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The worldwide prevalence of hypertension continues to rise, shortening lifespan and contributing to the risk of cardiovascular disease and stroke. Although there are numerous contributing risk factors, social adversity is believed to be one that may account, in part, for disparities in rates and treatment outcomes. We have recently begun to evaluate whether chronic social defeat stress (CSDS) can effectively model aspects of stress-induced cardiovascular disease. Adult male C57BL/6J mice were implanted with telemetry devices that recorded blood pressure, heart rate, core body temperature, and activity. Male CD1 mice were used to defeat C57BL/6J mice 1h before the onset of the dark cycle. Immediately following submission, the defeated mouse was housed in the same cage as the aggressor, physically separated by a perforated divider that allows for continuous visual, auditory, and olfactory contact. The C57BL/6J mouse was defeated and housed with a different CD1 mouse each day, to reduce the likelihood of habituation. Initial social defeat resulted in significant increases in blood pressure, activity, and temperature in comparison with control mice. Interestingly, while blood pressure returned to basal levels by the start of the light cycle for the first few days of defeat, chronic social defeat resulted in sustained elevations in blood pressure, lower activity and lower body temperature. Mice exposed to CSDS also exhibited anxiety-like behaviors, spending significantly more time in the closed arms of the elevated plus maze and less time in the center of an open field arena. At the end of the experiment, flow cytometry was performed on spleen and lung tissue from a subset of mice. Splenomegaly was observed in CSDS mice, with an increase in CD11b and monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs), consistent with chronic inflammation. There were significant increases in PMN-MDSCs isolated from lung, a strong predictor of pulmonary hypertension, as well as enhanced immunosuppressive capability of MDSCs which is associated with the severity of disease. CSDS also resulted in increases in body, heart and adrenal weights and fluid retention. These data suggest that CSDS may be useful for modeling aspects of hypertension and immune dysfunction induced by chronic social stress, thereby enabling us to better understand the mechanisms that contribute to cardiovascular disease. R35HL150750 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND Recurrent PCNSBLs represent a therapeutic challenge. Up to 60% of PCNSBL patients relapse to later face survival rates as low as 22%. Unfortunately, tumor heterogeneity and off-target effects have limited the success of immunotherapy against PCNSBL. METHODS We propose a novel immunotherapy to overcome PCNSBL heterogeneity and off-target effects in an exquisitely tumor specific manner using nanoparticle vaccination, capable of delivering personalized tumor derived mRNAs, that induces systemic orchestration of innate and adaptive immunity. We target tumor antigens derived from the B cell receptor (i.e., heavy chain immunoglobulin - IgH) clonotypes. IgH clonotypes are hypervariable gene rearrangements clonally generated by B cells. Tumor IgH clonotypes are unique for each malignant B cell clone and hence attractive immune targets, not shared by normal B cell clones avoiding undesirable off-target effects. RESULTS RNA-NPs can reprogram tumor microenvironment while activating the innate immunity via IFN type I (i.e., IFNα) and priming of hypervariable region clonotype specific T cell responses in naïve mice. We determined the rearranged IgH sequences (predominant clone 99% and nine additional clones with frequencies < 1%) of clinically relevant inbred murine PCNSBL models (BAL17 and A20) by PCR. The number of identified clonotypes confirmed the IgH variability observed in human B cell hematological malignancies. In preliminary experiments targeting lymphoma derived single clonotypes with RNA-NPs, we showed the feasibility of priming in-vivo T cells specific against hypervariable regions after 3 weekly i.v. RNA-NPs (median IFNγ: 58 pg/ml; range: 50-70 pg/ml vs controls < 30 pg/ml; p=0.008). Targeting of clonotype RNA-NPs was associated with decreased tumor growth (p=0.04). Interestingly, we have observed tumor reactive lymphangiogenesis that communicates with regional skull bone marrow observed in 3D microscopy that might direct future routes of RNA-NP administration. CONCLUSION Our RNA-NP systemic vaccination platform can induce PCNSBL clonotype specific T cell responses, sparing normal tissues.

BACKGROUND Although mRNA vaccines have been deployed with great success against COVID-19, unlocking this technology against glioblastoma will necessitate new lipid-nanoparticle formulations that overcome cancer tolerance and immunosuppression. OBJECTIVE/METHODS We sought to develop a novel mRNA vaccine system to make tolerogenic tumor antigens appear more dangerous through use of unmodified nucleosides (pathogen associated molecular patterns, PAMPs) and highly cationic lipid shells that elicit a systemic damage response against cancer antigens. RESULTS We developed a novel vaccine formulation that increases payload packaging of tumor amplified mRNA into multilamellar (onion-shaped) particles for systemic (intravenous) administration. We demonstrate significant immunogenicity and efficacy of multilamellar RNA-NPs in syngeneic murine models for high-grade glioma (KR158b-pp65), and diffuse midline glioma (H3K27M DMG). Remarkably, RNA-NPs significantly improve median survival outcomes of DMG bearing mice beginning therapy at endpoint (Day 35 after midline intracranial implantation). Unlike prototypical mRNA vaccines that activate endosomal toll-like receptors (i.e. TLR7), multilamellar RNA-NPs elicit immunologic response predominantly through intracellular pathogen recognition receptors (RIG-I); long-term survival benefits from RNA-NPs were completely abrogated in RIG-I knockout mice. In canines (pet dogs) with spontaneous gliomas, RNA-NPs elicit massive recruitment/activation of peripheral blood mononuclear cells (PBMCs) which correlate with their trafficking into lymphoreticular organs (in follow-up murine studies). In canines receiving neoadjuvant RNA-NPs, prior to glioma biopsy, we see significant reprogramming of the glioma microenvironment with increased gene signatures for antigen processing/presentation, interferon signaling and cytotoxicity. Upon translation into human clinical trials for glioblastoma patients (NCT04573140), RNA-NPs elicit rapid (within hours) release of cytokines (e.g. IL-1, IL-6, IL-12 TNF-α, interferons) and chemokines (e.g. MIP1α, MCP-1, IP-10), which correlate with mobilization of PBMCs and activation of dendritic cells/CD8 lymphocytes. CONCLUSION First-in-human application of systemic multilamellar RNA-NP vaccines results in significant biologic effects and rapid immunologic reprogramming.

BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is uniformly fatal. Upon diagnosis, DIPG cannot be safely debulked and systemic therapies have unproven benefit. Immunotherapy can overcome these obstacles, but objective responses are rare; and cancer antigens, even when tumor specific (i.e. H3K27M), are usually poorly immunogenic. OBJECTIVE We sought to develop a murine model of diffuse midline glioma (DMG) that better recapitulates human disease. In these models, we tested different iterations of mRNA loaded nanoparticle (NP) vaccines for their therapeutic effect. APPROACH: We implanted murine gliomas (expressing H3K27M mutation) midline into developing neonatal brains. We manufactured different mRNA constructs encoding for foreign proteins (e.g. GFP), H3K27M or wildtype H3K27 and evaluated immunogenicity and anti-tumor efficacy. These mRNA vaccines were layered into lipid particles (RNA-NP) and administered intravenously when animals became acutely symptomatic (similar to when patients are diagnosed). RESULTS Surprisingly, all mRNA constructs tested were sufficient to elicit anti-tumor efficacy against DMGs. This was true for both antigen specific (H3K27M) and non-antigen specific constructs (GFP, H3K27 wildtype). While H3K27M encoding RNA-NPs were superior to H3K27 wildtype encoding RNA-NPs (p=0.057), the adaptive immune effects appear marginal relative to the innate immune responses generated by all RNA-NP constructs. These innate responses are characterized by induction of type I interferon response from plasmacytoid DCs. Interestingly, these effects could not be recapitulated in murine models of adult type glioblastoma (KR158b) which required antigen specificity for induction of anti-tumor efficacy. CONCLUSION These data suggest that pediatric gliomas may be particularly sensitive to innate immunity. We have optimized development of a target H3K27M mRNA construct that balances innate and adaptive bi-directional immune induction. We are completing FDA-IND enabling data to support translation of H3K27M loaded RNA-NPs into human clinical trials.

INTRODUCTION Our group has developed a patented vaccine platform that combines an mRNA-nanoparticle with CXCL9 loaded polyethylene glycol (PEG) hydrogel. The HCM vaccine is given SQ and recruits a diverse immune cell population to deliver a large payload of mRNA. The objective of these studies was to evaluate the effects of HCM vaccination on the TME. METHODS C57BL/6 mice underwent intracranial implantation of KR158 or GL261 glioma cells. The HCM vaccine was formulated with total tumor mRNA, DOTAP, CXCL9 and PEG hydrogel created in collaboration with the College of Engineering. Survival studies were analyzed with the Mantel-Cox test. Flow cytometry and ELISA data was analyzed using ANOVA. Graphpad was used for statistical analysis. RESULTS Intracranial tumor bearing mice were treated with SQ or intracranial injection of the HCM vaccine (total tumor mRNA, DOTAP, CXCL9, PEG hydrogel). SQ HCM delivery resulted in significant survival benefit whereas intracranial delivery did not (p = 0.0012). In a separate experiment, tumors and spleens were collected for flow cytometry. A significant increase in antigen specific CD8 T cells was found. Separately, after SQ vaccination, tumors were collected for PCR analysis. Tumors treated with HCM vaccination demonstrated significant upregulation of CCL4 and CXCR6. Intracranial tumor bearing mice were treated with HCM vaccination with or without blockade of CCL4. CCL4 blockade resulted in no survival benefit of the vaccine. CONCLUSION The HCM vaccine results in migration of antigen specific CD8 T cells within the TME and significant increases in CCL4. HCM vaccine efficacy is dependent on CCL4.

INTRODUCTION: Immunotherapy for GBM has resulted in limited benefits to overall survival due to the targeting of limited antigens and the hostile TME. Our group has developed a patented vaccine platform that combines an mRNA-nanoparticle with CXCL9 loaded polyethylene glycol (PEG) hydrogel. The HCM vaccine is given SQ and recruits a diverse immune cell population to deliver a large payload of mRNA. The objective of these studies was to evaluate the efficacy and mechanisms of action of the HCM vaccine. METHODS: C57BL/6 mice underwent intracranial implantation of KR158 glioma cells. The HCM vaccine was formulated with total tumor mRNA, DOTAP, CXCL9 and PEG hydrogel created in collaboration with the College of Engineering. Survival studies were analyzed with the Mantel-Cox test. Flow cytometry and ELISA data was analyzed using ANOVA. Graphpad was used for statistical analysis. RESULTS: KR158-glioma intracranial tumor bearing mice were treated with SQ injection of the HCM vaccine (total tumor mRNA, DOTAP, CXCL9, PEG hydrogel). KR158 tumor bearing animals are resistant to treatment with radiation, temozolomide and other immunotherapy platforms in our laboratory. Treatment animals lived significantly longer compared to control animals (35 days versus 22 days, p < 0.0001). In a separate experiment, the fat pad was collected after vaccination and flow cytometry revealed recruitment of dendritic cells (DCs), CD4 and CD8 T cells and NK cells. Large numbers of antigen specific CD8 T cells were found in the spleen and within the TME after vaccination. When NK cells were blocked (NK 1.1 blocking antibody), the survival benefit from the vaccine was completely abrogated. NK cells were found to increase antigen presentation and available IFN-gamma within the PEG hydrogel scaffold. CONCLUSION: The HCM vaccine demonstrates efficacy in resistant murine glioma models and the efficacy is dependent on recruitment of NK cells at the site of vaccination.

