroma. Stromal‐derived factor (SDF)‐1α expression was negatively correlated with NK1R‐Tr expression in breast cancer cells. Secretion of SDF‐1α by HMSC‐bm may maintain the quiescent phenotype of breast cancer cells among bone marrow stroma by down‐regulating NK1R‐Tr expression. Transforming growth factor (TGF)‐β1 expression was positively associated with NK1R‐Tr expression in breast cancer cells. In a co‐culture system, MDA‐MB‐231‐TGF‐β1I (TGF‐β genes were suppressed using specific shRNA) cells were able to attach to HMSC‐bm quickly, indicating that TGF‐β1 was also a key factor for maintaining the quiescent phenotype of breast cancer cells in bone marrow stroma. However, the detailed mechanism still remained unclear and could involve other molecules, in addition to NK1R‐Tr.     

Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment

During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non‐neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin‐332 (LN‐332) as a principle paracrine factor in conditioned medium from mammary epithelium‐derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA‐MB‐231 cells. Then, we carried out a morphology‐based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA‐MB‐231 cells that were induced by conditioned medium from MCF10A cells and LN‐332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium‐treated MDA‐MB‐231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.     

Spontaneous bone metastases in a preclinical orthotopic model of invasive lobular carcinoma; the effect of pharmacological targeting TGFβ receptor I kinase

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80–90% and 10–15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E‐cadherin and p53 in a conditional K14cre;Cdh1(^F/F);Trp53(^F/F) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD‐208 (60 mg/kg per day), an orally available TGFβ receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD‐208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD‐208 treatment reduced TGFβ signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti‐tumour effects of TGFβ. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Redox state alters anti‐cancer effects of wedelolactone

Wedelolactone is one of the active plant polyphenolic compounds. Anti‐tumor effects of this drug have been demonstrated recently. We have described that wedelolactone acts as catalytic inhibitor of DNA topoisomerase IIα. The aim of this study was to further characterize the mechanism of its anti‐tumor effects. We showed that wedelolactone inhibits binding of DNA topoisomerase IIα to plasmid DNA and antagonizes formation of etoposide‐induced DNA cleavage complex. The inhibition of topoisomerase IIα by wedelolactone is reversible by excess of the enzyme but not DNA. The in vitro inhibitory effect of wedelolactone on the topoisomerase IIα activity is redox‐dependent as it diminished in the presence of reducing agents. Cytotoxicity of wedelolactone was partially inhibited by N‐acetylcysteine and glutathione ethyl ester in breast cancer MDA‐MB‐231 and MDA‐MB‐468 cells while the inhibitory effect of catalase was observed only in the former cell line. Finally, we found that wedelolactone can be oxidized in the presence of copper ions resulting in DNA strand break and abasic site formation in vitro. However, wedelolactone induced neither DNA damage in MDA‐MB‐231 cells nor mutations in bacterial cells detectable by Ames test suggesting that wedelolactone may not be an effective inducer of DNA damage. We conclude that the topoisomerase IIα inhibitory‐ and DNA damaging activities of wedelolactone in vitro depend on its redox state. Pro‐oxidant activity could, however, explain only part of wedelolactone‐induced cytotoxicity. Therefore, the major cellular target(s) of wedelolactone and the exact mechanism of wedelolactone‐induced cytotoxicity still remain to be identified. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of breast cancers and therapy response by MRS and quantitative gene expression profiling in the choline pathway

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non‐invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image‐guided ³¹P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high‐resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA‐mb‐231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3‐phosphate, are all raised in breast cancer lines relative to an immortalized non‐malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase‐mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A₂‐mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model. Copyright © 2008 John Wiley & Sons, Ltd.     

