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The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro. Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. Cancer Res; 76(8); 2094–104. ©2016 AACR.

Supplementary figure 1: Scheme of the imaging setup and representative in vivo images Supplementary figure 2: Representative in vivo images of GFP+ cells in middle and large tumors Supplementary figure 3: High expression of c-Met and Flt 1/4 correlate with metastasis-free survival in breast cancer patients

Table 1: Distribution of the GFP+ cells in whole tumor central vs peripheral area and tumor lobule central vs peripheral area. Table 2: RNAseq gene expressions of growth factor receptors in sorted RFP+ and GFP+ cells.

A representative video showing the Z�stack IVM images of a 2mm�diameter pure RFP+ cellinjected tumor in the wild type mouse. Sporadic GFP+ cells with cuboidal morphology were examined in the tumor.

supplementary figure legends

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PDF file - 1060K, CCSB derived signaling networks for distinct breast cancer metastases (S1); Chemical structures of the candidate repositioned drugs and their parameters for the "Rule of Five." (S2); Western Immunoblot analysis of RET, FYN and the down-stream pathway proteins in indicated cell lines (S3); Relationship between the brain metastasis core signaling network and ER, PR, HER2 status in different breast tumor cohorts (S4); Evaluation of p-FYN and p-RET activation and tumor loci surrounding edema and vessels in the brain metastatis mouse models (S5); Pharmacokinetic study of Sunitinib and Dasatinib accumulated in brain of the xenograft models (S6); The three-dimensional cubes for the best subtrees in the survival analysis (S7).

PDF file - 43K, Enriched signaling pathways for the differentially expressed genes in brain, lung and bone metastasis (S1); Repositioned drug candidates for lung metastasis of breast cancer (S2); Expression of p-RET and p-FYN in brain metastases tissues of breast cancer patient (S3).

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Little research has been done to address the huge opportunities that may exist to reposition existing approved or generic drugs for alternate uses in cancer therapy. In addition, there has been little work on strategies to reposition experimental cancer agents for testing in alternate settings that could shorten their clinical development time. Progress in each area has lagged, in part, because of the lack of systematic methods to define drug off-target effects (OTE) that might affect important cancer cell signaling pathways. In this study, we addressed this critical gap by developing an OTE-based method to repurpose drugs for cancer therapeutics, based on transcriptional responses made in cells before and after drug treatment. Specifically, we defined a new network component called cancer-signaling bridges (CSB) and integrated it with a Bayesian factor regression model (BFRM) to form a new hybrid method termed CSB-BFRM. Proof-of-concept studies were conducted in breast and prostate cancer cells and in promyelocytic leukemia cells. In each system, CSB-BFRM analysis could accurately predict clinical responses to more than 90% of drugs approved by the U.S. Food and Drug Administration and more than 75% of experimental clinical drugs that were tested. Mechanistic investigation of OTEs for several high-ranking drug–dose pairs suggested repositioning opportunities for cancer therapy, based on the ability to enforce retinoblastoma-dependent repression of important E2F-dependent cell-cycle genes. Together, our findings establish new methods to identify opportunities for drug repositioning or to elucidate the mechanisms of action of repositioned drugs. Cancer Res; 72(1); 33–44. ©2011 AACR.

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Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line–derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCβ-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells. Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052–64. ©2018 AACR.

