Evaluation of Facial Vitiligo Severity with a Mixed Clinical and Artificial Intelligence Approach.

Vitiligo is the most common depigmenting skin disorder. Given the ongoing development of new targeted therapies, it has become important to evaluate adequately the surface area involved. Assessment of vitiligo scores can be time consuming, with variations between investigators. Therefore, the aim of this study was to build an artificial intelligence system capable of assessing facial vitiligo severity. One hundred pictures of faces of patients with vitiligo were used to train and validate the artificial intelligence model. Sixty-nine additional pictures of facial vitiligo were then used as a final dataset. Three expert physicians scored the facial vitiligo on the same 69 pictures. Inter and intrarater performances were evaluated by comparing the scores between raters and artificial intelligence. Algorithm assessment achieved an accuracy of 93%. Overall, the scores reached a good agreement between vitiligo raters and the artificial intelligence model. Results demonstrate the potential of the model. It provides an objective evaluation of facial vitiligo and could become a complementary/alternative tool to human assessment in clinical practice and/or clinical research.

An EZH2 blocker sensitizes histone mutated diffuse midline glioma to cholesterol metabolism inhibitors through an off-target effect.

Background: Diffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target. Methods: We used a chemical EZH2 inhibitor, GSK126, small interfering RNAs, and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures, and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model. Results: GSK126 shows significant (P < .05-.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro, in DMG spheroid cultures, and in chick and mouse in vivo models (P < .05). All statistical tests were two-sided. Conclusions: Our results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity.

HDAC inhibition induces expression of scaffolding proteins critical for tumor progression in pediatric glioma: focus on EBP50 and IRSp53.

BACKGROUND: Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials. METHODS: BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization. RESULTS: Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo. CONCLUSION: Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment.

Rapid development of API nano-formulations from screening to production combining dual centrifugation and wet agitator bead milling.

Various wet ball nanomilling-screening tools for poorly soluble APIs are available which differ in their milling principle, batch size and number of samples. Here, the transferability of results from screening (small to medium-scale) to pharmaceutical production (largescale) was investigated. Wet ball milling in a dual centrifuge (DC) (10-100 mg API, 40 samples in parallel) was used to identify stable nanoformulations. In addition different sized agitator bead mills were used for scale-up to industrial scales. DC-and small-scale agitator milling (AM) resulted in small and virtually identical API-particles. Additionally, similar API-particles were obtained using two different sized agitator bead mills (batch size 1.5 and 30 kg) and applying comparable specific grinding energies (SGE). The SGE used in the trials represents the grinding limit for this API-suspension. Using lower SGEs, AM results in larger API-particles. All used milling tools had no influence on the APIs crystal structure and wear of grinding media (Zr/Y) is low. The study confirmed the importance to choose the right formulation and process parameters, which positively affect grinding efficacy, particle size distribution and wear contamination. The excellent comparability of results obtained from DC-milling and AM significantly reduces the duration for successful and predictable formulation development.

Tracking cellular and molecular changes in a species-specific manner during experimental tumor progression in vivo.

Hepatoblastoma (HBL) is a pediatric liver cancer with defined molecular alterations driving its progression. Here, we describe an animal model for HBL on the chick chorioallantoic membrane (CAM), which recapitulates relevant features of HBL in patients. Expression of classic tumor-associated proteins such as ß-catenin, EpCAM and CK19 was maintained in acini-like organized tumors on CAM, as was synthesis of AFP, a tumor marker used for monitoring patient response. RNA sequencing revealed an unexpected molecular evolution of HBL cells on the CAM, with significant deregulation of more than 6,000 genes including more than half of all HOX genes. Bioinformatic analysis distinguish between tumor cell-expressed genes and chick genes, thereby shedding new light on the complex interactions taking place during HBL progression. Importantly, human tumor suppressive ribosomal genes were downregulated after implantation, whereas mitochondrial genes encoding for anti-apoptotic peptides were strongly induced in vivo. Meprin-1α expression was increased during evolution of CAM tumors and confirmed by immunohistochemistry. Cisplatin, a commonly used chemotherapeutic agent for HBL, showed significant anti-tumoral effects. Our results broaden the understanding of the molecular adaptation process of human cancer cells to the microenvironment and might help to elaborate novel therapeutic concepts for the treatment of this pediatric liver tumor.

New insights into diagnosis and therapeutic options for proliferative hepatoblastoma.

