Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

The genetic basis of endometriosis and comorbidity with other pain and inflammatory conditions.

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention.

Gene expression of the endocannabinoid system in endometrium through menstrual cycle.

Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.

Correction: Dilated Thin-Walled Blood and Lymphatic Vessels in Human Endometrium: A Potential Role for VEGF-D in Progestin-Induced Break-Through Bleeding.

[This corrects the article DOI: 10.1371/journal.pone.0030916.].

Spatially Fractionated X-Ray Microbeams Elicit a More Sustained Immune and Inflammatory Response in the Brainstem than Homogenous Irradiation.

Synchrotron microbeam radiation therapy (MRT) is a preclinical irradiation technique which could be used to treat intracranial malignancies. The goal of this work was to discern differences in gene expression and the predicted regulation of molecular pathways in the brainstem after MRT versus synchrotron broad-beam radiation therapy (SBBR). Healthy C57BL/6 mice received whole-head irradiation with median acute toxic doses of MRT (241 Gy peak dose) or SBBR (13 Gy). Brains were harvested 4 and 48 h postirradiation and RNA was extracted from the brainstem. RNA-sequencing was performed to identify differentially expressed genes (false discovery rate < 0.01) relative to nonirradiated controls and significantly regulated molecular pathways and biological functions were identified (Benjamini-Hochberg corrected P < 0.05). Differentially expressed genes and regulated pathways largely reflected a pro-inflammatory response 4 h after both MRT and SBBR which was sustained at 48 h postirradiation for MRT. Pathways relating to radiation-induced viral mimicry, including HMGB1, NF-κB and interferon signaling cascades, were predicted to be uniquely activated by MRT. Local microglia, as well as circulating leukocytes, including T cells, were predicted to be activated by MRT. Our findings affirm that the transcriptomic signature of MRT is distinct from broad-beam radiotherapy, with a sustained inflammatory and immune response up to 48 h postirradiation.

The γH2AX DSB marker may not be a suitable biodosimeter to measure the biological MRT valley dose.

PURPOSE: γH2AX biodosimetry has been proposed as an alternative dosimetry method for microbeam radiation therapy (MRT) because conventional dosimeters, such as ionization chambers, lack the spatial resolution required to accurately measure the MRT valley dose. Here we investigated whether γH2AX biodosimetry should be used to measure the biological valley dose of MRT-irradiated mammalian cells. MATERIALS AND METHODS: We irradiated human skin fibroblasts and mouse skin flaps with synchrotron MRT and broad beam (BB) radiation. BB doses of 1-5 Gy were used to generate a calibration curve in order to estimate the biological MRT valley dose using the γH2AX assay. RESULTS: Our key finding was that MRT induced a non-linear dose response compared to BB, where doses 2-3 times greater showed the same level of DNA DSB damage in the valley in cell and tissue studies. This indicates that γH2AX may not be an appropriate biodosimeter to estimate the biological valley doses of MRT-irradiated samples. We also established foci yields of 5.9 ± 0.04 and 27.4 ± 2.5  foci/cell/Gy in mouse skin tissue and human fibroblasts respectively, induced by BB. Using Monte Carlo simulations, a linear dose response was seen in cell and tissue studies and produced predicted peak-to-valley dose ratios (PVDRs) of ∼30 and ∼107 for human fibroblasts and mouse skin tissue respectively. CONCLUSIONS: Our report highlights novel MRT radiobiology, attempts to explain why γH2AX may not be an appropriate biodosimeter and suggests further studies aimed at revealing the biological and cellular communication mechanisms that drive the normal tissue sparing effect, which is characteristic of MRT.

Elucidating the role of long intergenic non-coding RNA 339 in human endometrium and endometriosis.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however, the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterize the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing (RNA-seq), quantitative RT-PCR and in situ hybridization to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localization of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stromal cell lines and RNA-seq data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defence pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Peripheral, Central, and Cross Sensitization in Endometriosis-Associated Pain and Comorbid Pain Syndromes.