Ras GTPases are powerful drivers for tumorigenesis, but directly targeting Ras for treating cancer remains challenging. The growth and transforming activity of the aggressive basal-like breast cancer (BLBC) are driven by N-Ras. To target N-Ras in BLBC, this study screened existing pharmacologically active compounds for the new ability to induce N-Ras degradation, which led to the identification of flunarizine (FLN), previously approved for treating migraine and epilepsy. The FLN-induced N-Ras degradation was not affected by a 26S-proteasome inhibitor. Rather, it was blocked by autophagy inhibitors. Furthermore, N-Ras can be seen co-localized with active autophagosomes upon FLN treatment, suggesting that FLN alters the autophagy pathway to degrade N-Ras. Importantly, FLN treatment recapitulated the effect of N-RAS silencing in vitro by selectively inhibiting the growth of BLBC cells, but not that of breast cancer cells of other subtypes. In addition, in vivo FLN inhibited tumor growth of a BLBC xenograft model. In conclusion, this proof-of-principle study presents evidence that the autophagy pathway can be coerced by small molecule inhibitors, such as FLN, to degrade Ras as a strategy to treat cancer. FLN has low toxicity and should be further investigated to enrich the toolbox of cancer therapeutics.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although outcomes have improved in recent decades, new treatments are still needed to improve survival and reduce treatment-related complications. The MB subtypes groups 3 and 4 represent a particular challenge due to their intragroup heterogeneity, which limits the options for "rational" targeted therapies. Here, we report a systems biology approach to drug repositioning that integrates a nonparametric, bootstrapping-based simulated annealing algorithm and a 3D drug functional network to characterize dysregulated driver signaling networks, thereby identifying potential drug candidates. From more than 1300 drug candidates studied, we identified five members of the cardiac glycoside family as potentially inhibiting the growth of groups 3 and 4 MB and subsequently confirmed this in vitro. Systemic in vivo treatment of orthotopic patient-derived xenograft (PDX) models of groups 3 and 4 MB with digoxin, a member of the cardiac glycoside family approved for the treatment of heart failure, prolonged animal survival at plasma concentrations known to be tolerated in humans. These results demonstrate the power of a systematic drug repositioning method in identifying a potential treatment for MB. Our strategy could potentially be used to accelerate the repositioning of treatments for other human cancers that lack clearly defined rational targets.

Aims Cerebral ischemia is one of the common diseases treated by electro-acupuncture (EA). Although the clinical efficacy has been widely affirmed, the mechanisms of action leading to the health benefits are not understood. In this study, the role of EA in modulating the lactate energy metabolism and lactate transportation was explored on the middle cerebral artery occlusion (MCAO) ischemic rat model. Main methods Repeated EA treatments once daily for 7 days were applied to the MCAO rats and neurological function evaluation was performed. Brain tissues were harvested for lactate concentration examination, immunohistochemical staining, Western blot and qRT-PCR analyses for the expressions of lactate transporter (monocarboxylate transporter 1, MCT1) and glial fibrillary acidic protein (GFAP). Key findings The animal behavioral tests showed that the 7-day EA treatments significantly promoted the recovery of neurological deficits in the MCAO rats, which correlated with the enhanced lactate energy metabolism in the ischemic brain. In the cortical ischemic area of the MCAO rats, EA treatments led to the activation of astrocytes, and induced a further increase of lactate transporter (monocarboxylate transporter 1, MCT1) expression in astrocytes at both protein and mRNA levels. Significance Our results suggest that the EA treatments activated lactate metabolism in the resident astrocytes around the ischemic area and up-regulated the expression of MCT1 in these astrocytes which facilitated the transfer of intracellular lactate to extracellular domain to be utilized by injured neurons to improve the neurological deficit.

Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IκB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IκB signaling each prohibited the EMT and tumor progression.

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro. Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. Cancer Res; 76(8); 2094–104. ©2016 AACR.

A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.

mTOR inhibitor rapamycin and its analogs are lipophilic, demonstrate blood–brain barrier penetration, and have shown promising antitumor effects in several types of refractory tumors. We thus try to explore the therapeutic effects of mTOR inhibitors on brain metastasis models. We examined the effects of different dose of mTOR inhibitors (rapamycin, Temsirolimus-CCI-779) on cell invasion in two brain metastatic breast cancer cell lines (MDA-MB231-BR and CN34-BrM2). Antibody microarray and immunoblotting were applied to detect signaling pathways underlying the dose differential drug effects. The in vivo effects of single drug (CCI-779), and drug combination of CCI-779 with SL327 (a brain penetrant MEK inhibitor) to eliminate the unfavorable activation of MAPK pathway were evaluated in MDA-MB231-BR brain metastases xenograft mice. The two mTOR inhibitors, rapamycin and CCI-779, inhibited the invasion of brain metastatic cells only at a moderate concentration level, which was lost at higher concentrations secondary to activation of the MAPK signaling pathway. Pharmacological inhibition of ERK1/2 by PD98059 and SL327 restored the anti-invasion effects of mTOR inhibition in vitro. In vivo, a significant decrease was noted in the average number of micro and large metastatic lesions as well as the whole brain GFP expression in the CCI-779 1 mg/kg/day treated group compared with that in the vehicle group (P