The objective of this study was to develop and test a murine system for delivery of laser interstitial thermotherapy (LITT). A system was built that allows for stereotactic placement of the laser into a mouse brain and real-time thermometry with a custom-made thermometer holder. Tissue heating curves for Laserglow Technologies 1064nm laser experiments testing tissue heating were performed by placing the laser in tissue and temperature monitoring via thermal probe. The laser was adapted for use with a stereotactic frame utilizing a system with a Hamilton syringe and a holder for consistent and precise placement of the thermal probe. The murine experiments were performed with C57BL/6 mice and samples were analyzed with H&E staining. First, ideal laser output was established using chicken breast. Laser dial setting of 4.38 successfully achieved slow tissue heating while a setting of 4.43 and 4.50 accomplished it rapidly, reaching a thermal ablative temperature of 43C in three and a half minutes. Next, a system for laser placement into the murine brain using the stereotactic frame was built revealing that the thermometer probe would require a separate system for consistent and precise measurements of heating. A thermometer probe holder was designed and fashioned via trigonometric calculations that allowed for thermal monitoring within 3mm spacing during laser ablation. Mice were anesthetized and treated with laser ablation at laser output of 4.38 and 4.43. H&E staining of the brain tissue demonstrated clear evidence of localized ablation identifiable by visible lesions on the H&E slides. The successful development of the stereotactic system for acquisition of tissue heating data coupled with evidence of successful live brain tissue ablation in a murine model via LITT proves that our protocol is ready for research of therapeutic combinatorial effects on live tumor bearing mice.

Glioblastoma (GBM) is the most common and lethal malignant brain cancer in adults. Immunotherapy has emerged as a potentially powerful approach to achieve long-term survival in patients with GBM. Antigen presenting cells (APCs) play a central role in priming cancer-specific immune responses due to their ability to sample and present tumor neoantigens to the immune system. We hypothesis that antigen presenting cells could be induced by transdifferentiated from GBM in-situ so that these locally created APC will find themselves pre-planted in the tumor microenvironment (TME) with full access to tumor neoantigens. Top ten ranked candidate fate determinants for the GBM-DC transdifferentiation were predicted by NETZEN, an integrated deep-learning and gene network-based ranking artificial intelligence (AI) platform for precision medicine. We successfully transdifferentiated the murine GBM cell line GL261 into CD45+ immune cells accompanying morphological changes to less adhering cells by a combination of four factors (PU.1, IRF8, BATF3, ID2). Of these induced CD45 positive cells, a significant fraction also expresses high levels of the myeloid marker CD11b and antigen presenting molecules MHCII and MHCI by flow cytometry, suggesting these induced CD45+ cells are myeloid lineage APCs (iAPC).These iAPC exhibit phagocytic property, tested by incubating pHrodo Red bioparticles conjugated with Zymosan, an antigen found on the surface of fungi. More importantly, these induced iAPC appear to have lost their proliferative capacity characteristic of the parental GBM cells. Total live cell numbers were significantly reduced in 4F-induced culture compared to the EV control. In conclusion, we successfully transdifferentiated mouse GBM cells into APC-like cells based on NETZEN prediction and our work can potentially provide a novel therapeutic approach for developing an in-situ APC vaccine immunotherapy for GBM, and for other cancers.

BACKGROUND/OBJECTIVE Tumor treating Fields (TTFields) plus maintenance temozolomide is an approved standard treatment for newly diagnosed Glioblastoma (GBM). TTFields are alternating electric fields of low intensity and intermediate frequency delivered non-invasively via transducer arrays to tumor region. Immune checkpoints have not been studied widely in newly diagnosed GBM patients. TTFields combined with temozolomide elicit anti-mitotic effects on proliferative cancer cells and augment recruitment of immune effector cells specific for glioma cells to the tumor microenvironment where pembrolizumab further potentiates the immune reaction to achieve a synergistic therapeutic effect. This study [NCT03405792] will determine if adding pembrolizumab to TTFields and maintenance temozolomide (triple combination) increases progression-free survival (PFS) in patients with newly diagnosed GBM versus historical control (EF-14). METHODS This study will enroll patients (N=24) with pathologic diagnosis of newly diagnosed GBM WHO grade 4, > 18 years after maximal surgery or biopsy followed by radiation therapy with adjuvant temozolomide (Stupp protocol). The primary endpoint is increases in PFS compared to historical control data (EF-14). Secondary endpoints include: toxicity and tolerability of the triple combination; overall survival and response rates versus historical data; augmentation of TTFields-initiated glioma-specific immune reaction by pembrolizumab. Exploratory endpoints include: metabolomics signature of immune activation by TTFields and TTFields plus pembrolizumab in serum and urine, and correlation of mutation burden in primary tumor samples with response to pembrolizumab plus TTFields. We assumed an accrual period of 12 months, an accrual rate of 2 patients per month, an additional 18 months of follow-up, and proportional hazards. Per shape parameter estimate of k=0.88 with empirical 95% confidence interval (0.82, 0.95) obtained via simulation of historical control data, a sample size of 24 patients should detect an improvement in PFS of 6 to 8 months with 80% power and a 1-sided significance level of 0.05 (Wu and Xiong, 2014).