Endometriosis-associated pain and the mechanisms responsible for its initiation and persistence are complex and difficult to treat. Endometriosis-associated pain is experienced as dysmenorrhea, cyclical pain related to organ function including dysuria, dyschezia and dyspareunia, and persistent pelvic pain. Pain symptomatology correlates poorly with the extent of macroscopic disease. In addition to the local effects of disease, endometriosis-associated pain develops as a product of peripheral sensitization, central sensitization and cross sensitization. Endometriosis-associated pain is further contributed to by comorbid pain conditions, such as bladder pain syndrome, irritable bowel syndrome, abdomino-pelvic myalgia and vulvodynia. This article will review endometriosis-associated pain, its mechanisms, and its comorbid pain syndromes with a view to aiding the clinician in navigating the literature and terminology of pain and pain syndromes. Limitations of our current understanding of endometriosis-associated pain will be acknowledged. Where possible, commonalities in pain mechanisms between endometriosis-associated pain and comorbid pain syndromes will be highlighted.

Mesenchymal Niche-Derived Neuregulin-1 Drives Intestinal Stem Cell Proliferation and Regeneration of Damaged Epithelium.

Epidermal growth factor (EGF) maintains intestinal stem cell (ISC) proliferation and is a key component of organoid growth media yet is dispensable for intestinal homeostasis, suggesting roles for multiple EGF family ligands in ISC function. Here, we identified neuregulin 1 (NRG1) as a key EGF family ligand that drives tissue repair following injury. NRG1, but not EGF, is upregulated upon damage and is expressed in mesenchymal stromal cells, macrophages, and Paneth cells. NRG1 deletion reduces proliferation in intestinal crypts and compromises regeneration capacity. NRG1 robustly stimulates proliferation in crypts and induces budding in organoids, in part through elevated and sustained activation of mitogen-activated protein kinase (MAPK) and AKT. Consistently, NRG1 treatment induces a proliferative gene signature and promotes organoid formation from progenitor cells and enhances regeneration following injury. These data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of therapies for enhancing intestinal repair after injury.

Reproductive health research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2019.

The 2019 meeting of the Society for Reproductive Biology (SRB) provided a platform for the dissemination of new knowledge and innovations to improve reproductive health in humans, enhance animal breeding efficiency and understand the effect of the environment on reproductive processes. The effects of environment and lifestyle on fertility and animal behaviour are emerging as the most important modern issues facing reproductive health. Here, we summarise key highlights from recent work on endocrine-disrupting chemicals and diet- and lifestyle-induced metabolic changes and how these factors affect reproduction. This is particularly important to discuss in the context of potential effects on the reproductive potential that may be imparted to future generations of humans and animals. In addition to key summaries of new work in the male and female reproductive tract and on the health of the placenta, for the first time the SRB meeting included a workshop on endometriosis. This was an important opportunity for researchers, healthcare professionals and patient advocates to unite and provide critical updates on efforts to reduce the effect of this chronic disease and to improve the welfare of the women it affects. These new findings and directions are captured in this review.

Comparative toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model.

Synchrotron radiation can facilitate novel radiation therapy modalities such as microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR). Both of these modalities have unique physical properties that could be exploited for an improved therapeutic effect. While pre-clinical studies report promising normal tissue sparing phenomena, systematic toxicity data are still required. Our objective was to characterise the toxicity of SBBR and MRT and to calculate equivalent doses of conventional radiation therapy (CRT). A dose-escalation study was performed on C57BLJ/6 mice using total body and partial body irradiations. Dose-response curves and TD50 values were subsequently calculated using PROBIT analysis. For SBBR at dose-rates of 37 to 41 Gy/s, we found no evidence of a normal tissue sparing effect relative to CRT. Our findings also show that the MRT valley dose, rather than the peak dose, best correlates with CRT doses for acute toxicity. Importantly, longer-term weight tracking of irradiated animals revealed more pronounced growth impairment following MRT compared to both SBBR and CRT. Overall, this study provides the first in vivo dose-equivalence data between MRT, SBBR and CRT and presents systematic toxicity data for a range of organs that can be used as a reference point for future pre-clinical work.