Usually cell-based assay is performed using titer plates. Because of the large library of chemical compounds, robust and rapid methods are required to find, refine and test a potential drug candidate in an efficient manner. In this article, the drug effects analysis on human breast cancer cells with a droplet microfluidic chip is reported. Each droplet serves as a nanoliter-volume titer plate and contains a human breast cancer cell MDA-MB-231, Cytochalasin D drug solution and cell viability indicator such as Calcein AM, which emits cytoplasmic green fluorescence. The drug effects on each cell are monitored in real time using a fluorescence microscope and by analyzing the fluorescence image of each cell. Clear change of the cell shape and size has been observed after the drug treatment, which is similar to that of conventional petri dish technique, suggesting this approach is a potential viable technical platform for drug effect analysis and for high throughput drug screen and discovery.

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1alpha in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1alpha may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.

Object The study of tumor cell growth and invasion in cancer biology is often limited by the inability to visualize tumor cell behavior in real time in animal models. The authors provide evidence that glioma cells are heterogeneous, with a subset responsible for increased invasiveness. The use of bioluminescence (BL) imaging to investigate dynamic aspects of glioma progression are discussed. Methods Glioblastoma multiforme–initiating cells were generated under conditions typically used to sustain neural stem cells. The invasiveness potential was determined using a Matrigel chamber. The presence of an “invasiveness gene signature” that correlated with patient survival outcome was ascertained through microarray gene expression analysis. To measure invasiveness, the authors devised a method focussed on BL imaging and tested it in vitro and in vivo using a zebrafish xenograft model. Bioluminescence imaging signals were verified using known inhibitors of glioma growth: AEE788, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester, and compound E. Results The authors' data support the idea that glioblastoma multiforme–initiating cells are heterogeneous and possess an invasive subset; BL imaging was used as a readout method to assess this invasive subset. The in vitro data obtained using a known glioma growth inhibitor, AEE788, showed that BL imaging could detect cellular movement and invasion even before overall cell death was detectable on conventional viability assays. Further work using a zebrafish tumor xenograft model supported the efficacy of BL imaging in monitoring changes in tumor load. Conclusions The authors used optically transparent zebrafish and high-resolution confocal imaging to track tumor growth in vivo and demonstrate the efficacy of this model for screening antitumor and antiangiogenic compounds. The integration of zebrafish transgenic technology into human cancer biological studies may aid in the development of cancer models targeting specific organs, tissues, or cell types within tumors. Zebrafish could also provide a cost-effective means for the rapid development of therapeutic agents directed at blocking tumor growth and invasion.

Aim In the present study, we investigated the effects of pharmacological serum from the ovariectomized mice treated with Bushen Ningxin Decoction (BSNXD) on cell growth and etoposide-induced apoptosis of mouse osteoblasts (OBs). Methods The ovariectomized BALB/c mice serving as a postmenopausal osteoporosis model were treated with BSNXD for 12 weeks. The serum from the mice was collected and applied to the cultivation of the calvaria OBs of newborn BALB/c litters. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. Apoptosis rate was analyzed by Annexin V-FITC/PI flow cytometry (FCM) and the expression of pERK1/2 was measured by Western blot. Results The calvaria OBs treated with BSNXD serum showed more potent ability for cell proliferation than control groups (serum from the ovariectomized mice treated with saline). The BSNXD serum inhibited the etoposide-induced apoptosis of the OBs, with the effective concentrations of 10–20%. While being pretreated with the selective inhibitor of ERK1/2 U0126, the anti-apoptosis effect of the pharmacological serum on the OBs was almost completely blocked. Meanwhile, the expression of phosphor-pERK1/2 decreased significantly. Conclusion The BSNXD pharmacological serum promoted the proliferation and inhibited the apoptosis of the mouse OBs, which might be through the activation of MAPK signal transduction pathways and ERK1/2 activation via phosphoration of ERK.

