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Sepsis is a complex disease due to dysregulated host response to infection. Oxidative stress and mitochondrial dysfunction leading to metabolic dysregulation are among the hallmarks of sepsis. The transcription factor NRF2 (Nuclear Factor E2-related factor2) is a master regulator of the oxidative stress response, and the NRF2 mediated antioxidant response is negatively regulated by BTB and CNC homology 1 (BACH1) protein. This study tested whether Bach1 deletion improves organ function and survival following polymicrobial sepsis induced by cecal ligation and puncture (CLP). We observed enhanced post-CLP survival in Bach1

Microglia, the tissue-resident macrophage of the central nervous system (CNS), play a paramount role in brain health and disease status. Here, we describe a novel method for enriching and isolating primary microglia from mouse brain tissue. This isolation method yields a high number of cells from either young or adult mice, and importantly, maintains the health and function of the cells for subsequent cell culture. We also describe flow cytometry methods using novel cell surface markers, including CX3CR1 and Siglec-H, to specifically label microglia while avoiding other bone marrow and/or non-CNS derived macrophages and monocytes, which has been historically difficult to achieve. As microglia are crucial in multiple aspects of biology, such as in normal brain development/function, immune response, neurodegeneration, and cancer, this isolation technique could greatly benefit a wide range of studies in human CNS biology, health, and disease mechanisms. Being able to isolate a largely pure population of microglia could also allow for a more comprehensive understanding of their functional dynamics and role in disease mechanisms, advancement of potential biomarkers, and development of novel therapeutic targets to improve prognosis and quality of life in multiple diseases.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues—such as incomplete mechanistic knowledge and faulty clinical trial design—a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Cytosolic DNA sensing is an important process during the innate immune response that activates the stimulator of interferon genes (STING) adaptor and induces IFN-I. STING incites spontaneous immunity during immunogenic tumor growth and accordingly, STING agonists induce regression of therapy-resistant tumors. However DNA, STING agonists, and apoptotic cells can also promote tolerogenic responses via STING by activating immunoregulatory mechanisms such as indoleamine 2,3 dioxygenase (IDO). Here, we show that IDO activity induced by STING activity in the tumor microenvironment (TME) promoted the growth of Lewis lung carcinoma (LLC). Although STING also induced IDO in tumor-draining lymph nodes (TDLN) during EL4 thymoma growth, this event was insufficient to promote tumorigenesis. In the LLC model, STING ablation enhanced CD8+ T-cell infiltration and tumor cell killing while decreasing myeloid-derived suppressor cell infiltration and IL10 production in the TME. Depletion of CD8+ T cells also eliminated the growth disadvantage of LLC tumors in STING-deficient mice, indicating that STING signaling attenuated CD8+ T-cell effector functions during tumorigenesis. In contrast with native LLC tumors, STING signaling neither promoted growth of neoantigen-expressing LLC, nor did it induce IDO in TDLN. Similarly, STING failed to promote growth of B16 melanoma or to induce IDO activity in TDLN in this setting. Thus, our results show how STING-dependent DNA sensing can enhance tolerogenic states in tumors characterized by low antigenicity and how IDO inhibition can overcome this state by attenuating tumor tolerance. Furthermore, our results reveal a greater complexity in the role of STING signaling in cancer, underscoring how innate immune pathways in the TME modify tumorigenesis in distinct tumor settings, with implications for designing effective immunotherapy trials. Cancer Res; 76(8); 2076–81. ©2016 AACR.

STING ablation modifies inflammatory responses to LLC tumor growth.

Legends for Supplemental Figures S1-S3.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1’s importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells’ sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.

Introduction: Triple negative breast cancer (TNBC) subtype of breast cancer, which lacks molecular markers such as HER2 and estrogen/progesterone receptors, has limited treatment options. Exosomes are nano-sized (30-150 nm) spherical vesicles, derived from the endosomal system for intercellular communications, can be engineered to express targeting peptides to target specifically CD206 positive M2 macrophage. Investigators have used either synthetic nanoparticles or fusion protein to deliver Fc-IgG2b to initiate Antibody dependent cellular cytotoxicity (ADCC) but reports are showing a lack of ADCC following tagging with gold nanoparticles. To overcome this, in our lab, we developed engineered exosomes in non-tumorous cells, HEK293, to carry therapeutic Fc-mIgG2b to enhance ADCC and to carry peptide to deplete CD206+ M2 macrophages. We investigated the effectiveness of engineered therapeutic exosomes to target and deplete immunosuppressive CD206+ M2 macrophages in TNBC in combination with anti PD-1. Methods: In this study, we developed syngeneic metastatic TBNC model (luciferase + 4T1 cells in Balb/c) by implantation of 50K 4T1-luci cells in fat pad. On day 1 of tumor implantation, animals were treated with vehicle, engineered exosomes, anti-PD1 plus engineered exosomes, 2 dose/week for 3 weeks. They were euthanized 1 day after the last dose of treatment. Single-cell suspension was made from the primary TME, lung, and spleen. Using flow cytometry surfaces markers were investigated to determine different T-cell and myeloid cell populations. Results: We observed very surprising results following three weeks of therapies. Although the number of cytotoxic T-cells was significantly higher in the primary and metastatic TME in exosome-treated animals, there was also an increased number of proliferative T reg cells. The number of CD8+ cells was decreased following concurrent therapy with an anti-PD1 antibody. Most interestingly, lung tissue macrophages (CD11c) showed significantly increased PD-L1 expression following exosomes and anti PD1 with engineered exosomes therapies. Conclusion: This study demonstrates that treatment with engineered therapeutic exosome targeting M2 macrophages with anti PD-1 could be utilized effectively. This study will also make a greater impact in exosome engineering based targeted therapy and will open new arena to target and deplete pathogenic cell in different tumors using engineered exosome. Citation Format: Mahrima Parvin, Mohammad H. Rashid, Ahmet Alptekin, Ali S. Arbab. Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5492.

Myeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME). Along with the role of MDSC immunosuppression and antitumor immunity, MDSCs facilitate tumor growth, differentiation, and metastasis in several ways that are yet to be explored. Like any other cell type, MDSCs also release a tremendous number of exosomes, or nanovesicles of endosomal origin, that participate in intercellular communications by dispatching biological macromolecules. There have been no investigational studies conducted to characterize the role of MDSC-derived exosomes (MDSC exo) in modulating the TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant level of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed a higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper-activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that the immunosuppressive and tumor-promoting functions of MDSCs are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

Metastatic breast cancer (BC) is a highly invasive form of cancer that contributes greatly to patient mortality. Due to the complex mechanisms underlying cancer growth and metastasis, novel therapeutic targets must be found to increase patient survival. One such target is the cytochrome P450 4 (CYP4) metabolic pathway leading to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a lipid signaling molecule that promotes both tumor cell proliferation and neovascularization in tumors by inducing the proliferation and migration of endothelial cells, therefore promoting BC tumor survival. N-hydroxy-N’-(4-butyl-2 methyl phenyl) formamidine (HET0016) is a selective inhibitor of 20-HETE synthesis, thus impairing tumor growth and neovascularization. This study aims to investigate changes in BC (MDAMB231 and MCF7) cell lines overexpressing CYP4A11, a human CYP4 enzyme, with and without HET0016 treatment. To accomplish this, cells that overexpressed CYP4A11 were produced using lentivirus infection and confirmed via western blot. Tumor cell aggressiveness was assessed in vitro using migration and proliferation assays. Tumor growth with and without CYP4A11 overexpression was also measured in vivo using athymic nude mice. The results demonstrated that cells overexpressing CYP4A11 showed protein levels 4 times higher than the non-modified cells. MDAMB231 is a triple-negative BC (TNBC) that, when overexpressing CYP4A11, demonstrated increased proliferation capacity and significant increased tumor growth compared to non-modified cells. TNBC cells overexpressing CYP4A11 also showed increased motility. However, modified MCF7 cells did not show significant alterations when compared to non-modified MCF7 cells. Additionally, all cells treated with HET0016 showed decreased migration in the first 24 hours. In conclusion, these results demonstrate that the overexpression of CYP4A11 in TNBC cells increased tumor aggressiveness, proving this pathway to be an important target for therapeutic inhibition. Citation Format: Sehar Ali, Thaiz Borin, Ali Arbab. Evaluation of cytochrome P450 4A11 overexpression in breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-16-08.

CD73, an ecto-5′-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73hi population in human colorectal cancers (CRCs) and two CD73− murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A2A and A2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A2B-mediated ADO-CAF-CD73 feedforward circuit and A2A-mediated immune suppression is crucial for improving therapeutic outcomes.

Exosomes, a component of extracellular vesicles, are shown to carry important small RNAs, mRNAs, protein, and bioactive lipid from parent cells and are found in most biological fluids. Investigators have demonstrated the importance of mesenchymal stem cells derived exosomes in repairing stroke lesions. However, exosomes from endothelial progenitor cells have not been tested in any stroke model, nor has there been an evaluation of whether these exosomes target/home to areas of pathology. Targeted delivery of intravenous administered exosomes has been a great challenge, and a targeted delivery system is lacking to deliver naïve (unmodified) exosomes from endothelial progenitor cells to the site of interest. Pulsed focused ultrasound is being used for therapeutic and experimental purposes. There has not been any report showing the use of low-intensity pulsed focused ultrasound to deliver exosomes to the site of interest in stroke models. In this proof of principle study, we have shown different parameters of pulsed focused ultrasound to deliver exosomes in the intact and stroke brain with or without intravenous administration of nanobubbles. The study results showed that administration of nanobubbles is detrimental to the brain structures (micro bleeding and white matter destruction) at peak negative pressure of >0.25 megapascal, despite enhanced delivery of intravenous administered exosomes. However, without nanobubbles, pulsed focused ultrasound enhances the delivery of exosomes in the stroke area without altering the brain structures.

