BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression.

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.

Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization.

Microenvironment niches determine cellular fates of metastatic cancer cells. However, robust and unbiased approaches to identify niche components and their molecular profiles are lacking. We established Sortase A-Based Microenvironment Niche Tagging (SAMENT), which selectively labels cells encountered by cancer cells during metastatic colonization. SAMENT was applied to multiple cancer models colonizing the same organ and the same cancer to different organs. Common metastatic niche features include macrophage enrichment and T cell depletion. Macrophage niches are phenotypically diverse between different organs. In bone, macrophages express the estrogen receptor alpha (ERα) and exhibit active ERα signaling in male and female hosts. Conditional knockout of Esr1 in macrophages significantly retarded bone colonization by allowing T cell infiltration. ERα expression was also discovered in human bone metastases of both genders. Collectively, we identified a unique population of ERα+ macrophages in the metastatic niche and functionally tied ERα signaling in macrophages to T cell exclusion during metastatic colonization. HIGHLIGHTS: SAMENT is a robust metastatic niche-labeling approach amenable to single-cell omics.Metastatic niches are typically enriched with macrophages and depleted of T cells.Direct interaction with cancer cells induces ERα expression in niche macrophages. Knockout of Esr1 in macrophages allows T cell infiltration and retards bone colonization.

Bone mimetic environments support engineering, propagation, and analysis of therapeutic response of patient-derived cells, ex vivo and in vivo.

Bone metastases are the most common milestone in the lethal progression of prostate cancer and prominent in a substantial portion of renal malignancies. Interactions between cancer and bone host cells have emerged as drivers of both disease progression and therapeutic resistance. To best understand these central host-epithelial cell interactions, biologically relevant preclinical models are required. To achieve this goal, we here established and characterized tissue-engineered bone mimetic environments (BME) capable of supporting the growth of patient-derived xenograft (PDX) cells, ex vivo and in vivo. The BME consisted of a polycaprolactone (PCL) scaffold colonized by human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. PDX-derived cells were isolated from bone metastatic prostate or renal tumors, engineered to express GFP or luciferase and seeded onto the BMEs. BMEs supported the growth and therapy response of PDX-derived cells, ex vivo. Additionally, BMEs survived after in vivo implantation and further sustained the growth of PDX-derived cells, their serial transplant, and their application to study the response to treatment. Taken together, this demonstrates the utility of BMEs in combination with patient-derived cells, both ex vivo and in vivo. STATEMENT OF SIGNIFICANCE: Our tissue-engineered BME supported the growth of patient-derived cells and proved useful to monitor the therapy response, both ex vivo and in vivo. This approach has the potential to enable co-clinical strategies to monitor bone metastatic tumor progression and therapy response, including identification and prioritization of new targets for patient treatment.

Fast Burst-Sparsity Learning-Based Baseline Correction (FBSL-BC) Algorithm for Signals of Analytical Instruments.

Baseline correction is a critical step for eliminating the interference of baseline drift in spectroscopic analysis. The recently proposed sparse Bayesian learning (SBL)-based method can significantly improve the baseline correction performance. However, it has at least two disadvantages: (i) it works poorly for large-scale datasets and (ii) it completely ignores the burst-sparsity structure of the sparse representation of the pure spectrum. In this paper, we present a new fast burst-sparsity learning method for baseline correction to overcome these shortcomings. The first novelty of the proposed method is to jointly adopt a down-sampling strategy and construct a multiple measurements block-sparse recovery problem with the down-sampling sequences. The down-sampling strategy can significantly reduce the dimension of the spectrum; while jointly exploiting the block sparsity among the down-sampling sequences avoids losing the information contained in the original spectrum. The second novelty of the proposed method is introducing the pattern-coupled prior into the SBL framework to characterize the inherent burst-sparsity in the sparse representation of spectrum. As illustrated in the paper, burst-sparsity commonly occurs in peak zones with more denser nonzero coefficients. Properly utilizing such burst-sparsity can further enhance the baseline correction performance. Results on both simulated and real datasets (such as FT-IR, Raman spectrum, and chromatography) verify the substantial improvement, in terms of estimation accuracy and computational complexity.