The mass spectrometry (MS) technology in clinical proteomics is very promising for discovery of new biomarkers for diseases management. To overcome the obstacles of data noises in MS analysis, we proposed a new approach of knowledge-integrated biomarker discovery using data from Major Adverse Cardiac Events (MACE) patients. We first built up a cardiovascular-related network based on protein information coming from protein annotations in Uniprot, protein–protein interaction (PPI), and signal transduction database. Distinct from the previous machine learning methods in MS data processing, we then used statistical methods to discover biomarkers in cardiovascular-related network. Through the tradeoff between known protein information and data noises in mass spectrometry data, we finally could firmly identify those high-confident biomarkers. Most importantly, aided by protein–protein interaction network, that is, cardiovascular-related network, we proposed a new type of biomarkers, that is, network biomarkers, composed of a set of proteins and the interactions among them. The candidate network biomarkers can classify the two groups of patients more accurately than current single ones without consideration of biological molecular interaction.

We investigated the effects of visceral stimulation by colorectal distension (CRD) on mean arterial blood pressure (MAP) and renal sympathetic nerve activity (RSNA), the latter being an index of vasoconstrictor activity, in anesthetized central nervous system (CNS)-intact and C2 spinalized rats. The CRD stimulation was induced by the distention of a balloon inserted into the colorectum. In CNS-intact rats, there were significant reductions in MAP and RSNA in response to intraballoon pressures of 60 and 80 mmHg, but not to 20 and 40 mmHg. However, spinalized rats demonstrated significant increases in MAP in response to intraballoon pressures of 60 and 80 mmHg and increases in RSNA in response to intraballoon pressures of 40, 60, and 80 mmHg. These results suggest that noxious visceral stimulation at lower spinal levels reduces MAP by inhibiting sympathetic output in CNS-intact anesthetized rats. On the other hand, noxious visceral stimulation results in an increase in sympathetic-induced MAP in spinalized anesthetized rats.

Acupuncture treatment is one of the most desirable choices for the management of pain including chronic visceral pain, but its scientific evidence and laws of action are not very clear at this point. In this study, we examined the immediate and cumulative effects of electro-acupuncture (EA) on chronic visceral pain induced by colorectal distention (CRD) stimuli in rats using an irritable bowel syndrome (IBS) model (a chronic visceral hypersensitivity model). The results demonstrated that EA could significantly depress both abnormally increased abdominal withdrawal reflex (AWR) scores and the magnitude of electromyograms (EMGs) recorded from the rectus abdominis in response to CRD stimulation at strengths of 20, 40, 60, and 80 mmHg. Repeated EA treatment for 14 days showed that the effects of EA on both abnormal AWRs and EMGs appeared 2-4 days after the start of the EA application session, gradually enhanced to its maximum within 8-12 days, and lasted 5 days after EA treatment stopped. These data provide evidence that visceral pain associated with the rat IBS model can be effectively treated by EA and opens up the possibility of clinical treatment of chronic visceral pain with acupuncture.

Summary Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor with a very poor prognosis. Current therapies for GBM remain palliative and advances made in decades have resulted in only a slight improvement in treatment outcome. Exploring new therapeutic agents for GBM treatment, therefore, is of prime importance. In the present study, we performed a high-throughput screening for GBM cell growth and invasion, with an attempt to identify novel potential anti-GBM agents. An annotated compound library (LOPAC1280) of 1,280 pharmacologically active compounds was screened and ten compounds were validated and identified as inhibitors of GBM cell growth and invasion. Four of them, i.e., 6-nitroso-1,2-benzopyrone, S-(p-azidophenacyl) glutathione, phenoxybenzamine hydrochloride, and SCH-28080 have not been implicated in GBM cell growth and invasion previously, suggesting that they may serve as novel potential therapeutic agents for GBM treatment. In conclusion, novel inhibitors of GBM cell growth and invasion were identified in the present study, which provides a basis for the development of therapies for GBM, and may shed light on the molecular mechanisms underlying GBM cell behavior.