Activation of ERBB4 in Glioblastoma Can Contribute to Increased Tumorigenicity and Influence Therapeutic Response.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ErbB2 Receptor Tyrosine Kinase 4 (ERBB4) is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total ERBB4 mRNA was significantly lower in GBM than NNB samples, with the juxtamembrane JM-a and cytoplasmic CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0 ± 3.2 months) than was no p-ERBB4 (22.5 ± 9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low ERBB4 mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.

Differential Sensitivity of Human Hepatocellular Carcinoma Xenografts to an IGF-II Neutralizing Antibody May Involve Activated STAT3.

Hepatocellular carcinoma (HCC) is highly refractory to current therapeutics used in the clinic. DX-2647, a recombinant human antibody, potently neutralizes the action of insulin-like growth factor-II (IGF-II), a ligand for three cell-surface receptors (IGF-IR, insulin receptor A and B isoforms, and the cation-independent mannose-6-phosphate receptor) which is overexpressed in primary human HCC. DX-2647 impaired the growth of tumor xenografts of the HCC cell line, Hep3B; however, xenografts of the HCC cell line, HepG2, were largely unresponsive to DX-2647 treatment. Analysis of a number of aspects of the IGF signaling axis in both cell lines did not reveal any significant differences between the two. However, while DX-2647 abolished phospho (p)-IGF-IR, p-IR and p-AKT signaling in both cell lines, HepG2 showed high levels of p-STAT3, which was unaffected by DX-2647 treatment and was absent from the Hep3B cell line. The driver of p-STAT3 was found to be a secreted cytokine, and treatment of HepG2 cells with a pan- JAK kinase inhibitor resulted in a loss of p-STAT3. These findings implicate the activation of STAT3 as one pathway that may mediate resistance to IGF-II-targeted therapy in HCC.

Axitinib Has Antiangiogenic and Antitumorigenic Activity in Myxoid Liposarcoma.

Myxoid liposarcoma is a rare form of soft-tissue sarcoma. Although most patients initially respond well to treatment, approximately 21% relapse, highlighting the need for alternative treatments. To identify novel treatment regimens and gain a better understanding of myxoid liposarcoma tumor biology, we screened various candidate and approved targeted therapeutics and chemotherapeutics against myxoid liposarcoma cell lines. Therapeutics that target angiogenesis showed antitumor activity. The small molecule inhibitor axitinib, which targets angiogenesis by inhibiting the VEGFR and PDGFR families and c-Kit, inhibited cell cycle progression and induced apoptosis in vitro, as well as having significant antitumor activity against MLS 1765 myxoid liposarcoma xenografts in mice. Axitinib also displayed synergistic antitumor activity in vitro when combined with the potassium channel ionophore salinomycin or the BH3 mimetic ABT-737. Another angiogenesis-targeting therapeutic, 4EGI-1, which targets the oncoprotein eIF4E, significantly decreased angiogenic ligand expression by myxoid liposarcoma cells and reduced tumor cell growth. To verify this oncogenic addiction to angiogenic pathways, we utilized VEGFR-derived ligand traps and found that autocrine VEGFR signaling was crucial to myxoid liposarcoma cell survival. Overall, these findings suggest that autocrine angiogenic signaling through the VEGFR family is critical to myxoid liposarcoma cell survival and that further study of axitinib as a potential anticancer therapy is warranted.

Targeting Interleukin-11 Receptor-α Impairs Human Endometrial Cancer Cell Proliferation and Invasion In Vitro and Reduces Tumor Growth and Metastasis In Vivo.

