Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

Asiatic Acid Inhibits Nasopharyngeal Carcinoma Cell Viability and Migration via Suppressing STAT3 and Claudin-1.

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southeast Asia, but effective treatment options remain limited, and chemotherapy has a high resistance rate. Asiatic acid (AA), a triterpenoid found in Centella asiatica, has shown anticancer activity in various cancers. Therefore, this study aims to investigate the anticancer effects and mechanisms of AA in NPC cell lines. The effects of AA on NPC cytotoxicity, apoptosis, and migration were determined in TW-01 and SUNE5-8F NPC cell lines. Western blot analysis was performed to evaluate the protein expression levels affected by AA. The role of AA in proliferation and migration was investigated in STAT3 and claudin-1 knockdown cells. AA inhibited NPC cell viability and migration and induced cell death by increasing cleaved caspase-3 expression. Moreover, AA inhibited STAT3 phosphorylation and reduced claudin-1 expression in NPC cells. Although knockdown of STAT3 or claudin-1 slightly reduced cell viability, it did not enhance the anti-proliferative effect of AA. However, knockdown of STAT3 or claudin-1 increased the anti-migratory effect of AA in NPC cells. These results suggest that AA can be a promising candidate for drug development against NPC.

Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma.

Vasculogenic mimicry (VM) is the process where cancer cells adopt endothelial characteristics by forming tube-like structures and perfusing channels. This phenomenon has been demonstrated in several types of solid tumors and associated with the growth and survival of tumor cells. In this study, we investigated the presence of VM formation in human pancreatic ductal adenocarcinoma (PDAC) and elucidated the molecular mechanisms underlying the VM process. In human PDAC tissues, CD31-negative, periodic acid-Schiff (PAS)-positive channels were predominantly found in desmoplastic areas, which are generally also hypovascularized. We found a positive correlation of VM capacity to tumor size and NOTCH1 expression and nuclear localization with statistical significance, implicating that Notch activity is involved with VM formation. Additionally, our data showed that the presence of growth or angiogenic factors significantly increased Notch activity in PDAC cell lines and upregulated several mesenchymal marker genes, such as TWIST1 and SNAI1, which can be inhibited by a gamma-secretase inhibitor. Our data showed that Notch signaling plays an important role in inducing VM formation in PDAC by promoting the epithelial-to-mesenchymal transition process.

Inhibition of serine/arginine-rich protein kinase-1 (SRPK1) prevents cholangiocarcinoma cells induced angiogenesis.

The serine/arginine-rich protein kinase-1 (SRPK1) is an enzyme that has an essential role in regulating numerous aspects of mRNA splicing. SRPK1 has been reported to be overexpressed in multiple cancers, suggesting it as a promising therapeutic target in oncology. No previous studies reported the role of SRPK1 in cholangiocarcinoma (CCA) cells. This study aimed to examine the expression of SRPK1 and the effects of SRPK1 inhibition on the viability and angiogenesis activity of CCA cells using a selective SRPK1 inhibitor, SPHINX31. Here, we demonstrate that SPHINX31 (0.3-10 µM) had no inhibitory effects on CCA cells' viability and proliferation. However, SPHINX31 decreased the mRNA expression of pro-angiogenic VEGF-A165a isoform. In addition, SPHINX31 attenuated SRSF1 phosphorylation and nuclear localization, and increased the ratio of VEGF-A165b/total VEGF-A proteins. Moreover, when HUVECs were grown in conditioned medium from SPHINX31-treated CCA cells, migration slowed, and tube formation decreased. The present study demonstrates that targeting SRPK1 in CCA cells effectively attenuates angiogenesis by suppressing pro-angiogenic VEGF-A isoform splicing. These findings suggest a potential therapeutic treatment using SRPK1 inhibitors for the inhibition of angiogenesis in cholangiocarcinoma.

Enhanced Functional Properties of Three DNA Origami Nanostructures as Doxorubicin Carriers to Breast Cancer Cells.