Dehydroepiandrosterone (DHEA) is widely known for its beneficial effect on postmenopausal osteoporosis, although the underlying mechanisms remain mainly unclear. In this study, we tried to determine the activation of mitogen-activated protein kinase signal pathways during DHEA treatment and the indirect role of osteoblasts (OBs) on osteoclasts under the DHEA treatment of postmenopausal osteoporosis.Primary human OBs and osteoclast-like cells were cultured and, we pretreated OBs with or without U0126 (a highly selective inhibitor of both MEK1 and MEK2). The OBs were treated with DHEA. We then tested the effects of DHEA on human osteoblastic viability, osteoprotegerin production and the expression of phosphor-ERK1/2 (extracellular signal-regulated kinase). In the presence or absence of OBs, the function of osteoclastic resorption upon DHEA treatment was calculated.DHEA promoted the human osteoblastic proliferation and inhibited the osteoblastic apoptosis within the concentration range of 10(-8) - 10(-6) mol/L (P0.05, P0.01, respectively). Within the effective concentration range, the expression of phosphor-ERK1/2 and osteoprotegerin was increased by DHEA and blocked by U0126. In the presence of OBs, DHEA could significantly decrease the number and the area of bone resorption lacuna (P0.05 and P0.01, respectively). Without OBs, however, the effects of DHEA on the bone resorption lacuna were almost completely abolished.DHEA could indirectly inhibit the human osteoclastic resorption through promoting the osteoblastic viability and osteoprotegerin production, which is mediated by mitogen-activated protein kinases signal pathway involving the phosphor-ERK1/2.

Aberrant expression of epidermal growth factor receptor (EGFR; ErbB1) and HER2 (ErbB2) tyrosine kinases frequently occurs in glioblastoma multiforme (GBM) patients and is considered to be associated with tumor malignancy and poor patient prognosis. In the present study, a dual EGFR and HER2 inhibitor (GW2974) was evaluated for its effects in GBM in vitro and in vivo. Results showed that low-concentration GW2974 inhibited GBM cell invasion, whereas a high concentration of the same compound counteracted this effect. Similar results were observed in an intracranial GBM xenograft model, in which, although both doses of GW2974 slowed tumor growth, no improvement in survival was observed in mice treated with high-dose GW2974, presumably because of the augmentation of tumor invasion. By protein microarray and Western blot analyses, the p38 mitogen-activated protein kinase (MAPK) pathway was found to be activated in GBM cells under high-concentration GW2974. Additionally, blockage of the p38 MAPK pathway reproduced the inhibitory effect of low-concentration GW2974 on cell invasion. These data suggest that the p38 MAPK pathway might contribute to the differential regulation of cell invasion by GW2974. Taken together, our results indicate that GW2974 exerts different effects in GBM depending on drug dosage. This offers a new perspective on the role of GW2974 in tumor progression, providing a potential strategy for GBM treatment based on precise prescription. © 2012 Wiley Periodicals, Inc.

Little research has been done to address the huge opportunities that may exist to reposition existing approved or generic drugs for alternate uses in cancer therapy. In addition, there has been little work on strategies to reposition experimental cancer agents for testing in alternate settings that could shorten their clinical development time. Progress in each area has lagged, in part, because of the lack of systematic methods to define drug off-target effects (OTE) that might affect important cancer cell signaling pathways. In this study, we addressed this critical gap by developing an OTE-based method to repurpose drugs for cancer therapeutics, based on transcriptional responses made in cells before and after drug treatment. Specifically, we defined a new network component called cancer-signaling bridges (CSB) and integrated it with a Bayesian factor regression model (BFRM) to form a new hybrid method termed CSB-BFRM. Proof-of-concept studies were conducted in breast and prostate cancer cells and in promyelocytic leukemia cells. In each system, CSB-BFRM analysis could accurately predict clinical responses to more than 90% of drugs approved by the U.S. Food and Drug Administration and more than 75% of experimental clinical drugs that were tested. Mechanistic investigation of OTEs for several high-ranking drug–dose pairs suggested repositioning opportunities for cancer therapy, based on the ability to enforce retinoblastoma-dependent repression of important E2F-dependent cell-cycle genes. Together, our findings establish new methods to identify opportunities for drug repositioning or to elucidate the mechanisms of action of repositioned drugs. Cancer Res; 72(1); 33–44. ©2011 AACR.

