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Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology. more...

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs. more...

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical β-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124−/− embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124- regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors. [ABSTRACT FROM AUTHOR] more...

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis (1), (2). The liver possesses a rich sinusoidal capillary network with a higher degree [...] more...

Purpose: Targeted therapy with α-particle emitting radionuclides is a promising new option in cancer therapy. Stable conjugates of the vascular tumour-homing peptide F3 with the α-emitter Bi specifically target tumour cells. The aim of our study was to determine efficacy of combined Bi-diethylenetriaminepentaacetic acid (DTPA)-F3 and paclitaxel treatment compared to treatment with either Bi-DTPA-F3 or paclitaxel both in vitro and in vivo. Methods: Cytotoxicity of treatment with Bi-DTPA-F3 and paclitaxel, alone or in combination, was assayed towards OVCAR-3 cells using the alamarBlue assay, the clonogenic assay and flow cytometric analyses of the mode of cell death and cell cycle arrest. Therapeutic efficacy of the different treatment options was assayed after repeated treatment of mice bearing intraperitoneal OVCAR-3 xenograft tumours. Therapy monitoring was performed by bioluminescence imaging and histopathologic analysis. Results: Treatment of OVCAR-3 cells in vitro with combined Bi-DTPA-F3 and paclitaxel resulted in enhanced cytotoxicity, induction of apoptosis and G2/M phase arrest compared to treatment with either Bi-DTPA-F3 or paclitaxel. Accordingly, i.p. xenograft OVCAR-3 tumours showed the best response following repeated (six times) combined therapy with Bi-DTPA-F3 (1.85 MBq) and paclitaxel (120 μg) as demonstrated by bioluminescence imaging and histopathologic investigation of tumour spread on the mesentery of the small and large intestine. Moreover, mean survival of xenograft mice that received combined therapy with Bi-DTPA-F3 and paclitaxel was significantly superior to mice treated with either Bi-DTPA-F3 or paclitaxel alone. Conclusion: Combined treatment with Bi-DTPA-F3 and paclitaxel significantly increased mean survival of mice with peritoneal carcinomatosis of ovarian origin, thus favouring future therapeutic application. [ABSTRACT FROM AUTHOR] more...

Background: a-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for a-particle emitting isotopes facilitating selective tumor therapies. Principal Findings: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the a-emitter 213Bi (213Bi- DTPA-[F3]2). We found 213Bi-DTPA-[F3]2 to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of 213Bi-DTPA-[F3]2 we treated mice bearing intraperitoneally growing xenograft tumors with 213Bi-DTPA-[F3]2. In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6×1.85 MBq (50 mCi) of 213Bi-DTPA-[F3]2 were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6×1.85 MBq of 213Bi-DTPA-[F3]2 were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found 213Bi-DTPA-[F3]2 to accumulate in tumors but only low activities were found in control organs except for the kidneys, where 213Bi-DTPA-[F3]2 is found due to renal excretion. Conclusions/Significance: In conclusion we report that 213Bi-DTPA-[F3]2 is a novel tool for the targeted delivery of aemitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology. [ABSTRACT FROM AUTHOR] more...

In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter ²¹³Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of ²¹³Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with ²¹³Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. ²¹³Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of ²¹³Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with ²¹³Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of ²¹³Bi-induced toxicity. Preclinical treatment of MM with ²¹³Bi-anti-CD38-MAb turned out as an effective therapeutic option. more...