Previous studies have shown that chemotherapeutic efficacy could be enhanced with targeted drug delivery. Various DNA origami nanostructures have been investigated as drug carriers. Here, we compared drug delivery functionalities of three similar DNA origami nanostructures, Disc, Donut, and Sphere, that differ in structural dimension. Our results demonstrated that Donut was the most stable and exhibited the highest Dox-loading capacity. MUC1 aptamer modification in our nanostructures increased cellular uptake in MUC1-high MCF-7. Among the three nanostructures, unmodified Donut exerted the highest Dox cytotoxicity in MCF-7, and MUC1 aptamer modification did not further improve its effect, implicating that Dox delivery by Donut was efficient. However, all Dox-loaded nanostructures showed comparable cytotoxicity in MDA-MB-231 due to the innate sensitivity of this cell line to Dox. Our results successfully demonstrated that functional properties of DNA origami nanocarriers could be tuned by structural design, and three-dimensional Donut appeared to be the most efficient nanocarrier.

Tetrahedral DNA nanostructures as drug delivery and bioimaging platforms in cancer therapy.

Structural DNA nanotechnology enables DNA to be used as nanomaterials for novel nanostructure construction with unprecedented functionalities. Artificial DNA nanostructures can be designed and generated with precisely controlled features, resulting in its utility in bionanotechnological and biomedical applications. A tetrahedral DNA nanostructure (TDN), the most popular DNA nanostructure, with high stability and simple synthesis procedure, is a promising candidate as nanocarriers in drug delivery and bioimaging platforms, particularly in precision medicine as well as diagnosis for cancer therapy. Recent evidence collectively indicated that TDN successfully enhanced cancer therapeutic efficiency both in vitro and in vivo. Here, we summarize the development of TDN and highlight various aspects of TDN applications in cancer therapy based on previous reports, including anticancer drug loading, photodynamic therapy, therapeutic oligonucleotides, bioimaging platforms, and other molecules and discuss a perspective in opportunities and challenges for future TDN-based nanomedicine.

Downregulation of ABCA1 and ABCG1 transporters by simvastatin in cholangiocarcinoma cells.

Disturbances in cholesterol homeostasis of the bile duct epithelium, including transport interruption and the hyperaccumulation of intracellular cholesterol can lead to the initiation and progression of cholangiocarcinoma (CCA). Statins, which are lipid-lowering drugs, have been previously documented to exhibit anti-cancer properties. The role of statins in CCA cell cholesterol transport through the expression and function of ATP-binding cassette (ABC) A1 and ABCG1 was investigated in the current study. In four CCA cell lines, ABCA1 and ABCG1 expression was identified. However, neither ABCG5 nor ABCG8 expression was observed. Immunocytochemistry revealed that the expression of ABCA1 was localized in the proximity of the nucleus, while ABCG1 was more dispersed throughout the cytoplasm of KKU-100 cells. A cholesterol efflux assay was performed using bodipy cholesterol, and the translocation of cholesterol via ABCA1 and ABCG1 to Apo-A1 and high density lipoprotein was confirmed, respectively. Simvastatin and atorvastatin demonstrated the inhibitory effects on CCA cell viability. A reduction in intracellular lipid level and a lower expression of ABCA1 and ABCG1 were observed in KKU-100 cells under simvastatin treatment. The pre-exposure of KKU-100 cells to cholesterol diminished the statin effect. Furthermore, when KKU-100 cells were pre-loaded with cholesterol, ABCA1 and ABCG1-mediated exports were unaffected even though they were treated with simvastatin. The results of the current study indicated the limitations of the use of statin in CCA therapy, particularly under hypercholesterolemia conditions.

NOTCH1 regulates the viability of cholangiocarcinoma cells via 14-3-3 theta.