Real-time tagless monitoring of cell viability using patch-clamp microchips is reported and validated by using fluorescence imaging techniques for the first time. Specifically, four human breast cancer cell lines (MDA-MB231, MDA-MB231-brain metastatic subline (abbreviated as MB231-BR), MB231-BR over-expressing HER2 gene (MB231-BR-HER2), and MB231-BR-vector control for the HER2 (MB231-BR-vector)) have been used for these studies. Systematic experiments on these cells found that the seal impedance/resistance of cells captured by the micro-pipettes always decreases during the process when the cell loses its viability, and therefore it is a valid indicator of live or dead cells. Systematic experiments also found that the Mega-seal of patch-clamp microchip is sufficient for monitoring cell viability. Given its simplicity of direct electrical measurement of cells without fluorescence labeling, this technology may provide an efficient technical platform to monitor the drug effects on cells, thereby significantly benefiting high throughput drug screening and discovery process.

Cell migration is a key step for deterioration of many in situ or metastasis malignant tumours. Tumour anti-migration is a promising strategy to treat cancer, but corresponding drugs developed under such a strategy are still in dire poverty, partly due to the lengthly process of drug trials and approval required by the US Food and Drug Administration (FDA). Given there are thousands of FDA approved drugs in the market, we believe that drug repositioning may provide a fast and cost-effective way to identify potential anti-migration drugs. In this paper, an in-silico drug screening method using a genomic strategy is proposed for the goal, in which genomic signature identification combined with support vector machine modelling is adopted to estimate drug efficacy. And a high-throughput, sensitive, 3-dimensional invasion assay by quantitative bioluminescence imaging proved the performance of proposed method on in vitro disease models. © 2011 IEEE.

Engineered antibodies, with their high specificity and affinity for their target antigens, as well as their reduced size and multivalent design, can be tailored to carry radionuclide magnetic, luciferase or fluorescent probes for specific attachment to tissue cells, extracellularly or intracellularly, for PET, SPECT, MRI, optical and ultrasonic imaging. The antigen-specific imaging agents of engineered antibodies have deep tissue penetration, high tissue retention and fast blood clearance, which are desirable properties for the rapid imaging of tumors with high specificity and resolution in pre-clinical or clinical imaging studies.

Magnetic resonance imaging (MRI) is the imaging modality of choice by which to monitor patient gliomas and treatment effects, and has been applied to murine models of glioma. However, a major obstacle to the development of effective glioma therapeutics has been that widely used animal models of glioma have not accurately recapitulated the morphological heterogeneity and invasive nature of this very lethal human cancer. This deficiency is being alleviated somewhat as more representative models are being developed, but there is still a clear need for relevant yet practical models that are well-characterized in terms of their MRI features. Hence we sought to chronicle the MRI profile of a recently developed, comparatively straightforward human tumor stem cell (hTSC) derived glioma model in mice using conventional MRI methods. This model reproduces the salient features of gliomas in humans, including florid neoangiogenesis and aggressive invasion of normal brain. Accordingly, the variable, invasive morphology of hTSC gliomas visualized on MRI duplicated that seen in patients, and it differed considerably from the widely used U87 glioma model that does not invade normal brain. After several weeks of tumor growth the hTSC model exhibited an MRI contrast enhancing phenotype having variable intensity and an irregular shape, which mimicked the heterogeneous appearance observed with human glioma patients. The MRI findings reported here support the use of the hTSC glioma xenograft model combined with MRI, as a test platform for assessing candidate therapeutics for glioma, and for developing novel MR methods.

Diffusion tensor imaging (DTI) is an effective modality in studying the connectivity of the brain. To eliminate possible biases caused by fiber extraction approaches due to low spatial resolution of DTI and the number of fibers obtained, the fast marching (FM) algorithm based on the whole diffusion tensor information is proposed to model and study the brain connectivity network. Our observation is that the connectivity extracted from the whole tensor field would be more robust and reliable for constructing brain connectivity network using DTI data. To construct the connectivity network, in this paper, the arrival time map and the velocity map generated by the FM algorithm are combined to define the connectivity strength among different brain regions. The conventional fiber tracking-based and the proposed tensor-based FM connectivity methods are compared, and the results indicate that the connectivity features obtained using the FM-based method agree better with the neuromorphical studies of the human brain.