Endometrial cancer contributes to significant morbidity and mortality in women with advanced stage or recurrent disease. IL11 is a cytokine that regulates cell cycle, invasion, and migration, all hallmarks of cancer. IL11 is elevated in endometrial tumors and uterine lavage fluid in women with endometrial cancer, and alters endometrial epithelial cancer cell adhesion and migration in vitro, but its role in endometrial tumorigenesis in vivo is unknown. We injected mice subcutaneously with human-derived Ishikawa or HEC1A endometrial epithelial cancer cells (ectopic), or HEC1A cells into the uterus (orthotopic) to develop endometrial cancer mouse models. Administration of anti-human IL11 receptor (R) α blocking antibody dramatically reduced HEC1A-derived tumor growth in both models and reduced peritoneal metastatic lesion spread in the orthotopic model, compared with IgG. Anti-human IL11Rα retained a well-differentiated, endometrial epithelial phenotype in the HEC1A ectopic mice, suggesting it prevented epithelial-to-mesenchymal transition. Blockade of mouse IL11Rα with anti-mouse IL11Rα antibody did not alter tumor growth, suggesting that cancer epithelial cell IL11 signaling is required for tumor progression. In vitro, anti-human IL11Rα antibody significantly reduced Ishikawa and HEC1A cell proliferation and invasion and promoted apoptosis. Anti-human, but not anti-mouse, IL11Rα antibody reduced STAT3, but not ERK, activation in HEC1A cells in vitro and in endometrial tumors in xenograft mice. We demonstrated that targeted blockade of endometrial cancer epithelial cell IL11 signaling reduced primary tumor growth and impaired metastasis in ectopic and orthotopic endometrial cancer models in vivo Our data suggest that therapeutically targeting IL11Rα could inhibit endometrial cancer growth and dissemination. Mol Cancer Ther; 15(4); 720-30. ©2016 AACR.

Targeted drug delivery using genetically engineered diatom biosilica.

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.

A prenatally ascertained de novo terminal deletion of chromosomal bands 1q43q44 associated with multiple congenital abnormalities in a female fetus.

Terminal deletions in the long arm of chromosome 1 result in a postnatally recognizable disorder described as 1q43q44 deletion syndrome. The size of the deletions and the resulting phenotype varies among patients. However, some features are common among patients as the chromosomal regions included in the deletions. In the present case, ultrasonography at 22 weeks of gestation revealed choroid plexus cysts (CPCs) and a single umbilical artery (SUA) and therefore amniocentesis was performed. Chromosomal analysis revealed a possible terminal deletion in 1q and high resolution array CGH confirmed the terminal 1q43q44 deletion and estimated the size to be approximately 8 Mb. Following termination of pregnancy, performance of fetopsy allowed further clinical characterization. We report here a prenatal case with the smallest pure terminal 1q43q44 deletion, that has been molecularly and phenotypically characterized. In addition, to our knowledge this is the first prenatal case reported with 1q13q44 terminal deletion and Pierre-Robin sequence (PRS). Our findings combined with review data from the literature show the complexity of the genetic basis of the associated syndrome.

The identification of mitochondrial DNA variants in glioblastoma multiforme.

BACKGROUND: Mitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis. RESULTS: Using next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas. CONCLUSIONS: These novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.

A high-affinity ErbB4Fc fusion protein is a potent antagonist of heregulin-mediated receptor activation.

Ligand-mediated activation of ErbB3 and ErbB4 is implicated in the pathogenesis of several human malignancies including cancer of the ovary and melanoma. We have used the broad ErbB ligand specificity of ErbB4 to assemble and express an ErbB4 fusion protein comprising the first 497 amino acids of the mature ErbB4 ectodomain fused to the human IgG Fc constant region. The purified fusion protein, designated sErbB4.497.Fc, binds the ErbB receptor ligands betacellulin and heregulin-ß1 (HRG-ß1) with high affinity (K(D) = 130 pM), an increase in affinity of 10- to 20-fold, respectively, compared with sErbB4.615.Fc. sErbB4.497.Fc inhibited ligand-stimulated phosphorylation of epidermal growth factor receptor and ErbB2, and blocked HRG-ß1 activation of the IKB/MAP/JNK/AKT signalling pathways. sErbB4.497.Fc inhibited HRG-ß1-stimulated proliferation in MCF7 cells. In a mouse tumour xenograft model, sErbB4.497.Fc as a monotherapy modestly inhibited the growth of MDA-MB-231 breast cancer cells. sErbB4.497.Fc may be useful in an adjuvant setting in combination with conventional therapeutic agents.

Non-agonistic bivalent antibodies that promote c-MET degradation and inhibit tumor growth and others specific for tumor related c-MET.

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519-561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed ß-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.