Notch signaling has been reported to correlate with tumor progression and metastasis in several types of cancer. In cholangiocarcinoma (CCA), it has recently been shown that NOTCH1 is overexpressed in both nucleus and cytoplasm of CCA cells; however, the complete understanding of Notch signaling in CCA is still lacking. Here, we aimed to understand the functions of NOTCH1 in CCA cells and the molecular mechanisms that underlie those functions. We used retroviral vectors to overexpress active forms of NOTCH1, the NOTCH1 intracellular domain (N1ICD) and N1ICD that lacks the RBP-J-associated module (RAM), in human CCA cell lines RMCCA-1 and HuCCA-1. Our results showed that activation of Notch signaling by both N1ICD variants enhanced CCA cell proliferation and survival via upregulation of pro-survival protein Mcl-1 and Bcl-xL. Moreover, our LC-MS/MS proteomic studies demonstrated that NOTCH1 may cooperate with 14-3-3 theta to promote CCA cell survival. Knockdown of 14-3-3 theta in RMCCA-1 cells overexpressing N1ICD, diminished pro-survival effects of N1ICD under gemcitabine treatment. In conclusion, these data demonstrated that NOTCH1 plays a role in CCA cell proliferation and survival via the regulation of 14-3-3 theta in a RAM-independent fashion.

Terrein inhibits migration of human breast cancer cells via inhibition of the Rho and Rac signaling pathways.

Breast cancer is the most common cancer in women worldwide. Progression and aggressiveness of breast cancer is usually associated with its migration and invasion abilities. Recently, natural products with potential anticancer activity have become attractive candidates for alternative treatment of cancer. A fungal metabolite, terrein, isolated from the Aspergillus terreus has been revealed to exhibit selective anticancer activity; although this molecule has a variety of biological activities. The inhibitory effect on cell proliferation in hepatoma, keratinocytes, and lung cancer cells was due to cell cycle arrest without induction of apoptosis. In contrast, its effects on cervical and breast cancer cells were mediated through activation of the apoptotic process. However, the effect of terrein on cell migration and invasion has not been explored. In the present study we analyzed the molecular effects of terrein on cell adhesion, cell migration, and cell invasion using two breast cancer cell lines, MCF-7 and MDA-MB-231, which exhibit different levels of invasiveness. Terrein induced apoptosis in both breast cancer cell lines in a dose-dependent manner. In addition, at a non-toxic concentration terrein exhibited a weak inhibition of cell adhesion, using either fibronectin or type IV collagen as substrates. Notably, terrein significantly inhibited both the migration and invasion abilities of MDA-MB-231 cells at the same non-toxic concentration. A marked decrease in MMP-2 and MMP-9 transcripts, as evaluated by real-time PCR, confirmed the anti-invasion effect of terrein at the transcriptional level. Western blot analyses revealed that terrein treatment suppressed RhoB expression and reduced Rac1 phosphorylation, leading to Rho GTPase inhibition. In addition, terrein-treated MCF-7 and MDA-MB-231 cells both displayed a scattered pattern of migration, suggesting that the suppression of RhoB and Rac1 disturbed the collective migration processes of breast cancer cells.

Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-α.

Lupeol and stigmasterol, major phytosterols in various herbal plants, possess anti-inflammatory activities and have been proposed as candidates for anti-cancer agents, but their molecular mechanisms are still unclear. Here, we investigated the effects of lupeol and stigmasterol on tumor and endothelial cells in vitro and their anti-cancer activities in vivo. Our results demonstrated that lupeol and stigmasterol suppressed cell viability, migration, and morphogenesis of human umbilical vein endothelial cells (HUVECs) but not cholangiocarcinoma (CCA) cells. Expression analyses showed that the treatment of both compounds significantly reduced the transcript level of tumor necrosis factor-α (TNF-α), and Western blot analyses further revealed a decrease in downstream effector levels of VEGFR-2 signaling, including phosphorylated forms of Src, Akt, PCL, and FAK, which were rescued by TNF-α treatment. In vivo, lupeol and stigmasterol disrupted tumor angiogenesis and reduced the growth of CCA tumor xenografts. Immunohistochemical analyses confirmed a decrease in CD31-positive vessel content and macrophage recruitment upon treatment. These findings indicate that lupeol and stigmasterol effectively target tumor endothelial cells and suppress CCA tumor growth by their anti-inflammatory activities and are attractive candidates for anti-cancer treatment of CCA tumors.

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRß+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRßlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

DNA origami applications in cancer therapy.