Background Cancer and Alzheimer's disease (AD) are two seemingly distinct diseases and rarely occur simultaneously in patients. To explore molecular determinants differentiating pathogenic routes towards AD or cancer, we investigate the role of amyloid β protein (Aβ) on multiple tumor cell lines that are stably expressing luciferase (human glioblastoma U87; human breast adenocarcinoma MDA-MB231; and mouse melanoma B16F). Results Quantification of the photons emitted from the MDA-MB231 or B16F cells revealed a significant inhibition of cell proliferation by the conditioning media (CM) derived from amyloid precursor protein (APP) over-expressing cells. The inhibition of U87 cells was observed only after the media was conditioned for longer than 2 days with APP over-expressing cells. Conclusion Our results suggest that Aβ plays an inhibitory role in tumor cell proliferation; this effect could depend on the type of tumor cells and amount of Aβ.

In vivo bioluminescence imaging (BLI) has the advantages of high sensitivity and low background. By counting the number of photons emitted from a specimen, BLI can quantify biological events such as tumour growth, gene expression and drug response. The intensities and kinetics of the BL signal are affected by many factors and may confound the quantitative results acquired from consecutive imaging sessions or different specimens. We used three different mouse models of tumours to examine whether anaesthetics, positioning and tumour growth may affect the consistency of the BL signal. The results showed that BLI signal could be affected by different anaesthetics and repetitive positioning. Using the same anaesthetics produced consistent peak times, while other factors were held constant. However, as the tumours grew the peak times shifted and the time course of BL signals had different shapes, depending on the positioning of the mice. The data indicate that a carefully designed BLI experiment is required to generate optimal and consistent results.

Engineered antibodies, with their high specificity and affinity for their target antigens, as well as their reduced size and multivalent design, can be tailored to carry radionuclide magnetic, luciferase or fluorescent probes for specific attachment to tissue cells, extracellularly or intracellularly, for PET, SPECT, MRI, optical and ultrasonic imaging. The antigen-specific imaging agents of engineered antibodies have deep tissue penetration, high tissue retention and fast blood clearance, which are desirable properties for the rapid imaging of tumors with high specificity and resolution in pre-clinical or clinical imaging studies.

Anti-migratory drugs constitute a promising strategy for cancer treatments. In present study, we proposed a multi-scale high-throughput anti-migratory assay by bioluminescent imaging, including in vitro 3D cell assay, in vivo anti-migratory assay on Zebrafish embryo xenograft, and mouse metastatic tumor model. The combined assay by bioluminescent imaging is less labor intensive, faster, sensitive, and easily repeatable, which make it attractive for high throughput screening for anti-migration compounds.

Electroacupuncture (EA) was applied bilaterally to the acupoints of Zu-san-li (ST-36) and Kun-lun (BL-60) in the hindlimbs of mice. The therapeutic effect of EA on inflammatory pain induced by an ipsilateral injection of complete Freund's adjuvant (CFA) into the right paw of the mouse was investigated in this study. The time of paw-withdrawal latency (PWL) was used as an indicator for judging the intensity of the pain induced by the CFA injection. The EA effects were divided into immediate (PWL tests within 2 h after EA treatment) and cumulative (PWL tests during and after repetitive EA treatments for 3 weeks) effects. As immediate effects, PWL was significantly shortened in the CFA-injected paw, but was again prolonged 20 min after an EA treatment and lasted until 30 min after. As cumulative effects, PWL was significantly shortened in the CFA-injected paw, but recovered from the 2nd to the 8th day during repetitive EA treatments. No such effects could be observed after sham EA treatment, which resulted in behavior similar to that in untreated animals. These results demonstrate that the CFA-induced inflammatory pain in mice is an ideal model system for the investigation of EA effects and may serve as a valuable reference for the clinical treatment of inflammatory pain in human beings. Furthermore, the mouse pain model opens the possibility to apply the investigation also to transgenic mice.