OBJECTIVES Tumor Treating Fields (TTFields) are approved in combination with temozolomide for newly diagnosed glioblastoma (GBM). The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly. However, most GBM patients eventually develop resistance to TTFields. The mechanism of TTFields resistance remains largely unexplored. Understanding how GBM cells circumvent the biophysical forces of TTFields and their downstream effects will improve therapeutic efficacy of this novel anti-cancer treatment modality. METHODS A panel of GBM cell lines were treated continuously with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system until cells with relative resistance to the cytotoxic effects of TTFields. A systems approach aided by innovative network ranking computational algorithms were utilized to analyze global gene expression profiles and identify resistance pathways, which were subsequently validated experimentally. RESULTS TTFields-induced chromosomal instability such as the formation of cytoplasmic micronuclei is preserved in resistant cells, indicating that TTFields resistance is mediated through a non-biophysical mechanism. This acquired TTFields resistance phenotype is associated with a transition of GBM cells to a stem-like state as determined by a neurosphere assay. Using an innovative computational platform, we methodically dissected this stemness program in resistant cells. Mechanistically, Prostaglandin E Receptor 3 (PTGER3) is the top ranked master regulator responsible for resistance. PTGER3 is rapidly upregulated in GBM cells upon exposure to TTFields and further increases with prolonged treatment as resistance sets in. Pharmacological inhibition of PTGER3 either using aspirin to reduce prostaglandin E production or PTGER3-specific inhibitors resensitized cells to TTFields. CONCLUSIONS We have identified a novel pathway with PTGER3 at the apex that plays a critical role in TTFields resistance. This pathway is a potential therapeutic target to reduce resistance to TTFields therapy in GBM.

OBJECTIVES Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults. Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly. In many patients, a transient stage of increased peritumoral edema is often observed early during TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. We hypothesize that TTFields activate the immune system by triggering pyroptosis and type I Interferon (IFN) response. METHODS A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Cells were analyzed for the production of micronuclei and activation of both pyroptosis and STING pathways using immunostaining, quantitative PCR, ELISA and cytometry. RESULTS TTFields resulted in a significantly higher rate of micronuclei structures released into the cytoplasm, which were co-localized with two upstream dsDNA sensors AIM2 and cGAS. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway leading to the release of type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. In a co-culture experiment of bone marrow cells with cells/supernatants obtained from GBM cells treated with TTFields, GBM cells depleted of AIM2 and STING failed to induce bone marrow cells. CONCLUSIONS These results provide compelling evidence that TTFields activates the innate immune system in GBM cells, and a strong rationale for combining TTFields with immune checkpoint inhibitors to create a therapeutic synergy.

Objectives: Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults. Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules' assembly. In many patients, a transient stage of increased peritumoral edema is often observed early during TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. We hypothesize that TTFields activate the immune system by triggering pyroptosis and type I Interferon (IFN) response. Methods: A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Cells were analyzed for the production of micronuclei and activation of both pyroptosis and STING pathways using immunostaining, quantitative PCR, ELISA and cytometry. Pre-treated mouse GBM cells were injected into B6 mouse brain, IVIS and immunophenotyping were performed post implantation. Results: TTFields resulted in a significantly higher rate of micronuclei structures released into the cytoplasm, which were co-localized with two upstream dsDNA sensors AIM2 and cGAS. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by LDH release assay, and through AIM2-recruited caspase1 activation and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway leading to the release of type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. In a co-culture experiment of bone marrow cells with cells/supernatants obtained from GBM cells treated with TTFields, GBM cells depleted of AIM2 and STING failed to induce bone marrow cells. In mouse model, double knocking down of STING/AIM2 eliminated the tumor suppression effects caused by TTFields. TTFields pretreated wild type cells successfully elevated dendritic cell level in mouse cervical lymph nodes which can be reversed by double knocking down. Conclusions: These results provide compelling evidence that TTFields activates the innate immune system in GBM cells, and a strong rationale for combining TTFields with immune checkpoint inhibitors to create a therapeutic synergy. Citation Format: Dongjiang Chen, Nagheme Thomas, Jie Ren, Son Le, Mathew Sebastian, Changwang Deng, Dan Jin, David Tran. Co-activation of STING pathway and immunogenic cell death by tumor treating fields produces effective antitumor immunity in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5524.

Glioblastoma (GBM) is the most prevalent and aggressive brain cancer in adults. GBM is enriched with glioma stemlike cells (GSCs) that contribute to tumor initiation and treatment resistance and thus a natural focus for therapeutic development. However, targeting GSCs has been a challenge because of the dearth of knowledge of master regulators specific to GSCs and not to normal brain cells. Therefore systematic elucidation of the GSC specific core regulatory program will improve our understanding of GSC biology and provide an opportunity to develop novel GSC specific therapy with maximal efficacy and minimal toxicity. To accomplish these objectives, we have recently developed two innovative computational platforms, GeneRep and nSCORE and experimentally validated them in several published datasets. With this platforms, a cluster of 6 interrelated master subnetworks emerged, which were functionally grouped into the stemness and cancer pathways, some of which are known Olig and Myc factors. By enforcing the expression of this cluster in normal Human Astrocytes, we were able to reprogram these normal cells into GSC-like cells as measured by the neurosphere formation and in vivo tumorigenesis assays, confirming the contribution of this cluster for the GSC development. Furthermore, this cluster was present and functioned cooperatively to maintain GSCs in patient derived GSC lines. Functionally blockage individually or two of them by shRNA significantly reduces neurosphere formation and promotes cell death. Intracranial Injection of those gene-modified-GSCs into NSG mouse, dramatically double the mouse survival time. Most promising, preliminary data shows that in vivo tumor growth could be inhibited by either modulation of gene expression or via cancer vaccine. Our data establish a novel set of genes controlling GSCs survival and provide a compelling rationale for its therapeutic targeting to reduce tumor growth.