Surgery and cisplatin-based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%-80% of patients. However, some important challenges remain in diagnosing high-risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16-gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four-gene signature: hydroxysteroid 17-beta dehydrogenase 6, integrin alpha 6, topoisomerase 2-alpha, and vimentin, with topoisomerase 2-alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT-PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration-approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway-associated double-strand DNA repair, and significantly impedes HB growth in vivo. CONCLUSION: The highly proliferating C2A subtype is characterized by topoisomerase 2-alpha gene up-regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89-102).

Dual centrifugation - A new technique for nanomilling of poorly soluble drugs and formulation screening by an DoE-approach.

The development of nanosuspensions of poorly soluble APIs takes a lot of time and high amount of active material is needed. In this publication the use of dual centrifugation (DC) for an effective and rapid API-nanomilling is described for the first time. DC differs from normal centrifugation by an additional rotation of the samples during centrifugation, resulting in a very fast and powerful movement of the samples inside the vials, which - in combination with milling beads - result in effective milling. DC-nanomilling was compared to conventional wet ball milling and results in same or even smaller particle sizes. Also drug concentrations up to 40% can be processed. The process is fast (typical 90min) and the temperature can be controlled. DC-nanomilling appears to be very gentle, experiments showed no change of the crystal structure during milling. Since batch sizes are very small (100-1000mg) and since 40 sample vials can be processed in parallel, DC is ideal for the screening of suitable polymer/surfactant combinations. Fenofibrate was used to investigate DC-nanomilling for formulation screening by applying a DoE-approach. The presented data also show that the results of DC-nanomilling experiments are highly comparable to the results obtained by common agitator mills.

New tumor suppressor microRNAs target glypican-3 in human liver cancer.

Glypican-3 (GPC3) is an oncogene, frequently upregulated in liver malignancies such as hepatocellular carcinoma (HCC) and hepatoblastoma and constitutes a potential molecular target for therapy in liver cancer. Using a functional screening system, we identified 10 new microRNAs controlling GPC3 expression in malignant liver cells, five of them e.g. miR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v reduce GPC3 expression. These 5 microRNAs were significantly downregulated in tumoral compared to non-tumoral liver and inhibited tumor cell proliferation. Interestingly, miR-4510 inversely correlated with GPC3 mRNA and protein in HCC samples. This microRNA also induced apoptosis of hepatoma cells and blocked tumor growth in vivo in the chick chorioallantoic membrane model. We further show that the tumor suppressive effect of miR-4510 is mediated through direct targeting of GPC3 mRNA and inactivation of Wnt/ß-catenin transcriptional activity and signaling pathway. Moreover, miR-4510 up-regulated the expression of several tumor suppressor genes while reducing the expression of other pro-oncogenes. In summary, we uncovered several new microRNAs targeting the oncogenic functions of GPC3. We provided strong molecular, cellular and in vivo evidences for the tumor suppressive activities of miR-4510 bringing to the fore the potential value of this microRNA in HCC therapy.

MicroRNA therapy inhibits hepatoblastoma growth in vivo by targeting ß-catenin and Wnt signaling.

Hepatoblastoma (HBL) is the most common pediatric liver cancer. In this malignant neoplasm, beta-catenin protein accumulates and increases Wnt signaling due to recurrent activating mutations in the catenin-beta 1 (CTNNB1) gene. Therefore, beta-catenin is a key therapeutic target in HBL. However, controlling beta-catenin production with therapeutic molecules has been challenging. New biological studies could provide alternative therapeutic solutions for the treatment of HBL, especially for advanced tumors and metastatic disease. In this study, we identified microRNAs (miRNAs) that target beta-catenin and block HBL cell proliferation in vitro and tumor growth in vivo. Using our dual-fluorescence-FunREG system, we screened a library of 1,712 miRNA mimics and selected candidates inhibiting CTNNB1 expression through interaction with its untranslated regions. After validating the regulatory effect of nine miRNAs on beta-catenin in HBL cells, we measured their expression in patient samples. Let-7i-3p, miR-449b-3p, miR-624-5p, and miR-885-5p were decreased in tumors compared to normal livers. Moreover, they inhibited HBL cell growth and Wnt signaling activity in vitro partly through beta-catenin down-regulation. Additionally, miR-624-5p induced cell senescence in vitro, blocked experimental HBL growth in vivo, and directly targeted the beta-catenin 3'-untranslated region. Conclusion: Our results shed light on how beta-catenin-regulating miRNAs control HBL progression through Wnt signaling inactivation. In particular, miR-624-5p may constitute a promising candidate for miRNA replacement therapy for HBL patients. (Hepatology Communications 2017;1:168-183).