All bone metastases are not created equal: Revisiting treatment resistance in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common malignancy of the kidney, representing 80-90% of renal neoplasms, and is associated with a five-year overall survival rate of approximately 74%. The second most common site of metastasis is bone. As patients are living longer due to new RCC targeting agents and immunotherapy, RCC bone metastases (RCCBM) treatment failure is more prevalent. Bone metastasis formation in RCC is indicative of a more aggressive disease and worse prognosis. Osteolysis is a prominent feature and causes SRE, including pathologic fractures. Bone metastasis from other tumors such as lung, breast, and prostate cancer, are more effectively treated with bisphosphonates and denosumab, thereby decreasing the need for palliative surgical intervention. Resistance to these antiresportives in RCCBM reflects unique cellular and molecular mechanisms in the bone microenvironment that promote progression via inhibition of the anabolic reparative response. Identification of critical mechanisms underlying RCCBM induced anabolic impairment could provide needed insight into how to improve treatment outcomes for patients with RCCBM, with the goals of minimizing progression that necessitates palliative surgery and improving survival.

Statins reduce castration-induced bone marrow adiposity and prostate cancer progression in bone.

A fraction of patients undergoing androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) will develop recurrent castrate-resistant PCa (CRPC) in bone. Strategies to prevent CRPC relapse in bone are lacking. Here we show that the cholesterol-lowering drugs statins decrease castration-induced bone marrow adiposity in the tumor microenvironment and reduce PCa progression in bone. Using primary bone marrow stromal cells (BMSC) and M2-10B4 cells, we showed that ADT increases bone marrow adiposity by enhancing BMSC-to-adipocyte transition in vitro. Knockdown of androgen receptor abrogated BMSC-to-adipocyte transition, suggesting an androgen receptor-dependent event. RNAseq analysis showed that androgens reduce the secretion of adipocyte hormones/cytokines including leptin during BMSC-to-adipocyte transition. Treatment of PCa C4-2b, C4-2B4, and PC3 cells with leptin led to an increase in cell cycle progression and nuclear Stat3. RNAseq analysis also showed that androgens inhibit cholesterol biosynthesis pathway, raising the possibility that inhibiting cholesterol biosynthesis may decrease BMSC-to-adipocyte transition. Indeed, statins decreased BMSC-to-adipocyte transition in vitro and castration-induced bone marrow adiposity in vivo. Statin pre-treatment reduced 22RV1 PCa progression in bone after ADT. Our findings with statin may provide one of the mechanisms to the clinical correlations that statin use in patients undergoing ADT seems to delay progression to "lethal" PCa.

Multiple pathways coordinating reprogramming of endothelial cells into osteoblasts by BMP4.

Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3ß-ßcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and ß-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that ß-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.

Cabozantinib Reverses Renal Cell Carcinoma-mediated Osteoblast Inhibition in Three-dimensional Coculture In Vitro and Reduces Bone Osteolysis In Vivo.

Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.

A Novel Edge Effect Detection Method for Real-Time Cellular Analyzer Using Functional Principal Component Analysis.

Real-time cellular analyzer (RTCA) has been generally applied to test the cytotoxicity of chemicals. However, several factors impact the experimental quality. A non-negligible factor is the abnormal time-dependent cellular response curves (TCRCs) of the wells located at the edge of the E-plate which is defined as edge effect. In this paper, a novel statistical analysis is proposed to detect the edge effect. First, TCRCs are considered as observations of a random variable in a functional space. Then, functional principal component analysis (FPCA) is adopted to extract the principal component (PC) functions of the TCRCs, and the first and second PCs of these curves are selected to distinguish abnormal TCRCs. The average TCRC of the inner wells with the same culture environment is set as the standard. If the distance between the scoring point of the standard curve and one designated scoring point exceeds the defined threshold, the corresponding TCRC of the designated point should be removed automatically. The experimental results demonstrate the effectiveness of the proposed algorithm. This method can be used as a standard method to resolve general time-dependent series issues.

Bone secreted factors induce cellular quiescence in prostate cancer cells.

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.

Osteoblast-Secreted Factors Mediate Dormancy of Metastatic Prostate Cancer in the Bone via Activation of the TGFßRIII-p38MAPK-pS249/T252RB Pathway.

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFß2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFß2 and GDF10 induced dormancy through TGFßRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both in vitro and in vivo Lower TGFßRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFßRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone.Significance: These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Cancer Res; 78(11); 2911-24. ©2018 AACR.