2098 Background: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effects against radiation-induced lipid peroxidation. Also in vitro antiproliferative effects have been reported against glioblastoma cell lines. The aim of this study was to use a liposomal formulation to evaluate LAZ radioprotective and antiproliferative effects in a glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed at the University of Houston, College of Pharmacy. Glioblastoma cell line U87-expressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. There were 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation 2Gy weekly (M+R), Lipo G at 5 mg/kg dose IP twice per week (M+L) and radiation with Lipo G (M+R+L). Treatment lasted three weeks. Tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse. Mice were sacrificed after 3 weeks. Brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. Survival was evaluated using Kaplan Meier analysis at P= 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to control . For the radiation treated groups (M+R and M+R+L), there was no significant difference in tumor size compared to control group. MDA brain concentration in M+L and M+R+L groups was significantly less than in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. Mean survival of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group Conclusions: LAZ liposomal formulations administered at 5 mg/kg dose reduced tumor growth by 65%. LAZ also protected brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration by 50%. These provocative data warrant further investigation of LAZ as a radiation protectant and chemotherapeutic agent.

Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2011-5460

Brain metastasis is the worst stage of breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cells get through blood-brain barrier and grow in brain tissue. To provide information in this direction, we developed a microfluidic single-cell patch clamp microdevice to study the electrophysiological and biomechanical properties of human breast cancer brain metastatic cells for the first time. The results show that the brain metastatic cells possess higher membrane resistance and greater membrane stiffness compared to non-brain metastatic cells. The over-expression of HER2 and EGFR decrease the cell membrane resistance, while increase the cell stiffness, which correlates with the enhanced cell invasion through the in vitro blood-brain barrier and increased formation of brain metastatic loci in the xenograft mice. Gene expression analysis of the brain metastatic cells identifies cell membrane protein PCDH7 mediates the increased cell mechanics property functionally, and PCDH7 is also an enhancer of blood-brain barrier crossing and brain colonization for the brain metastatic cells, through the cytoskeleton rearrangement mechanism. This cell mechanics-cytoskeleton-PCDH7 protein loop mechanism may prove to be an attraction therapeutic target to block the brain metastasis of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2011-5161

Phase I/II clinical studies with rapamycin analogs in breast and other cancers have demonstrated favorable responses. However, little is known on the effects of the mTOR inhibitor on breast cancer cell metastasis, which is a major cause of morbidity and death. We developed a highly sensitive 3-dimensional (3D) proliferation/invasion assay using quantitative bioluminescence (BL) imaging and applied this assay to evaluate the effects of rapamycin on the triple negative breast cancer cell line MDA-MB231. Without cytotoxicity of rapamycin on this cell line, rapamycin at 10nM inhibited the cell migration/invasion, but not at 1nM and 100nM, which was confirmed by the time-lapse single cell tracking analysis. The quantification of cytoskeleton changes showed most potent effects of 10nM rapamycin on the MDA-MB231 cells, with the formation and rearrangement of specialized cell membrane structures and actin fiber implicated in cell motility. Then, the Panorama Cell Signaling Antibody Microarray, enabling the global comparative analysis of cell signal proteins simultaneously, was exploited to analyze the effects of rapamycin on the cellular signaling network of the MDA-MB231 breast cancer cell line. 100nM rapamycin activated the MAPK pathway obviously, through the attenuated negative feedback of activated S6K1 to PI3K-Raf, which increased the expressions of activated Jun N-terminus kinase (JNK), Erk1/2, MEK-1, Raf-pS621, and MAPK-activated protein kinase 2 (MAPKAPK2) in the cells exposed to 100nM rapamycin. MEK inhibitor U0126 or PD98059 could restore the anti-migration effects of 100nM rapamycin on the MDA-MB231 cells. Furthermore, the combination of MEK inhibitors and rapamycin performed synergism on inhibiting the cell proliferation and migration/invasion. Accordingly, rapamycin at a certain dose suppresses MDA-MB231 cell migration/invasion, and the co-targeting of mTOR and MAPK pathways enhances the inhibition on cell proliferation and migration/invasion, underscoring the potential therapeutic utility of rapamycin, and rapamycin combining with MAPK inhibitors in triple negative breast cancer progression, and the results highlight the cross-talk homeostasis of mTOR and MAPK pathways in cancer treatment. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5080.