Integration of zebrafish fin regeneration genes with expression data of human tumors in silico uncovers potential novel melanoma markers.

Tissue regeneration requires expression of a large, unknown number of genes to initiate and maintain cellular processes such as proliferation, extracellular matrix synthesis, differentiation and migration. A unique model to simulate this process in a controlled manner is the re-growth of the caudal fin of zebrafish after amputation. Within this tissue stem cells differentiate into fibroblasts, epithelial and endothelial cells as well as melanocytes. Many genes implicated in the regeneration process are deregulated in cancer. We therefore undertook a systematic gene expression study to identify genes upregulated during the re-growth of caudal fin tissue. By applying a high stringency cut-off value of 4-fold change, we identified 54 annotated genes significantly overexpressed in regenerating blastema. Further bioinformatics data mining studies showed that 22 out of the 54 regeneration genes where overexpressed in melanoma compared to normal skin or other cancers. Whereas the role of TNC (tenascin C) and FN1 (fibronectin 1) in melanoma development is well documented, implication of MARCKS, RCN3, BAMBI, PEA3/ETV4 and the FK506 family members FKBP7, FKBP10 and FKBP11 in melanoma progression is unclear. Corresponding proteins were detected in melanoma tissue but not in normal skin. High expression of FKBP7, DPYSL5 and MDK was significantly associated with poor survival. We discuss a potential role of these novel melanoma genes, which have promising potential as new therapeutic targets or diagnostic markers.

Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

Biological outcome and mapping of total factor cascades in response to HIF induction during regenerative angiogenesis.

Hypoxia Inducible Factor (HIF) is the main transcription factor that mediates cell response to hypoxia. Howeverthe complex factor cascades induced by HIF during regenerative angiogenesis are currently incompletely mapped and the biological outcome mediated by chronic HIF induction during vessel regeneration are not well known. Here, we investigated the biological impact of HIF induction on vascular regeneration and identified the differentially regulated genes during regeneration, HIF induction and hypoxic regeneration. The use of the fin zebrafish regeneration model revealed that exposure to HIF inducer (cobalt chloride) prevents vessel differentiation by maintaining their vascular plexuses in an immature state. The regenerated fins are easily breakable, lacking completely endochondral ossification. Gene expression arrays combined to gene functional enrichment analysis revealed that regenerative process and HIF induction shared the regulation of common genes mainly involved in DNA replication and proteasome complex. HIF induction during regeneration affected the expression of exclusive genes involved in cell differentiation and communication, consistent with the observed immature vascular plexuses of the regenerated fins during HIF induction. The use of morpholino (MO) knockdown strategy revealed that the expression of some of these genes such as tubulin and col10a1 are required for fin regeneration. Taken together, this study revealed the impact of HIF induction on regenerative angiogenesis and provided a framework to develop a gene network leading to regenerative process during HIF expression.

Impaired angiogenesis and tumor development by inhibition of the mitotic kinesin Eg5.

Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors.

PRCP regulates angiogenesis in vivo.

In this issue of Blood, Adams et al provide evidence for an important novel function of prolylcarboxypeptidase (PRCP), or angiotensinase C, in endothelial cell physiology and angiogenesis.

IGFBP-4 anti-angiogenic and anti-tumorigenic effects are associated with anti-cathepsin B activity.

Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.

The experimental renal cell carcinoma model in the chick embryo.

The clear cell subtype of renal carcinoma (CCRCC) is highly vascularized and despite a slow progression rate, it is potentially a highly aggressive tumor. Although a doubling of median progression-free survival in CCRCC patients treated by targeted therapies has been observed, the fact that tumors escape after anti-VEGF treatment suggests alternative pathways. The chick chorioallantoic membrane (CAM) is a well-established model, which allows in vivo studies of tumor angiogenesis and the testing of anti-angiogenic molecules. However, only a few data exist on CCRCC grafted onto CAM. We aimed to validate herein the CAM as a suitable model for studying the development of CCRCC and the interactions with the surrounding stroma. Our study uses both CCRCC cell lines and fresh tumor samples after surgical resection. We demonstrate that in both cases CCRCC can be grafted onto the CAM, to survive and to induce an angiogenic process. We further provide insights into the transcriptional regulation of the model by performing a differential analysis of tumor-derived and stroma-derived transcripts.

Inhibition of angiogenesis and the angiogenesis/invasion shift.

Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.

A short-term in vivo model for giant cell tumor of bone.

BACKGROUND: Because of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose. METHODS: Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH). RESULTS: The suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells which were generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries. CONCLUSIONS: A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.