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PDF file - 2418K, MYOD is expressed across different molecular subgroups of medulloblastoma.

PDF file - 112K, Supplementary Figure legends, primer sequences and Western blot antibody details.

PDF file - 4148K, Loss of MyoD does not influence its canonical targets in the myogenic differentiation program.

While medulloblastoma, a pediatric tumor of the cerebellum, is characterized by aberrations in developmental pathways, the majority of genetic determinants remain unknown. An unbiased Sleeping Beauty transposon screen revealed MyoD as a putative medulloblastoma tumor suppressor. This was unexpected, as MyoD is a muscle differentiation factor and not previously known to be expressed in cerebellum or medulloblastoma. In response to deletion of one allele of MyoD, two other Sonic hedgehog-driven mouse medulloblastoma models showed accelerated tumor formation and death, confirming MyoD as a tumor suppressor in these models. In normal cerebellum, MyoD was expressed in the proliferating granule neuron progenitors that are thought to be precursors to medulloblastoma. Similar to some other tumor suppressors that are induced in cancer, MyoD was expressed in proliferating medulloblastoma cells in three mouse models and in human medulloblastoma cases. This suggests that although expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis. Cancer Res; 73(22); 6828–37. ©2013 AACR.

PDF file - 12536K, MyoD expression is higher in the tumor periphery compared to the inner core.

PDF file - 1235K, MyoD is expressed in proliferating tumor cells in SmoA1, SmoA2 and Ptch conditional knock out medulloblastoma mouse models.

PDF file - 25456K, Comparative analysis of MyoD +/+; SmoA2, MyoD +/-; SmoA2 and MyoD-/-; SmoA2 mice.

We introduce a new, robust method to map the numbers of proteins in living cells. The method can be applied to laser scanning, spinning disk, and lattice light-sheet microscopes in a robust, reproducible, and scalable fashion. The method uses calibrated EGFP solutions that are imaged with the appropriate microscope modality to create a calibration curve that is then applied to convert the fluorescence intensities from 3D microscope images into molecule numbers. We applied this method to human induced pluripotent stem cells in which proteins representing key cellular structures were endogenously tagged with mEGFP. We used the ratio of mEGFP-tagged proteins to total proteins to create 3D maps of live cells showing the density of total proteins measured in molecules per µm3. The method opens the door to new quantitative single cell analyses of cellular protein numbers in the context of single cell gene expression, associations with cellular complexes, and changes in cellular behaviors. The method is capable of quantifying protein numbers, over three orders of magnitude, in the cytoplasm or within various cellular structures while offering the unique advantages of each microscopy modality.

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient-derived xenograft (PDX) model, CDX-STS2, was established. The CDX-STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug-specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX-STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.

Purpose To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in a patient's tumor. To increase the flexibility of this approach beyond histological readouts of response, contrast-enhanced MRI was evaluated for the detection of cell death in living tumors after injection. Methods A six-needle arrayed microinjection device designed to provide head-to-head comparisons of chemotherapy responses in living tumors was used. Xenografted non-Hodgkin lymphoma tumors in athymic Nude-Foxn1nu mice were injected either with different doses of vincristine or with one needle each of vincristine, doxorubicin, bendamustine, prednisolone, mafosfamide, and a vehicle control. To assess drug responses, measurements of enhancement by T1-weighted contrast-enhanced MRI were made for individual sites at 24, 48, and 72 h after injection. For comparison, histological evaluations of cell death were obtained after tumor resection. Results Measurements of MRI enhancement at injection sites showed a significant (P

The vision of a precision medicine–guided approach to novel cancer drug development is challenged by high intratumor heterogeneity and interpatient diversity. This complexity is rarely modeled accurately during preclinical drug development, hampering predictions of clinical drug efficacy. To address this issue, we developed Comparative In Vivo Oncology (CIVO) arrayed microinjection technology to test tumor responsiveness to simultaneous microdoses of multiple drugs directly in a patient's tumor. Here, in a study of 18 canine patients with soft tissue sarcoma (STS), CIVO captured complex, patient-specific tumor responses encompassing both cancer cells and multiple immune infiltrates following localized exposure to different chemotherapy agents. CIVO also classified patient-specific tumor resistance to the most effective agent, doxorubicin, and further enabled assessment of a preclinical autophagy inhibitor, PS-1001, to reverse doxorubicin resistance. In a CIVO-identified subset of doxorubicin-resistant tumors, PS-1001 resulted in enhanced antitumor activity, increased infiltration of macrophages, and skewed this infiltrate toward M1 polarization. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors and across a diverse immunocompetent patient population may provide a foundation from which to make informed drug development decisions. This method also represents a viable functional approach to complement current precision oncology strategies. Cancer Res; 77(11); 2869–80. ©2017 AACR.