Investigating intracranial tumour growth patterns with multiparametric MRI incorporating Gd‐DTPA and USPIO‐enhanced imaging

High grade and metastatic brain tumours exhibit considerable spatial variations in proliferation, angiogenesis, invasion, necrosis and oedema. Vascular heterogeneity arising from vascular co‐option in regions of invasive growth (in which the blood–brain barrier remains intact) and neoangiogenesis is a major challenge faced in the assessment of brain tumours by conventional MRI. A multiparametric MRI approach, incorporating native measurements and both Gd‐DTPA (Magnevist) and ultrasmall superparamagnetic iron oxide (P904)‐enhanced imaging, was used in combination with histogram and unsupervised cluster analysis using a k‐means algorithm to examine the spatial distribution of vascular parameters, water diffusion characteristics and invasion in intracranially propagated rat RG2 gliomas and human MDA‐MB‐231 LM2–4 breast adenocarcinomas in mice. Both tumour models presented with higher ΔR₁ (the change in transverse relaxation rate R₁ induced by Gd‐DTPA), fractional blood volume (fBV) and apparent diffusion coefficient than uninvolved regions of the brain. MDA‐MB‐231 LM2–4 tumours were less densely cellular than RG2 tumours and exhibited substantial local invasion, associated with oedema, whereas invasion in RG2 tumours was minimal. These additional features were reflected in the more heterogeneous appearance of MDA‐MB‐231 LM2–4 tumours on T₂‐weighted images and maps of functional MRI parameters. Unsupervised cluster analysis separated subregions with distinct functional properties; areas with a low fBV and relatively impermeable blood vessels (low ΔR₁) were predominantly located at the tumour margins, regions of MDA‐MB‐231 LM2–4 tumours with relatively high levels of water diffusion and low vascular permeability and/or fBV corresponded to histologically identified regions of invasion and oedema, and areas of mismatch between vascular permeability and blood volume were identified. We demonstrate that dual contrast MRI and evaluation of tissue diffusion properties, coupled with cluster analysis, allows for the assessment of heterogeneity within invasive brain tumours and the designation of functionally diverse subregions that may provide more informative predictive biomarkers.     

Quantitative T2* imaging of metastatic human breast cancer to brain in the nude rat at 3 T

This study uses quantitative T₂* imaging to track ferumoxides–protamine sulfate (FEPro)‐labeled MDA‐MB‐231BR‐Luc (231BRL) human breast cancer cells that metastasize to the nude rat brain. Four cohorts of nude rats were injected intracardially with FEPro‐labeled, unlabeled or tumor necrosis factor‐related apoptosis‐inducing ligand(TRAIL)‐treated (to induce apoptosis) 231BRL cells, or saline, in order to develop metastatic breast cancer in the brain. The heads of the rats were imaged serially over 3–4 weeks using gradient multi‐echo and turbo spin‐echo pulse sequences at 3 T with a solenoid receive‐only 4‐cm‐diameter coil. Quantitative T₂* maps of the whole brain were obtained by the application of single‐exponential fitting to the signal intensity of T₂* images, and the distribution of T₂* values in brain voxels was calculated. MRI findings were correlated with Prussian blue staining and immunohistochemical staining for iron in breast cancer and macrophages. Quantitative analysis of T₂* from brain voxels demonstrated a significant shift to lower values following the intracardiac injection of FEPro‐labeled 231BRL cells, relative to animals receiving unlabeled cells, apoptotic cells or saline. Quartile analysis based on the T₂* distribution obtained from brain voxels demonstrated significant differences (p < 0.0083) in the number of voxels with T₂* values in the ranges 10–35 ms (Q1), 36–60 ms (Q2) and 61–86 ms (Q3) from 1 day to 3 weeks post‐infusion of labeled 231BRL cells, compared with baseline scans. There were no significant differences in the distribution of T₂* obtained from serial MRI in rats receiving unlabeled or TRAIL‐treated cells or saline. Histologic analysis demonstrated isolated Prussian blue‐positive breast cancer cells scattered in the brains of rats receiving labeled cells, relative to animals receiving unlabeled or apoptotic cells. Quantitative T₂* analysis of FEPro‐labeled metastasized cancer cells was possible even after the hypointense voxels were no longer visible on T₂*‐weighted images. Published in 2010 by John Wiley & Sons, Ltd.     