Due to the complexity and heterogeneity of cancer, the development of cancer diagnosis and therapy is still progressing, and a complete understanding of cancer biology remains elusive. Recently, cancer nanomedicine has gained much interest as a promising diagnostic and therapeutic strategy, as a wide range of nanomaterials possess unique physical properties that can render drug delivery systems safer and more effective. Also, targeted drug delivery and precision medicine have now become a new paradigm in cancer therapy. With nanocarriers, chemotherapeutic drugs could be directly delivered into target cancer cells, resulting in enhanced efficiency with fewer side-effects. DNA, a biomolecule with molecular self-assembly properties, has emerged as a versatile nanomaterial to construct multifunctional platforms; DNA nanostructures can be modified with functional groups to improve their utilities as biosensors or drug carriers. Such applications have become possible with the advent of the scaffolded DNA origami method. This breakthrough technique in structural DNA nanotechnology provides an easier and faster way to construct DNA nanostructures with various shapes. Several experiments proved that DNA origami nanostructures possess abilities to enhance efficacies of chemotherapy, reduce adverse side-effects, and even circumvent drug resistance. Here, we highlight the principles of the DNA origami technique and its applications in cancer therapeutics and discuss current challenges and opportunities to improve cancer detection and targeted drug delivery.

Notch suppresses angiogenesis and progression of hepatic metastases.

The Notch pathway plays multiple key roles in tumorigenesis, and its signaling components have therefore aroused great interest as targets for emerging therapies. Here, we show that inhibition of Notch, using a soluble receptor Notch1 decoy, unexpectedly caused a remarkable increase in liver metastases from neuroblastoma and breast cancer cells. Increased liver metastases were also seen after treatment with the γ-secretase inhibitor PF-03084014. Transgenic mice with heterozygous loss of Notch1 demonstrated a marked increase in hepatic metastases, indicating that Notch1 signaling acts as metastatic suppressor in the liver microenvironment. Inhibition of DLL1/4 with ligand-specific Notch1 decoys increased sprouting of sinusoidal endothelial cells into micrometastases, thereby supporting early metastatic angiogenic growth. Inhibition of tumor-derived JAG1 signaling activated hepatic stellate cells, increasing their recruitment to vasculature of micrometastases, thereby supporting progression to macrometastases. These results demonstrate that inhibition of Notch causes pathologic activation of liver stromal cells, promoting angiogenesis and growth of hepatic metastases. Our findings have potentially serious implications for Notch inhibition therapy.

NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.

UNLABELLED: A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.

Notch functions in developmental and tumour angiogenesis by diverse mechanisms.

The Notch signalling pathway is a key regulator of developmental and tumour angiogenesis. Inhibition of Delta-like 4 (Dll4)-mediated Notch signalling results in hyper-sprouting, demonstrating that Notch regulates tip-stalk cell identity in developing tissues and tumours. Paradoxically, Dll4 blockade leads to reduced tumour growth because the newly growing vessels are poorly perfused. To explore the potential for targeting Notch, we developed Notch inhibitors, termed the Notch1 decoys. A Notch1 decoy variant containing all 36 epidermal growth factor (EGF)-like repeats of the extracellular domain of rat Notch1 has been shown to inhibit both Dll and Jagged class Notch ligands. Thus this Notch1 decoy functions differently than Dll4-specific blockade, although it has the potential to inhibit Dll4 activity. Expression of the Notch1 decoy in mice disrupted tumour angiogenesis and inhibited tumour growth. To understand the mechanism by which Notch blockade acts, it is important to note that Notch can function in multiple cell types that make up the vasculature, including endothelial cells and perivascular cells. We investigated Notch function in retinal microglia and determined how myeloid-expressed Notch can influence macrophages and angiogenesis. We found that myeloid-specific loss of Notch1 reduced microglia recruitment and led to improper microglia localization during retinal angiogenesis. Thus either pharmacological inhibition of Notch signalling or genetic deficiencies of Notch function in microglia leads to abnormal angiogenesis.

Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis.