Glioblastoma (GBM) is the most prevalent and aggressive brain cancer in adults. GBM is enriched with glioma stem-like cells (GSCs) that contribute to tumor initiation and treatment resistance and thus a natural focus for therapeutic development. However, targeting GSCs has been a challenge because of the dearth of knowledge of master regulators specific to GSCs and not to normal brain cells. Therefore systematic elucidation of the GSC-specific core regulatory program will improve our understanding of GSC biology and provide an opportunity to develop novel GSC-specific therapy with maximal efficacy and minimal toxicity. To accomplish these objectives, we have recently developed two innovative computational platforms, GeneRep and nSCORE and experimentally validated them in several published datasets. The tandem platforms complement each other and allow us to 1) interrogate large-scale gene expression profiles of GSCs, GBM-differentiated cells, normal neuronal precursor cells (NPCs) and astrocytes (NAs) to extract candidate GSC-specific gene regulatory networks, and 2) functionally rank GSC-specific core master regulators, respectively. A cluster of 8 interrelated master subnetworks emerged, which were functionally grouped into the stemness and cancer pathways, some of which belonging to the Sox, Olig and Myc families. By enforcing the-expression of this cluster in NAs, we were able to reprogram these normal cells into GSCs as measured by the neurosphere formation and in vivo tumorigenesis assays, confirming the critical requirement of this cluster for the GSC identity. Furthermore this network cluster was present and functioned cooperatively to maintain GSCs in several patient-derived GSC lines examined to date. Survival and neurosphere formation rates of GSCs were profoundly suppressed when at least two nodes in this cluster were knock down by siRNA compared to depletion of individual nodes. Our data establish a novel gene regulatory cluster controlling GSCs and provide a compelling rationale for its therapeutic targeting to reduce tumor recurrence.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite surgery and aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) have been approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation and cell death. However, treatment resistance develops in many TTFields responders. The mechanism of TTFields resistance remains largely unexplored. Understanding how GBM cells circumvent the biophysical forces of TTFields and their downstream effects will provide new opportunities to improve therapeutic efficacy of this novel anti-cancer treatment. To accomplish these objectives, we have developed several human GBM cell lines that demonstrated relative resistance to the cytotoxic effects of TTFields compared to the parental cells. Importantly TTFields-induced chromosomal instability such as the formation of cytoplasmic micronuclei was preserved in resistant cells compared to their sensitive counterparts, indicating resistance to TTFields is mediated through a non-bioiphysical mechanism. Indeed, TTFields-induced inflammatory response was severely suppressed in resistant cells, supporting the hypothesis that that resistance to TTFields is conferred by a selective loss of the deleterious effects induced by the biophysical insults. Importantly, this acquired TTFields resistance phenotype was associated with a transition to a stem-like state as determined by a standard neurosphere assay. Using a systems approach aided by a suite of innovative computational platforms, we methodically dissected this stemness program in resistant cells to identify master regulators of the resistance mechanism. Interestingly, 3 networks were found disrupted, including nervous system developmental regulation, inflammatory response and cell-cell adhesion, all of which play critical roles in GBM stem-like cells, thus confirming our initial hypothesis. Utilizing a unique master regulator ranking system, we successfully identified Prostaglandin E Receptor 3 (PTGER3) as a key master regulator at the apex of these pathways and responsible for the resistant phenotype. PTGER3 is rapidly upregulated in GBM cells when exposed to TTFields and appears to channel treated cells away from the beneficial inflammatory pathways that TTFields also activates in parallel. Ongoing experiments are aimed at understanding the molecular mechanism of PTGER3-dependent TTFields resistance. Our goal is to develop targeted therapies to overcome resistance to TTFields. Note: This abstract was not presented at the meeting. Citation Format: Dongjiang Chen, Son Le, Nagheme Thomas, Changwang Deng, Dan Jin, Mathew Sebstian, Jie Ren, David Tran. Prostaglandin E receptor 3 mediates resistance to tumor treating fields in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2094.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) was recently approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early in the course of TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. However, the mechanism underlying these observations remains unclear. Here we report results on a panel of GBM cell lines treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Our data showed 24 hrs TTFields-treated GBM cells had a significantly higher rate (19.9% vs. 4.3%, p=0.0032) of micronuclei structures released into the cytoplasm as a result of TTFields-induced chromosomal instability. Nearly 40% of these micronuclei were co-localized with two upstream dsDNA sensors: absent in melanoma 2 (AIM2)andInterferon (IFN)-inducible proteinCyclic GMP-AMP synthase (cGAS), compared to absence of co-localization in untreated cells. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway components including Type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. GBM cell-specific shRNA depletion of either AIM2 or STING or both in a co-culture experiment of bone marrow cells or splenocytes with supernanants obtained from knockdown GBM cells was able to reverse the inducement of immune cells. These results provide compelling evidence that TTFields function as an activator of the immune system in GBM cells, and a strong rationale for combining TTFields with immunotherapy aimed at augmenting an anti-tumor immune response such as immune checkpoint inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Dongjiang Chen, Nagheme Thomas, David D. Tran. TTFields induces immunogenic cell death and STING pathway activation through cytoplasmic double-stranded DNA in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3280.

