Anti-Tumorigenic Effect of a Novel Derivative of 2-Hydroxyoleic Acid and the Endocannabinoid Anandamide on Neuroblastoma Cells.

Modulation of the endogenous cannabinoid system has been suggested as a potential anticancer strategy. In the search for novel and less toxic therapeutic options, structural modifications of the endocannabinoid anandamide and the synthetic derivative of oleic acid, Minerval (HU-600), were done to obtain 2-hydroxy oleic acid ethanolamide (HU-585), which is an HU-600 derivative with the anandamide side chain. We showed that treatment of SK-N-SH neuroblastoma cells with HU-585 induced a better anti-tumorigenic effect in comparison to HU-600 as evidenced by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and migration assay. Moreover, HU-585 demonstrated pro-apoptotic properties shown by increased levels of activated caspase-3 following treatment and a better senescence induction effect in comparison to HU-600, as demonstrated by increased activity of lysosomal ß-galactosidase. Finally, we observed that combined treatment of HU-585 with the senolytic drugs ABT-263 in vitro, and ABT-737 in vivo resulted in enhanced anti-proliferative effects and reduced neuroblastoma xenograft growth in comparison to treatment with HU-585 alone. Based on these results, we suggest that HU-585 is a pro-apoptotic and senescence-inducing compound, better than HU-600. Hence, it may be a beneficial option for the treatment of resistant neuroblastoma especially when combined with senolytic drugs that enhance its anti-tumorigenic effects.

Ex Vivo Expanded 3D Human Kidney Spheres Engraft Long Term and Repair Chronic Renal Injury in Mice.

End-stage renal disease is a worldwide epidemic requiring renal replacement therapy. Harvesting tissue from failing kidneys and autotransplantation of tissue progenitors could theoretically delay the need for dialysis. Here we use healthy and end-stage human adult kidneys to robustly expand proliferative kidney epithelial cells and establish 3D kidney epithelial cultures termed "nephrospheres." Formation of nephrospheres reestablishes renal identity and function in primary cultures. Transplantation into NOD/SCID mice shows that nephrospheres restore self-organogenetic properties lost in monolayer cultures, allowing long-term engraftment as tubular structures, potentially adding nephron segments and demonstrating self-organization as critical to survival. Furthermore, long-term tubular engraftment of nephrospheres is functionally beneficial in murine models of chronic kidney disease. Remarkably, nephrospheres inhibit pro-fibrotic collagen production in cultured fibroblasts via paracrine modulation, while transplanted nephrospheres induce transcriptional signatures of proliferation and release from quiescence, suggesting re-activation of endogenous repair. These data support the use of human nephrospheres for renal cell therapy.

NCAM1/FGF module serves as a putative pleuropulmonary blastoma therapeutic target.

Pleuropulmonary blastoma (PPB) is a rare pediatric lung neoplasm that recapitulates developmental pathways of early embryonic lungs. As lung development proceeds with highly regulated mesenchymal-epithelial interactions, a DICER1 mutation in PPB generates a faulty lung differentiation program with resultant biphasic tumors composed of a primitive epithelial and mesenchymal stroma with early progenitor blastomatous cells. Deciphering of PPB progression has been hampered by the difficulty of culturing PPB cells, and specifically progenitor blastomatous cells. Here, we show that in contrast with in-vitro culture, establishment of PPB patient-derived xenograft (PDX) in NOD-SCID mice selects for highly proliferating progenitor blastoma overexpressing critical regulators of lung development and multiple imprinted genes. These stem-like tumors were sequentially interrogated by gene profiling to show a FGF module that is activated alongside Neural cell adhesion molecule 1 (NCAM1). Targeting the progenitor blastoma and these transitions with an anti-NCAM1 immunoconjugate (Lorvotuzumab mertansine) inhibited tumor growth and progression providing new paradigms for PPB therapeutics. Altogether, our novel in-vivo PPB xenograft model allowed us to enrich for highly proliferating stem-like cells and to identify FGFR and NCAM1 as two key players that can serve as therapeutic targets in this poorly understood and aggressive disease.

In Vivo Expansion of Cancer Stemness Affords Novel Cancer Stem Cell Targets: Malignant Rhabdoid Tumor as an Example.

Cancer stem cell (CSC) identification relies on transplantation assays of cell subpopulations sorted from fresh tumor samples. Here, we attempt to bypass limitations of abundant tumor source and predetermined immune selection by in vivo propagating patient-derived xenografts (PDX) from human malignant rhabdoid tumor (MRT), a rare and lethal pediatric neoplasm, to an advanced state in which most cells behave as CSCs. Stemness is then probed by comparative transcriptomics of serial PDXs generating a gene signature of epithelial to mesenchymal transition, invasion/motility, metastasis, and self-renewal, pinpointing putative MRT CSC markers. The relevance of these putative CSC molecules is analyzed by sorting tumorigenic fractions from early-passaged PDX according to one such molecule, deciphering expression in archived primary tumors, and testing the effects of CSC molecule inhibition on MRT growth. Using this platform, we identify ALDH1 and lysyl oxidase (LOX) as relevant targets and provide a larger framework for target and drug discovery in rare pediatric cancers.