BIGH3 Promotes Osteolytic Lesions in Renal Cell Carcinoma Bone Metastasis by Inhibiting Osteoblast Differentiation.

BACKGROUND: Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS: We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS: We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

Cabozantinib-induced osteoblast secretome promotes survival and migration of metastatic prostate cancer cells in bone.

Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

Effective treatment via early cranioplasty for intractable contralateral subdural effusion after standard decompressive craniectomy in patients with severe traumatic brain injury.

OBJECTIVE: This study aimed to introduce an effective treatment for intractable contralateral subdural effusion after standard decompressive craniectomy in patients with severe traumatic brain injury (TBI) and to analyze the underlying mechanism. METHODS: A retrospective analysis was performed in 13 patients with severe traumatic craniocerebral injury showing complicated intractable contralateral subdural effusion after standard decompressive craniectomy, in whom satisfactory results were not obtained from treatments, including compression bandaging, head-down bed rest (HDBR), continuing lumbar drainage, and Ommaya catheter drainage. Among these patients, 6 underwent temporal muscle sticking, while 7 underwent early cranioplasty. The postoperative changes in the subdural effusion were observed. RESULTS: In the 6 patients who underwent temporal muscle sticking and the 7 who underwent early cranioplasty, the subdural effusion completely resolved or was significantly reduced within one month, and no recurrence was observed in the 6-month follow-up period. However, secondary bilateral cranioplasty was still necessary in the postoperative 3-6 months for the patients who underwent temporal muscle sticking. In the early cranioplasty group, there were three total operations, and the average length of stay (ALOS) was 76days. In the temporal muscle sticking group, there were four total operations, and the ALOS was 56.1days. A retrospective analysis of surgical pain showed that 100% of the patients in the first group experienced unacceptable suffering, while 14.3% of the patients in the second group experienced pain. CONCLUSION: Early cranioplasty is an effective, economical, and less painful treatment for intractable contralateral subdural effusion after standard decompressive craniectomy.

Technique of ICP Monitored Stepwise Intracranial Decompression Effectively Reduces Postoperative Complications of Severe Bifrontal Contusion.

BACKGROUND: Bifrontal contusion is a common clinical brain injury. In the early stage, it is often mild, but it progresses rapidly and frequently worsens suddenly. This condition can become life threatening and therefore requires surgery. Conventional decompression craniectomy is the commonly used treatment method. In this study, the effect of intracranial pressure (ICP) monitored stepwise intracranial decompression surgery on the prognosis of patients with acute severe bifrontal contusion was investigated. METHOD: A total of 136 patients with severe bifrontal contusion combined with deteriorated intracranial hypertension admitted from March 2001 to March 2014 in our hospital were selected and randomly divided into two groups, i.e., a conventional decompression group and an ICP monitored stepwise intracranial decompression group (68 patients each), to conduct a retrospective study. The incidence rates of acute intraoperative encephalocele, delayed hematomas, and postoperative cerebral infarctions and the Glasgow outcome scores (GOSs) 6 months after the surgery were compared between the two groups. RESULTS: (1) The incidence rates of acute encephalocele and contralateral delayed epidural hematoma in the stepwise decompression surgery group were significantly lower than those in the conventional decompression group; the differences were statistically significant (P < 0.05); (2) 6 months after the surgery, the incidence of vegetative state and mortality in the stepwise decompression group were significantly lower than those in the conventional decompression group (P < 0.05); the rate of favorable prognosis in the stepwise decompression group was also significantly higher than that in the conventional decompression group (P < 0.05). CONCLUSION: The ICP monitored stepwise intracranial decompression technique reduced the perioperative complications of traumatic brain injury through the gradual release of ICP and was beneficial to the prognosis of severe traumatic brain injury treatment.

Predicting GHS toxicity using RTCA and discrete-time Fourier transform.