Movement representations in the motor cortex can reorganize to support motor skill learning during young adulthood. However, little is known about how motor representations change during aging or whether their change is influenced by continued practice of a skill after it is learned. We used intracortical microstimulation to characterize the organization of the forelimb motor cortex in young and aged C57/BL6 mice after short (2-4 weeks) or long (8 weeks) durations of training on a skilled reaching task or control procedures. In young mice, a short duration of reach training increased the area of proximal forelimb movement representations at the expense of distal representations. Following a longer training duration, ratios of proximal to distal movements returned to baseline, even with ongoing practice and skill maintenance. However, lingering changes were evident in thresholds for eliciting distal forelimb movements, which declined over the longer training period. In aged mice, movement representations and movement thresholds failed to change after either duration of training. Furthermore, there was an age-related loss of digit representations and performance decrements on other sensorimotor tests. Nevertheless, in quantitative measures of reaching success, aged mice learned and performed the skilled reaching task at least as well as younger mice. These results indicate that experience-driven topographical reorganization of motor cortex varies with age, as well as time, and is partially dissociable from behavioral performance. They also support an enduring capacity to learn new manual skills during aging, even as more youthful forms of cortical plasticity and sensorimotor function are lost.

Cell-based immunotherapy has shown encouraging results in solid cancers including glioblastoma (GBM). However, major limitations of cell-based immunotherapy in GBM include difficulty with isolation and generation of effective antigen-presenting or immune effector cells, inadequate migration of immune cells across the blood brain barrier, and the high cost of cell-based vaccines. One approach to overcome these barriers is to convert GBM cells directly into immune cells of interest. Transdifferentiation, the transformation of one differentiated cell type directly into another without an intermediate pluripotent stage, has shown early promise in regenerative medicine, but has had a limited role in cancer therapy, especially for solid tumors. This is in large part due to the laborious and time-consuming process of identifying experimentally master fate determinants of various cell types. To enable this novel application, we have developed and experimentally validated two tandem computational platforms, GeneRep and nSCORE. Applying this novel tool to large quantities of published datasets, we successfully predicted cell fate determinants of tens of different cell types with several having been validated in transdifferentiation experiments by others and us, such as the conversions of astrocytes to neuronal stem cells and of fibroblasts to macrophages, etc. Here, we describe the process of combining a core set of hematopoietic stem cell, myeloid, and macrophage or dendritic cell (DC) fate determinants as predicted by GeneRep-nSCORE to efficiently transdifferentiate GBM cells into functional macrophages or dendritic cells, respectively, while neutralizing their malignant phenotype. This novel transdifferentiation has the potential for transformative impacts in many areas including DC vaccine immunotherapy and cancer therapy in general, and pioneers the concept that solid tumor cells can be converted into another cell type, and not just any cell type, but one that potentially can elicit a therapeutic response against itself.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Recently, Tumor Treating Fields (TTFields) were approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early in the course of TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. However, the mechanism underlying these observations remains unclear. A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using the inovitro system. Our data showed TTFields-treated GBM cells had a significantly higher rate (19.9% vs. 4.3%, p=0.0032) of micronuclei structures released into the cytoplasm as a result of TTFields-induced chromosomal instability. Nearly 40% of these micronuclei were co-localized with two upstream dsDNA sensors Interferon (IFN)-inducible protein absent in melanoma 2 (AIM2) and Cyclic GMP-AMP synthase (cGAS), compared to absence of co-localization in untreated cells. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway components including Type I IFNs and pro-inflammatory cytokines in GBM cells. These results provide compelling evidence that TTFields function as an activator of the immune system in GBM, and present a strong rationale for combining TTFields with immunotherapy aimed at augmenting an anti-tumor immune response such as immune checkpoint inhibitors.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Recently, Tumor Treating Fields (TTFields) were approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation and cell death. However, many TTFields responders eventually develop progression. The mechanism of TTFields resistance remains largely unexplored. Understanding how cancer cells gain the ability to circumvent the biophysical forces of TTFields and their downstream effects will provide new opportunities to improve therapeutic efficacy of this novel anti-cancer treatment. To accomplish these objectives, we have developed several human GBM cell lines that demonstrated relative resistance to the cytotoxic effects of TTFields compared to the parental cells. Importantly TTFields-induced chromosomal instability such as the formation of micronuclei was unchanged in resistant cells compared to their sensitive counterparts. In contrast, TTFields-induced inflammatory response was severely suppressed in resistant cells, suggesting that resistance to TTFields may be conferred by a selective loss of the deleterious effects downstream of the biophysical insults. Importantly, this acquired TTFields resistance phenotype of GBM cells was associated with a transition to a stem-like state. Using a systems approach aided by a suite of innovative computational platforms, we methodically dissected this renewed stemness program in resistant cells to identify master regulators of the resistance mechanism. Our long-term goal is to develop targeted therapies that prevent tumor’s resistance to TTFields.