Pancreatic cancer ascites xenograft-an expeditious model mirroring advanced therapeutic resistant disease.

Pancreatic ductal adenocarcinoma has limited treatment options. There is an urgent need for developing appropriate pre-clinical models recapitulating metastatic disease, the most common clinical scenario at presentation. Ascites accumulation occurs in up to 20-30% of patients with pancreatic cancer; this milieu represents a highly cellular research resource of metastatic peritoneal spread. In this study, we utilized pancreatic ascites/pleural effusion cancer cells to establish patient derived xenografts.Ascites/pleural effusion-patient derived xenografts were established from twelve independent cases. Xenografts were serially passed in nude mice and tissue bio-specimen banking has been established. Histopathology of emergent tumors demonstrates poorly to moderately differentiated, glandular and mucin producing tumors, mirroring morphology of primary pancreatic cancer tumors. Whole genome sequencing of six patient derived xenografts samples demonstrates common mutations and structural variations similar to those reported in primary pancreatic cancer. Xenograft tumors were dissociated to single-cells and in-vitro drug sensitivity screen assays demonstrated chemo-resistance, correlating with patient clinical scenarios, thus serving as a platform for clinically relevant translational research.Therefore, establishment of this novel ascites/pleural effusion patient derived xenograft model, with extensive histopathology and genomic characterization, opens an opportunity for the study of advanced aggressive pancreatic cancer. Characterization of metastatic disease and mechanisms of resistance to therapeutics may lead to the development of novel drug combinations.

Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML-xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ-pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor-initiating capacity, via a TGFB-mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ-activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.

Dissecting Stages of Human Kidney Development and Tumorigenesis with Surface Markers Affords Simple Prospective Purification of Nephron Stem Cells.

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1(+)CD133(-) marks SIX2(+) multipotent renal stem cells transiting to NCAM1(+)CD133(+) differentiating segment-specific SIX2(-) epithelial progenitors and NCAM1(-)CD133(+) differentiated nephron cells. In tumorigenesis, NCAM1(+)CD133(-) marks SIX2(+) blastema that includes the ALDH1(+) WT cancer stem/initiating cells, while NCAM1(+)CD133(+) and NCAM1(-)CD133(+) specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1(+) nephron stem cells in normal and malignant nephrogenesis.

Wilms' tumor blastemal stem cells dedifferentiate to propagate the tumor bulk.

An open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms' tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1(+)) aldehyde dehydrogenase 1-positive (ALDH1(+)) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1(+) WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema.

Identification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease.

Identification of tissue-specific renal stem/progenitor cells with nephrogenic potential is a critical step in developing cell-based therapies for renal disease. In the human kidney, stem/progenitor cells are induced into the nephrogenic pathway to form nephrons until the 34 week of gestation, and no equivalent cell types can be traced in the adult kidney. Human nephron progenitor cells (hNPCs) have yet to be isolated. Here we show that growth of human foetal kidneys in serum-free defined conditions and prospective isolation of NCAM1(+) cells selects for nephron lineage that includes the SIX2-positive cap mesenchyme cells identifying a mitotically active population with in vitro clonogenic and stem/progenitor properties. After transplantation in the chick embryo, these cells-but not differentiated counterparts-efficiently formed various nephron tubule types. hNPCs engrafted and integrated in diseased murine kidneys and treatment of renal failure in the 5/6 nephrectomy kidney injury model had beneficial effects on renal function halting disease progression. These findings constitute the first definition of an intrinsic nephron precursor population, with major potential for cell-based therapeutic strategies and modelling of kidney disease.

Reactivation of NCAM1 defines a subpopulation of human adult kidney epithelial cells with clonogenic and stem/progenitor properties.

The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1(+) cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial-mesenchymal transition. NCAM1(+) cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1(+) cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases.

The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.

There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated-NCAM1 expressing-"blastema" phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.

Avastin treatment reduces retinal neovascularization in a mouse model of retinopathy of prematurity.

