IL-13Rα2/TGF-ß bispecific CAR-T cells counter TGF-ß-mediated immune suppression and potentiate anti-tumor responses in glioblastoma.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies targeting glioblastoma (GBM)-associated antigens such as interleukin-13 receptor subunit alpha-2 (IL-13Rα2) have achieved limited clinical efficacy to date, in part due to an immunosuppressive tumor microenvironment (TME) characterized by inhibitory molecules such as transforming growth factor-beta (TGF-ß). The aim of this study was to engineer more potent GBM-targeting CAR-T cells by countering TGF-ß-mediated immune suppression in the TME. METHODS: We engineered a single-chain, bispecific CAR targeting IL-13Rα2 and TGF-ß, which programs tumor-specific T cells to convert TGF-ß from an immunosuppressant to an immunostimulant. Bispecific IL-13Rα2/TGF-ß CAR-T cells were evaluated for efficacy and safety against both patient-derived GBM xenografts and syngeneic models of murine glioma. RESULTS: Treatment with IL-13Rα2/TGF-ß CAR-T cells leads to greater T-cell infiltration and reduced suppressive myeloid cell presence in the tumor-bearing brain compared to treatment with conventional IL-13Rα2 CAR-T cells, resulting in improved survival in both patient-derived GBM xenografts and syngeneic models of murine glioma. CONCLUSION: Our findings demonstrate that by reprogramming tumor-specific T-cell responses to TGF-ß, bispecific IL-13Rα2/TGF-ß CAR-T cells resist and remodel the immunosuppressive TME to drive potent anti-tumor responses in GBM.

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Inhibition of metastatic brain cancer in Sonic Hedgehog medulloblastoma using caged nitric oxide albumin nanoparticles.

Medulloblastoma is a tumor of the cerebellum that metastasizes to the leptomeninges of the central nervous system (CNS), including to forebrain and to spinal cord. The inhibitory effect of polynitroxylated albumin (PNA), a caged nitroxide nanoparticle, on leptomeningeal dissemination and metastatic tumor growth was studied in a Sonic Hedgehog transgenic mouse model. PNA treated mice showed an increased lifespan with a mean survival of 95 days (n = 6, P<0.05) compared with 71 days in controls. In primary tumors, proliferation was significantly reduced and differentiation was significantly increased (P<0.001) as shown by Ki-67+ and NeuN+ immunohistochemistry, while cells in spinal cord tumors appeared unaffected. Yet, histochemical analysis of metastatic tumor in spinal cord showed that the mean total number of cells in spinal cord was significantly reduced in mice treated with PNA compared to albumin vehicle (P<0.05). Examination of various levels of the spinal cord showed that PNA treated mice had significantly reduced metastatic cell density in the thoracic, lumbar and sacral spinal cord levels (P<0.05), while cell density in the cervical region was not significantly changed. The mechanism by which PNA may exert these effects on CNS tumors is discussed.

Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements.

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.

Transcription factor TAp73 and microRNA-449 complement each other to support multiciliogenesis.

Motile cilia serve vital functions in development, homeostasis, and regeneration. We recently demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3, axonemal dyneins Dnali1 and Dnai1, plays a pivotal role in the generation of MCCs in male and female reproductive ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be preserved despite the loss of TAp73, and robust activity of cilia-related networks is maintained in the absence of TAp73. Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To address whether increased miR449 levels in the brain maintain the multiciliogenesis program in the absence of TAp73, we deleted both TAp73 and miR449 in mice. Although loss of miR449 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus were observed in the brain lacking both TAp73 and miR449. In contrast, miR449 loss in other MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female reproductive ducts, whereas miR449 and TAp73 complement each other to support multiciliogenesis and CP development in the brain.

ATOH1 Promotes Leptomeningeal Dissemination and Metastasis of Sonic Hedgehog Subgroup Medulloblastomas.

Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/- mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766-77. ©2017 AACR.

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells.

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

Antidepressant treatment can normalize adult behavioral deficits induced by early-life exposure to methylphenidate.

BACKGROUND: Methylphenidate (MPH) is prescribed for the treatment of attention-deficit/hyperactivity disorder. Exposure to MPH before adulthood causes behavioral deficits later in life, including anxiety- and depression-like behaviors and decreased responding to natural and drug rewards. We examined the ability of fluoxetine (FLX), a selective serotonin reuptake blocker, to normalize these MPH-induced behavioral deficits. METHODS: Male rats received MPH (2.0 mg/kg) or saline (VEH) during preadolescence (postnatal day [PD] 20-35). When adults, rats were divided into groups receiving no treatment, acute or chronic FLX, and behavioral reactivity to several emotion-eliciting stimuli were assessed. RESULTS: The MPH-treated rats were significantly less responsive to natural (i.e., sucrose) and drug (i.e., morphine) rewards and more sensitive to stress- and anxiety-eliciting situations. These MPH-induced deficits were reversed by exposure to FLX. CONCLUSIONS: These results indicate that exposure to MPH during preadolescence leads to behavioral alterations that endure into adulthood and that these behavioral deficits can be normalized by antidepressant treatment. These results highlight the need for further research to better understand the effects of stimulants on the developing nervous system and the potential enduring effects resulting from early-life drug exposure.