The organization of forelimb representation areas of the monkey, cat, and rat motor cortices has been studied in depth, but its characterization in the mouse lags far behind. We used intracortical microstimulation (ICMS) and cytoarchitectonics to characterize the general organization of the C57BL/6 mouse motor cortex, and the forelimb representation in more detail. We found that the forelimb region spans a large area of frontal cortex, bordered primarily by vibrissa, neck, shoulder, and hindlimb representations. It included a large caudal forelimb area, dominated by digit representation, and a small rostral forelimb area, containing elbow and wrist representations. When the entire motor cortex was mapped, the forelimb was found to be the largest movement representation, followed by head and hindlimb representations. The ICMS-defined motor cortex spanned cytoarchitecturally identified lateral agranular cortex (AGl) and also extended into medial agranular cortex. Forelimb and hindlimb representations extended into granular cortex in a region that also had cytoarchitectural characteristics of AGl, consistent with the primary motor–somatosensory overlap zone (OL) characterized in rats. Thus, the mouse motor cortex has homologies with the rat in having 2 forelimb representations and an OL but is distinct in the predominance of digit representations.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Recently, Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules' assembly, leading to disrupted chromosomal segregation and cell death. However, the majority of GBM are not sensitive to TTFields and most of TTFields responders eventually develop resistance. However the mechanism of resistance remains largely unexplored. Understanding how cancer cells gain the ability to circumvent the biophysical forces of TTFields and their downstream effects will provide new opportunities to improve therapeutic efficacy of this novel anti-cancer treatment modality. To accomplish these objectives, we have developed several human GBM cell lines that demonstrated relative resistance to the cytotoxic effects of TTFields compared to the parental cells. Importantly TTFields-induced chromosomal instability such as the formation of micronuclei was unchanged in resistant cells compared to their sensitive counterparts, suggesting that resistance to TTFields may be conferred by a selective loss of deleterious downstream reactions to the biophysical damages. To systematically identify the mechanism of resistance, we have recently developed two innovative computational platforms, GeneRep and nSCORE and experimentally validated them in several published datasets. The tandem platforms complemented each other and allowed us to 1) interrogate large-scale gene expression profiles of pairs of TTFields resistant cells vs. sensitive counterparts to extract candidate resistance-specific gene regulatory networks, and 2) functionally rank specific core factors responsible for resistance, respectively. Once identified, biological confirmation of these factors using cell lines, mouse models and samples of patient's derived TTFields sensitive and resistant GBM. Our long-term goal is to develop targeted therapy to prevent tumor's resistance to TTFields therapy. Citation Format: Dongjiang Chen, Son Le, Nagheme Thomas, David D. Tran. A systems approach for determining the mechanism of resistance to tumor treating fields in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5898.

Cell-based immunotherapy has shown encouraging results in solid cancers. However, major limitations of this approach include difficulty with isolation and generation of effective antigen-presenting or immune effector cells and the high cost of cell-based vaccines. In glioblastoma (GBM), the most common and lethal brain cancer in adults, inadequate migration of immune cells across the blood-brain barrier represents another major obstacle to cell-based immunotherapy. One approach to overcome these barriers is to convert GBM cells directly into immune cells of interest. Transdifferentiation, the transformation of one cell type other than stem cells directly into another cell type without an intermediate pluripotent stage, has shown early promise in regenerative medicine, but has had a limited role as a cancer therapy, especially for solid tumors. This is in large part due to the laborious and time-consuming process of identifying master fate determinants of the cell types of interest in the transdifferentiation. To enable this novel application, we have developed and experimentally validated two tandem computational platforms, GeneRep and nSCORE. Applying this novel algorithm to large quantities of published and in-house gene expression datasets, we successfully predicted cell fate determinants of tens of different cell types with several having been validated in transdifferentiation experiments by others and us, such as the conversions of astrocytes to neuronal stem cells, of astrocytes to glioma stem-like cells, of fibroblasts to macrophages, etc. Here, we describe the process of combining a unique core set of hematopoietic stem cell, myeloid, and macrophage or dendritic cell (DC) fate determinants—some have not been described as critical in these cell fates—as predicted and highly ranked by GeneRep-nSCORE to efficiently transdifferentiate GBM cells into functional macrophages or dendritic cells, respectively, while neutralizing their malignant phenotype. This novel transdifferentiation has the potential for transformative impacts in many areas, including DC vaccine immunotherapy and cancer therapy in general, and pioneers the concept that solid tumor cells can be converted into another cell type, and not just any cell type, but one that potentially can elicit a therapeutic response against itself. Citation Format: Mathew Sebastian, Son B. Le, Changwang Deng, Nagheme Thomas, David D. Tran. Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4072.

Background. Cortical electrical stimulation of the motor cortex in combination with rehabilitative training (CS/RT) has been shown to enhance motor recovery in animal models of focal cortical stroke, yet in clinical trials, the effects are much less robust. The variability of stroke location in human patient populations that include both cortical and subcortical brain regions may contribute to the failure to find consistent effects clinically. Objective. This study sought to determine whether infarct location influences the enhanced motor recovery previously observed in response to CS/RT. The efficacy of CS/RT to promote improvements in motor function was examined in 2 different rat models of stroke that varied the amount and location of cortical and subcortical damage. Methods. Ischemic infarctions were induced by injecting the vasoconstricting peptide endothelin-1 either (1) onto the middle cerebral artery (MCA) producing damage to the frontal cortex and lateral striatum or (2) into a subcortical region producing damage to the posterior thalamus and internal capsule (subcortical capsular ischemic injury [SCII]). Daily CS/RT or RT alone was then given for 20 days, during which time performance on a skilled reaching task was assessed. Results. Animals with MCA occlusion infarctions exhibited enhanced improvements on a skilled reaching task in response to CS/RT relative to RT alone. No such enhancement was observed in animals with SCII infarctions across the 20 days of treatment. Conclusions. The efficacy of CS for enhancing motor recovery after stroke may depend in part on the extent and location of the ischemic infarct.