In order to promote the acceptance of cell-based toxicity testings, the accuracy of cytotoxicity test must be determined when compared to in vivo results. Traditional methods of cytotoxicity analysis, such as LC[Formula: see text] (concentration where 50% of the cells are killed) can be problematic since they have been found to vary with time. Technological advances in cytotoxicity testing make it easy to record the dynamic data on changes in cell proliferation, morphology, and damage. To effectively and reasonably analyze the dynamic data, we present a new in vitro toxicity assessed method using the discrete-time Fourier transform (DTFT) which maps the measured cell index from the time domain to the frequency domain. The direct current (DC) component of the DTFT is extracted as a feature which reflects the intensity of cytotoxicity. The smaller the value, the higher the cytotoxicity. Then, a novel toxicity index, as expressed in terms of DC[Formula: see text], is calculated. Results generated with selected test chemicals are compared favorably with data obtained from The Interagency Coordinating Committee on the Validation of Alternative Method (ICCVAM) report concerning the prediction of acute systemic toxicity in rodents. The method can be applied with the standard and high throughput to estimate acute rodent oral toxicity which reduces the number of animals required in subsequent pharmacological/toxicological studies.

Cadherin-11 endocytosis through binding to clathrin promotes cadherin-11-mediated migration in prostate cancer cells.

Cadherin-11 (Cad11) cell adhesion molecule plays a role in prostate cancer cell migration. Because disassembly of adhesion complexes through endocytosis of adhesion proteins has been shown to play a role in cell migration, we examined whether Cad11 endocytosis plays a role in Cad11-mediated migration. The mechanism by which Cad11 is internalized is unknown. Using a GST pulldown assay, we found that clathrin binds to the Cad11 cytoplasmic domain but not to that of E-cadherin. Using deletion analysis, we identified a unique sequence motif, VFEEE, in the Cad11 membrane proximal region (amino acid residues 11-15) that binds to clathrin. Endocytosis assays using K(+)-depletion buffer showed that Cad11 internalization is clathrin dependent. Proximity ligation assays showed that Cad11 colocalizes with clathrin, and immunofluorescence assays showed that Cad11 localizes in vesicles that stain for the early endosomal marker Rab5. Deletion of the VFEEE sequence from the Cad11 cytoplasmic domain (Cad11-cla-Δ5) leads to inhibition of Cad11 internalization and reduces Cad11-mediated cell migration in C4-2B and PC3-mm2 prostate cancer cells. These observations suggest that clathrin-mediated internalization of Cad11 regulates surface trafficking of Cad11 and that dynamic turnover of Cad11 regulates the migratory function of Cad11 in prostate cancer cells.

Technique of Stepwise Intracranial Decompression Combined with External Ventricular Drainage Catheters Improves the Prognosis of Acute Post-Traumatic Cerebral Hemispheric Brain Swelling Patients.

BACKGROUND: Acute post-traumatic cerebral hemispheric brain swelling (ACHS) is a serious disorder that occurs after traumatic brain injury, and it often requires immediate treatment. The aim of our clinical study was to assess the effects of stepwise intracranial decompression combined with external ventricular drainage (EVD) catheters on the prognosis of ACHS patients. METHODS: A retrospective study was performed on 172 cases of severe craniocerebral trauma patients with ACHS. The patients were divided into two groups: unilateral stepwise standard large trauma craniectomy (S-SLTC) combined with EVD catheter implants (n = 86) and unilateral routine frontal temporal parietal SLTC (control group, n = 86). RESULT: No significant differences in age, sex, or pre-operative Glasgow Coma Scale score were observed between groups (P < 0.05). There were no significant differences in the ipsilateral subdural effusion incidence rates between the S-SLTC + EVD treatment group and the routine SLTC group. However, the incidence rates of intraoperative acute encephalocele and contralateral epidural and subdural hematoma in the S-SLTC + EVD group were significantly lower than those in the SLTC group (17.4 and 3.5 vs. 37.2 and 23.3%, respectively). The mean intracranial pressure (ICP) values of patients in the S-SLTC + EVD group were also lower than those in the SLTC group at days 1 through 7 (P < 0.05). A positive neurological outcome [Glasgow Outcome Scale (GOS) score 4-5, 50.0%] and decreased mortality (15.1%) was observed in the S-SLTC + EVD group compared to the neurological outcome (GOS score 4-5, 33.8%; 36.0%) in the SLTC group (P < 0.05). CONCLUSION: Our data suggest that S-SLTC + EVD is more effective for controlling ICP, improving neurological outcome, and decreasing mortality rate compared with routine SLTC.

Three-dimensional (3D) culture of bone-derived human 786-O renal cell carcinoma retains relevant clinical characteristics of bone metastases.

Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems.