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Stem cell–replacement therapies have been proposed as a potential tool to treat sensorineural hearing loss by aiding the regeneration of spiral ganglion neurons (SGNs) in the inner ear. However, transplantation procedures have yet to be explored thoroughly to ensure proper cell differentiation and optimal transplant procedures. We hypothesized that the aggregation of human embryonic stem cell (hESC)–derived otic neuronal progenitor (ONP) cells into a multicellular form would improve their function and their survival in vivo post-transplantation. We generated hESC–derived ONP spheroids—an aggregate form conducive to differentiation, transplantation, and prolonged cell survival—to optimize conditions for their transplantation. Our findings indicate that these cell spheroids maintain the molecular and functional characteristics similar to those of ONP cells, which are upstream in the SGN lineage. Moreover, our phenotypical, electrophysiological, and mechanical data suggest an optimal spheroid transplantation point after 7 days of in vitro three-dimensional (3D) culture. We have also developed a feasible transplantation protocol for these spheroids using a micropipette aided by a digital microinjection system. In summary, the present work demonstrates that the transplantation of ONP cells in spheroid form into the inner ear through micropipette 7 days after seeding for 3D spheroid culture is an expedient and viable method for stem cell replacement therapies in the inner ear. IMPACT STATEMENT: Sensorineural hearing loss affects millions of people worldwide. Although inner ear stem cell replacement therapies offer a promising method to mitigate this hearing loss by aiding in the regeneration of spiral ganglion neurons, few in vivo studies have been performed in this area. By providing detailed cell spheroid characterization, determining an optimal development stage for transplantation, and establishing a reliable and reproducible transplantation protocol, we aimed to provide the necessary details to bridge the gap between in vitro experiments and in vivo studies of stem cell replacement therapies in the inner ear.

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro . Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development.

ABT-700 is a therapeutic antibody against the hepatocyte growth factor receptor (MET). At doses or regimens that lead to exposures exceeding optimum in vivo, the efficacy of ABT-700 is unexpectedly reduced. We hypothesized that this reduction in efficacy was due to a “prozone-like” effect in vivo. A prozone-like effect, which is a reduction in efficacy beyond optimum exposure, is caused due a mechanism similar to the generation of false negative flocculation tests by excessive antibody titres. In vitro, we demonstrate that at higher ABT-700 concentrations, this “prozone-like” effect is mediated by a progressive conversion from bivalent to ineffective monovalent binding of the antibody. In vivo, the efficacy of ABT-700 is dependent on an optimum range of exposure as well. Our data suggest that the “prozone-like” effect is operative and independent of target expression. ABT-700 dose, regimen, exposure, and tumor burden are interdependent variables influencing the “prozone-like” effect and mediating and in vivo efficacy. By optimization of dosage and regimen we demonstrate that the “prozone-like” effect can be alleviated and ABT-700 efficacy at varying tumor loads can be further extended in combination with cisplatin. Our results suggest that optimization of exposure taking tumor burden into account may alleviate “prozone-like” effects without compromising efficacy.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (P

We describe a series of potent and selective oxindole–pyridine-based protein kinase B/Akt inhibitors. The most potent compound 11n in this series demonstrated an IC50 of 0.17 nM against Akt1 and more than 100-fold selectivity over other Akt isozymes. The selectivity against other protein kinases was highly dependent on the C-3 substitutions at the oxindole scaffold, with unsubstituted 9e or 3-furan-2-ylmethylene (11n) more selective and 3-(1H-pyrrol-2-yl)methylene (11f) or 3-(1H-imidazol-2-yl)methylene (11k) less selective. In a mouse xenograft model, 9d, 11f, and 11n inhibited tumor growth but with accompanying toxicity.

The structure–activity relationships of a series of isoquinoline–pyridine-based protein kinase B/Akt antagonists have been investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in several species (e.g., mouse iv t1/2 = 0.3 h, po F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacokinetic property in mice (iv t1/2 = 5.0 h, po F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft model, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.

Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654). Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.