Bone resorption increases tumour growth in a mouse model of osteosclerotic breast cancer metastasis

Osteosclerotic metastases account for 20% of breast cancer metastases with the remainder osteolytic or mixed. In mouse models, osteolytic metastases are dependent on bone resorption for their growth. However, whether the growth of osteosclerotic bone metastases depends on osteoclast or osteoblast actions is uncertain. In this study, we investigate the effects of high and low bone resorption on tumour growth in a mouse model of osteosclerotic metastasis. We implanted human breast cancer, MCF-7, cells into the tibiae of mice. Low and high levels of bone resorption were induced by osteoprotegerin (OPG) treatment or calcium deficient diet respectively. We demonstrate that OPG treatment significantly reduces tumour area compared to vehicle (0.42 +/- 0.06 vs. 1.27 +/- 0.16 mm2, P < 0.01) in association with complete inhibition of osteoclast differentiation. In contrast, low calcium diet increases tumour area compared to normal diet (0.90 +/- 0.30 vs. 0.58 +/- 0.20 mm2, P < 0.05) in association with increased osteoclast numbers (84.44 +/- 5.18 vs. 71.11 +/- 3.56 per mm2 bone lesion area, P < 0.05). Osteoblast surfaces and new woven bone formation were similarly increased within the tumour boundaries in all treatment groups. Tumour growth in this model of osteosclerotic metastasis is dependent on ongoing bone resorption, as has been observed in osteolytic models. Bone resorption, rather than bone formation, apparently mediates this effect as osteoblast surfaces in the tumour mass were unchanged by treatments. Treatment of breast cancer patients through correction of calcium deficiency and/or with anti-resorptive agents such as OPG, may improve patient outcomes in the adjuvant as well as palliative settings.     

CD169+ macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune‐competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169⁺ macrophages using a suicide gene knock‐in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer‐induced bone changes. In human bone metastasis specimens, CD68⁺ macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune‐competent mouse model, CD169⁺ macrophage ablation significantly inhibited prostate cancer‐induced woven bone formation, suggesting that CD169⁺ macrophages within pathological woven bone are integral to tumour‐induced bone formation. In contrast, pan‐phagocytic cell, but not targeted CD169⁺ macrophage depletion resulted in increased tumour mass, indicating that CD169^? macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour‐induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Phase contrast MRI is an early marker of micrometastatic breast cancer development in the rat brain

The early growth of micrometastatic breast cancer in the brain often occurs through vessel co‐option and is independent of angiogenesis. Remodeling of the existing vasculature is an important step in the evolution of co‐opting micrometastases into angiogenesis‐dependent solid tumor masses. The purpose of this study was to determine whether phase contrast MRI, an intrinsic source of contrast exquisitely sensitive to the magnetic susceptibility properties of deoxygenated hemoglobin, could detect vascular changes occurring independent of angiogenesis in a rat model of breast cancer metastases to the brain. Twelve nude rats were administered 10⁶ MDA‐MB‐231BRL ‘brain‐seeking’ breast cancer cells through intracardiac injection. Serial, multiparametric MRI of the brain was performed weekly until metastatic disease was detected. The results demonstrated that images of the signal phase (area under the receiver operating characteristic curve, 0.97) were more sensitive than T₂* gradient echo magnitude images (area under the receiver operating characteristic curve, 0.73) to metastatic brain lesions. The difference between the two techniques was probably the result of the confounding effects of edema on the magnitude of the signal. A region of interest analysis revealed that vascular abnormalities detected with phase contrast MRI preceded tumor permeability measured with contrast‐enhanced MRI by 1–2 weeks. Tumor size was correlated with permeability (R² = 0.23, p < 0.01), but phase contrast was independent of tumor size (R² = 0.03). Histopathologic analysis demonstrated that capillary endothelial cells co‐opted by tumor cells were significantly enlarged, but less dense, relative to the normal brain vasculature. Although co‐opted vessels were vascular endothelial growth factor‐negative, vessels within larger tumor masses were vascular endothelial growth factor‐positive. In conclusion, phase contrast MRI is believed to be sensitive to vascular remodeling in co‐opting brain tumor metastases independent of sprouting angiogenesis, and may therefore aid in preclinical studies of angiogenic‐independent tumors or in the monitoring of continued tumor growth following anti‐angiogenic therapy. Published 2011. This article is a US Government work and is in the public domain in the USA.