PURPOSE: This study was designed to evaluate the effect of one intraperitoneal (IP) injection of bevacizumab (Avastin) on the severity of oxygen-induced retinopathy (OIR) in a mouse model. MATERIALS AND METHODS: Twenty-eight eyes of 14 mice with OIR were studied. There were nine mice in the bevacizumab-treated group (study group) and five mice in the saline-treated group (controls). The mouse OIR model consisted of a 5-day exposure to 75% oxygen. On postnatal day 12 (P12), Avastin 2.5 mg/kg was administered IP to the study group and 2.5 mg/kg normal saline was administered IP to the controls. All 14 mice underwent fluorescein angiography of the retinal vasculature on P17 and the following parameters were scored (Modified Retinopathy Scoring System, MRSS): blood vessel growth, formation of blood vessel tufts, extraretinal neovascularization, degree of central constriction, and tortuosity of vessels. In addition, the neovascular vessels were quantified on the hematoxylin and eosin (H&S)-stained paraffin sections of the eyes in a masked fashion. RESULTS: The MRSS score in the Avastin-treated mice was significantly lower than that of the saline-treated mice (3.06 ± 1.63 versus 7.1 ± 2.01, respectively, p = 0.0021). The neovascularization count was also significantly lower in the study group (3.44 ± 1.81 versus 9.34 ± 3.23 for the controls, p = 0.0013). CONCLUSIONS: IP Avastin treatment reduced the extent of oxygen-induced retinopathy in a mouse model of retinopathy of prematurity.

The effect of thalidomide on neovascularization in a mouse model of retinopathy of prematurity.

PURPOSE: Abnormal angiogenesis is the hallmark feature of retinopathy of prematurity (ROP), and contributes to the severe visual loss that accompanies this disease. Thalidomide is a well-known anti-angiogenic drug. We tested the assumption that injection of intraperitoneal thalidomide could reduce the severity of oxygen-induced retinopathy (OIR) in a mouse model. METHODS: Forty-three baby wild type mice were used in this study. The mouse model of oxygen-induced retinopathy consisted of a 5-day exposure to 75% oxygen from postnatal day 7 to 12 (P12) followed by 5 days in room air (relative hypoxia). Control mice were those with normally developing retinal vasculature exposed to room air from birth until postnatal day 17 (P17). Thalidomide (200 mg/Kg) was administered daily intraperitoneally to control and ROP mice in two protocols: (1) from P12 to P16, and (2) from P11 to P15 . Fluorescein-conjugated dextran angiography of retinal vasculature was performed on P17, and retinal whole mounts were prepared to score features of retinopathy. The parameters that were scored in a masked fashion included blood vessel growth, blood vessel tufts formation, extra retinal neovascularization, degree of central constriction, and tortuosity of vessels. These parameters constitute the Modified Retinopathy Scoring System (MRSS). In addition, quantification of the number of blood vessel tufts was performed in a masked fashion with hematoxylin & eosin (H&S) staining of paraffin-embedded eye sections. RESULTS: The retinopathy score by MRSS in the thalidomide treated mice was similar to that of untreated mice that were exposed to oxygen (9.3 +/- 1.9 vs 10.15 +/- 1.6; p = 0.21). The neovascularization count was also similar between the two groups (10.4 +/- 5.6 vs 9.6 +/- 4.8; p = 0.56). In the control group left in the room air, the retinopathy score was 0.19 +/- 0.37 (p = 0) and the neovascularization count was also very low (2.92 +/- 2.14; p = 0). CONCLUSIONS: Although thalidomide might have a proven anti-angiogenic and anti-inflammatory effect, our model did not show a significant effect on the retinopathy. The reason might be an ineffective level of the drug in the retina due to ineffective metabolism of the drug, or due to blockage of the drug by the blood-retina barrier, or the involvement of other factors besides those influenced by thalidomide in the process.

Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2.

Rat stromal bone-marrow cells cultured in the presence of dexamethasone, ascorbic acid, beta-glycerophosphate, and fibroblast growth factor-2 (FGF-2) express the osteogenic phenotype (Pitaru et al., J. Bone Miner. Res. 8:919-929, 1993). The purpose of this study was to establish a long-term homogeneous culture expressing the osteogenic phenotype. The cultures were routinely passaged every 5 days in the absence or presence of either or both dexamethasone and FGF-2, and the cumulative doubling number and the expression of the osteogenic phenotype were determined. Cultures treated with dexamethasone (10(-7) M) ceased proliferation and only upon addition of FGF-2 (3 ng/ml) was a spontaneous immortalization achieved, as expressed by sustained proliferation for about 1 year, with a doubling time of 22 h and more than 300 doublings in 72 passages. Both FGF-2 and dexamethasone are required and act synergistically to maintain cell propagation, alkaline phosphatase expression, and osteocalcin secretion; however, protein content was FGF-2 dependent and the mineralization was dexamethasone dependent. Repetitive single-cell cloning tested the homogeneity and stability of the cells expressing the osteogenic phenotype in these long-term cultures. It was shown that 25% to 50% of subclones derived from clones with an osteogenic phenotype do not further express the osteogenic phenotype. In conclusion, we have established a spontaneously immortalized dexamethasone- and FGF-2-dependent rat stromal bone-marrow-derived long-term culture expressing the osteogenic phenotype. The cultures tend to lose the osteogenic phenotype, and dexamethasone supports the long-term preservation of the osteogenic phenotype.