Background. The incidence of stroke in adulthood increases with advancing age, but there is little understanding of how poststroke treatment should be tailored by age. Objective. The goal of this study was to determine if age and task specificity of rehabilitative training affect behavioral improvement and motor cortical organization after stroke. Methods. Young and aged mice were trained to proficiency on the Pasta Matrix Reaching Task prior to lesion induction in primary motor cortex with endothelin-1. After a short recovery period, mice received 9 weeks of rehabilitative training on either the previously learned task (Pasta Matrix Reaching), a different reaching task (Tray Reaching), or no training. To determine the extent of relearning, mice were tested once weekly on the Pasta Matrix Reaching Task. Mice then underwent intracortical microstimulation mapping to resolve the remaining forelimb movement representations in perilesion motor cortex. Results. Although aged mice had significantly larger lesions compared with young mice, Pasta Matrix Reaching served as effective rehabilitative training for both age-groups. Young animals also showed improvement after Tray Reaching. Behavioral improvement in young mice was associated with an expansion of the rostral forelimb area (“premotor” cortex), but we failed to see reorganization in the aged brain, despite similar behavioral improvements. Conclusions. Our results indicate that reorganization of motor cortex may be limited by either aging or greater tissue damage, but the capacity to improve motor function via task-specific rehabilitative training continues to be well maintained in aged animals.

The present study determined the differential effects of unilateral striatal dopamine depletion on cranial motor versus limb motor function. Forty male Long Evans rats were first trained on a comprehensive motor testing battery that dissociated cranial versus limb motor function and included: cylinder forepaw placement, single pellet reaching, vermicelli pasta handling; sunflower seed opening, pasta biting acoustics, and a licking task. Following baseline testing, animals were randomized to either a 6-hydroxydopamine (6-OHDA) (n = 20) or control (n = 20) group. Animals in the 6-OHDA group received unilateral intrastriatal 6-OHDA infusions to induce striatal dopamine depletion. Six-weeks following infusion, all animals were re-tested on the same battery of motor tests. Near infrared densitometry was performed on sections taken through the striatum that were immunohistochemically stained for tyrosine hydroxylase (TH). Animals in the 6-OHDA condition showed a mean reduction in TH staining of 88.27%. Although 6-OHDA animals were significantly impaired on all motor tasks, limb motor deficits were more severe than cranial motor impairments. Further, performance on limb motor tasks was correlated with degree of TH depletion while performance on cranial motor impairments showed no significant correlation. These results suggest that limb motor function may be more sensitive to striatal dopaminergic depletion than cranial motor function and is consistent with the clinical observation that therapies targeting the nigrostriatal dopaminergic system in Parkinson’s disease are more effective for limb motor symptoms than cranial motor impairments.

Stroke is the leading cause of disability in the Unites States resulting in upper extremity motor impairments. The present study examined the efficacy of systemically administered hematopoietic endothelial progenitor cells (EPCs) for ameliorating motor impairments and cortical dysfunction following cerebral ischemia. Methods: Baseline motor performance of Forty one male adult rats was established on a single pellet reaching task. Animals were assigned to one of five experimental conditions that counterbalanced baseline motor performance. Animals in the Stroke condition (n=7) received infusion of endothelin-1 onto the middle cerebral artery contralateral to the preferred paw. Animals in the PRE (n=7) and POST (n=7) conditions received the same infusion of endothelin-1 one but also received ten million EPCs injected into the tail vein either thirty minutes before (PRE) or immediately after (POST) infusion. The EPC’s were enriched from the bone marrow of a separate group of donor rats using nanoparticles tagged with LIN negative and CD90 markers. SHAM (n=9) animals only received EPC infusion and CONTROLS received no stroke or EPC infusion. One, three and five weeks following stroke all animals were tested on the skilled reaching task after which intracortical microstimulation was used to derive maps of forelimb movement representations within the motor cortex contralateral to the preferred paw. All assessments were performed in a blinded manner. Results: showed POST animals performed significantly better on single pellet reaching than animals in all other stroke conditions on weeks 3 and 5. Control animals scored a reaching accuracy of 41±8.1% and 46±11% at 3 and 5 weeks, respectively. Stroke decreased this task to 12±2.1% and 18±2.8%, respectively (p

Rehabilitation-dependent motor recovery after cerebral ischemia is associated with functional reorganization of residual cortical tissue. Recovery is thought to occur when remaining circuitry surrounding the lesion is “retrained” to assume some of the lost function. This reorganization is in turn supported by synaptic plasticity within cortical circuitry and manipulations that promote plasticity may enhance recovery. Activation of the cAMP/CREB pathway is a key step for experience-dependent neural plasticity. Here we examined the effects of the prototypical phosphodiesterase inhibitor 4 (PDE4) rolipram and a novel PDE inhibitor (HT-0712), known to enhance cAMP/CREB signaling and cognitive function, on restoration of motor skill and cortical function after focal cerebral ischemia. Adult male rats were trained on a skilled reaching task to establish a baseline level of motor performance. Intracortical microstimulation was then used to derive high-resolution maps of forelimb movement representations within the caudal forelimb area of motor cortex contralateral to the trained paw. A focal ischemic infarct was created within approximately 30% of the caudal forelimb area. The effects of administering either rolipram or the novel PDE4 inhibitor HT-0712 during rehabilitation on motor recovery and restoration of movement representations within residual motor cortex were examined. Both compounds significantly enhanced motor recovery and induced an expansion of distal movement representations that extended beyond residual motor cortex. The expansion beyond the initial residual cortex was not observed in vehicle injected controls. Furthermore, the motor recovery seen in the HT-0712 animals was dose dependent. Our results suggest that PDE4 inhibitors during motor rehabilitation facilitate behavioral recovery and cortical reorganization after ischemic insult to levels significantly greater than that observed with rehabilitation alone.