While medulloblastoma, a pediatric tumor of the cerebellum, is characterized by aberrations in developmental pathways, the majority of genetic determinants remain unknown. An unbiased Sleeping Beauty transposon screen revealed MyoD as a putative medulloblastoma tumor suppressor. This was unexpected, as MyoD is a muscle differentiation factor and not previously known to be expressed in cerebellum or medulloblastoma. In response to deletion of one allele of MyoD, two other Sonic hedgehog-driven mouse medulloblastoma models showed accelerated tumor formation and death, confirming MyoD as a tumor suppressor in these models. In normal cerebellum, MyoD was expressed in the proliferating granule neuron progenitors that are thought to be precursors to medulloblastoma. Similar to some other tumor suppressors that are induced in cancer, MyoD was expressed in proliferating medulloblastoma cells in three mouse models and in human medulloblastoma cases. This suggests that although expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis. Cancer Res; 73(22); 6828–37. ©2013 AACR.

Background & Aims The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo , and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. Methods CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. Results HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r 2 =0.88, p ⩽0.001). Conclusions While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures.

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.

Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

The enzyme horseradish peroxidase is routinely used in immunohistochemistry to facilitate the chromogenic oxidation of 3,3'-diaminobenzidine, producing an insoluble brown precipitate marking the location and quantity of a tissue protein. In an effort to develop non-protein reagents for tissue diagnostics, we have developed a peroxidase DNAzyme construct that can be used as a chromogenic functional group in immunohistochemistry assays. The DNAzyme is based on a reported 18-mer G-quadruplex-forming DNA sequence, PS2.M, and has been covalently linked to its catalytically active moiety, hemin, to avoid the high background signal associated with the use of free hemin in histochemical studies. The activity of the covalent construct is maintained under conditions where G-quadruplex formation is unfavored and where the noncovalent DNAzyme-hemin complex has no activity.

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, notoriously challenging to treat with standard chemotherapy options, and therefore is an area of intense focus for discovery of novel effective combination therapies. Here we used a previously described technology platform called CIVO, which enables assessment of multiple drugs and drug combinations simultaneously in living tumors, to identify drug combinations that result in synergistic anti-tumor activity in the HCC1187 model of TNBC. Our study focused on agents that combine with Voruciclib, a novel clinical stage oral CDK inhibitor with potent activity ( Citation Format: Joyoti Dey, Joseph Casalini, Sally Ditzler, Matt Biery, Angela Merrell, Derek Thirstrup, Marc Grenley, Richard Klinghoffer. Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2835.

In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug ‘tumor necrosis factor-related apoptosis inducing ligand’ (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular β-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment.

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.

Introduction Chronic hepatitis C is characterized by a persistent hepatic inflammatory response and the recruitment of immune effector cells to the liver by pro-inflammatory chemokines. The chemokine CXCL10 is induced by HCV infection in vitro and in vivo , and is correlated with the outcome of Interferon (IFN)-based therapies. Therefore, we investigated how sensing of HCV infection by the pathogen recognition receptors (PRRs) TLR3 and Retinoic Acid Inducible Gene 1 (RIG-I) led to expression of CXCL10 in hepatocytes. Methods Primary human hepatocytes were infected in vitro with the HCV clone JFH-1. CXCL10 production was measured via real-time RT-PCR, Luminex Bead Array, and immunofluorescence. Type I and type III interferon induction was measured directly by real-time PCR and indirectly by IFIT1 (ISG56) activation. CXCL10 production was also measured in response to RIG-I-specific (Sendai Virus) and TLR3-specific (polyI:C) stimuli as well as siRNA knockdown of RIG-I and TLR3 in PH5CH8 hepatocyte cultures. Transcription factors involved in CXCL10 induction were identified using CXCL10 promoter-Luciferase reporter constructs. Experiments were also performed in Huh7 hepatoma-derived cell lines that expressed either one, both, or neither PRR. Results CXCL10 mRNA and protein expression were detected following HCV infection of both human hepatoma cell lines and primary human hepatocytes. siRNA knockdown of TLR3 and RIG-I protein abrogated CXCL10 induction in response to PRR-specific agonists. Huh7-derived cells expressing both TLR3 and RIG-I produced maximal CXCL10 mRNA with negligible induction of type I or III IFN. Furthermore, neutralization of type I and type III IFN did not impact CXCL10 induction during HCV infection while IFIT1 activation was completely abolished. Immunofluorescence studies revealed a direct positive correlation between intracellular HCV core and CXCL10 protein expression. Finally, TLR3 and RIG-I-specific agonists activated CXCL10 transcription in a NF- κ B-dependent manner. Conclusion Collectively, the data suggest that CXCL10 induction during HCV infection derives directly from PRR signaling to NF- κ B activation and is marginally influenced by paracrine/autocrine IFNs. Direct induction via both TLR3 and RIG-I pathways may indicate the existence of uncharacterized crosstalk within the canonical innate immune signaling cascade and may reveal host-directed drug targets for anti-inflammatory therapies.

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.