BACKGROUND: Anti-angiogenesis is a validated strategy to treat cancer, with efficacy in controlling both primary tumor growth and metastasis. The role of the Notch family of proteins in tumor angiogenesis is still emerging, but recent data suggest that Notch signaling may function in the physiologic response to loss of VEGF signaling, and thus participate in tumor adaptation to VEGF inhibitors. METHODS: We asked whether combining Notch and VEGF blockade would enhance suppression of tumor angiogenesis and growth, using the NGP neuroblastoma model. NGP tumors were engineered to express a Notch1 decoy construct, which restricts Notch signaling, and then treated with either the anti-VEGF antibody bevacizumab or vehicle. RESULTS: Combining Notch and VEGF blockade led to blood vessel regression, increasing endothelial cell apoptosis and disrupting pericyte coverage of endothelial cells. Combined Notch and VEGF blockade did not affect tumor weight, but did additively reduce tumor viability. CONCLUSIONS: Our results indicate that Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis, and show that concurrent blockade disrupts primary tumor vasculature and viability further than inhibition of either pathway alone.

Inhibition of Notch uncouples Akt activation from hepatic lipid accumulation by decreasing mTorc1 stability.

Increased hepatic lipid content is an early correlate of insulin resistance and can be caused by nutrient-induced activation of mammalian target of rapamycin (mTor). This activation of mTor increases basal Akt activity, leading to a self-perpetuating lipogenic cycle. We have previously shown that the developmental Notch pathway has metabolic functions in adult mouse liver. Acute or chronic inhibition of Notch dampens hepatic glucose production and increases Akt activity and may therefore be predicted to increase hepatic lipid content. Here we now show that constitutive liver-specific ablation of Notch signaling, or its acute inhibition with a decoy Notch1 receptor, prevents hepatosteatosis by blocking mTor complex 1 (mTorc1) activity. Conversely, Notch gain of function causes fatty liver through constitutive activation of mTorc1, an effect that is reversible by treatment with rapamycin. We demonstrate that Notch signaling increases mTorc1 complex stability, augmenting mTorc1 function and sterol regulatory element binding transcription factor 1c (Srebp1c)-mediated lipogenesis. These data identify Notch as a therapeutically actionable branch point of metabolic signaling at which Akt activation in the liver can be uncoupled from hepatosteatosis.

Keratinocyte stem cells and the targets for nonmelanoma skin cancer.

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.

Notch signaling in developmental and tumor angiogenesis.

The discovery that Notch, a key regulator of cell fate determination, is functional in the vasculature has greatly improved our understanding of differentiation and specialization of vessels. Notch signaling has been proven to be critical for arterial specification, sprouting angiogenesis, and vessel maturation. In newly forming vascular sprouts, Notch promotes the distinction between the leading "tip" endothelial cell and the growing "stalk" cell, the endothelial cells that eventually form a new capillary. Notch signaling has also been implicated in vessel stability by regulating vascular mural cell function. More recently, macrophages carrying an activated Notch have been implicated in shaping the course of new sprout formation. Tumor vessels abide by similar principles and use Notch signaling in similar ways. An exciting discovery, made by several researchers, shows that blocking Notch function in tumor vasculature provides a means by which to suppress tumor growth. The authors discuss the developmental and physiological role of Notch in the vasculature and apply this knowledge to an overview of how Notch targeting in the tumor environment can affect tumor angiogenesis and growth.

Bone morphogenetic protein 5 regulates the number of keratinocyte stem cells from the skin of mice.

Understanding keratinocyte stem cell regulation is important in understanding the pathogenesis of wound healing and nonmelanoma skin cancer. We previously used a sensitive and quantitative assay for in vitro keratinocyte colony formation and mapped the keratinocyte stem cell locus (Ksc1) on mouse chromosome 9. Examination of the candidate genes in this locus disclosed a sequence variant in the gene for bone morphogenetic protein 5 (Bmp5). In this report, we used a naturally occurring mouse with a null mutation in this gene to probe stem cell properties in mouse epidermis. We found that the mutant keratinocytes had a significant reduction in the size and number of clonogenic keratinocytes. The mutant mice had a 50% reduction in the number of label-retaining cells when compared with their littermates. Addition of exogenous Bmp5 protein increased the number and size of keratinocyte colonies in the mutant as well as their wild-type littermates. Surprisingly, the mutant mice showed at least a 2-fold increase in skin tumor susceptibility over their littermates. We conclude that a naturally occurring mutation in Bmp5 affects keratinocyte stem cell proliferation, and skin tumor susceptibility, and is a candidate stem cell regulatory gene in the Ksc1 locus.