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFβ1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFβ1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFβ1 signaling require initial interaction of regDCs EXO with TGFβ1R followed by internalization of regDCs EXO with TGFβ1-TGFβ1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFβ1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.

Formation of neutrophil extracellular traps worsens cerebrovascular outcomes after traumatic brain injury., Traumatic brain injury (TBI) is a major cause of mortality and morbidity. Preventative measures reduce injury incidence and/or severity, yet one-third of hospitalized patients with TBI die from secondary pathological processes that develop during supervised care. Neutrophils, which orchestrate innate immune responses, worsen TBI outcomes via undefined mechanisms. We hypothesized that formation of neutrophil extracellular traps (NETs), a purported mechanism of microbial trapping, exacerbates acute neurological injury after TBI. NET formation coincided with cerebral hypoperfusion and tissue hypoxia after experimental TBI, while elevated circulating NETs correlated with reduced serum deoxyribonuclease-1 (DNase-I) activity in patients with TBI. Functionally, Toll-like receptor 4 (TLR4) and the downstream kinase peptidylarginine deiminase 4 (PAD4) mediated NET formation and cerebrovascular dysfunction after TBI. Last, recombinant human DNase-I degraded NETs and improved neurological function. Thus, therapeutically targeting NETs may provide a mechanistically innovative approach to improve TBI outcomes without the associated risks of global neutrophil depletion.

Angiogenesis is a hallmark of glioblastoma (GBM) and remains an important therapeutic target in its treatment, especially for recurrent GBM. GBMs are characterized by the release of vascular endothelial growth factor (VEGF), an important regulator and promoter of angiogenesis. Therefore, antiangiogenic therapies (AATs) targeting VEGF or VEGF receptors (VEGFRs) were designed and thought to be an effective tool for controlling the growth of GBM. However, recent results of different clinical trials using humanized monoclonal antibodies against VEGF (bevacizumab), as well as tyrosine kinase inhibitors (TKIs) that target different VEGFRs alone or in combination with other therapeutic agents demonstrated mixed results, with the majority of reports indicating that GBM developed resistance against antiangiogenic treatments.

Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early detection and intervention of CD206-positive M2-macrophages may boost the clinical outcome. To determinein vivodistribution of M2-macrophages, we adopted111In-oxine-based radiolabeling of the targeted exosomes. When injected these radiolabeled targeted exosomes into breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, spleen, and liver.Ex vivoquantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed adherence of exosomes to the CD206-positive M2-macrophages onex vivofluorescent microscopy imaging. In addition, we utilized these engineered exosomes to carry the Fc portion of IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages bothin vitroandin vivo, and reduce tumor burden increasing survival in a metastatic breast cancer model.

Myeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Ourin vitrodata suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cellsin vivoby treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

Different effects of anticancer drugs between mouse and human have caused increasing concerns. A better understanding of cancer initiation between the two species is needed. We have previously identified an oncoprotein GT198 (PSMC3IP) in human breast cancer. In this report, we investigated GT198 in MMTV-PyMT mouse mammary gland tumors and found a reconcilable mechanism in human and mouse. Specifically, distinct tumor initiating stimuli in human and mouse result in a common GT198-mediated tumorigenic pathway in both species. Here we show, similar to human breast cancer even before a tumor appears, GT198 has overexpressed in mouse tumor stroma including pericyte stem cells, descendent adipocytes, fibroblasts, and myoepithelial cells. Using recombinant GT198 protein as an antigen, we vaccinated MMTV-PyMT mice and found that the GT198 vaccine delayed mouse tumor growth and reduced lung metastasis. The antitumor effects in vaccinated mice were linearly correlated with serum titers of GT198 antibody, which can recognize cell surface GT198 protein on viable tumor cells confirmed by FACS. Furthermore, tumor cells isolated from MMTV-PyMT mice were re-implanted into normal FVB/N mice, GT198+tumor cells induced faster tumor growths than GT198-tumor cells. Together, this first study of GT198 vaccine in mouse showed its effectiveness in antitumor and anti-metastasis. The finding may accelerate future development of GT198 immunotherapy in human cancer. Our finding also indicates that even though distinct cancer-initiation stimuli exist between mouse and human, a common tumorigenic pathway mediated by oncoprotein GT198 is shared in both species.

Glioblastoma (GBM) is a hypervascular and aggressive primary malignant tumor of the central nervous system. Recent investigations showed that traditional therapies along with antiangiogenic therapies failed due to the development of post-therapy resistant and recurrent GBM. Our investigations show that there are changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and we need to rethink how to treat GBM.We hypothesize that the composition of TME-associated cells will be different based on the therapy and therapeutic agents, and TME-targeting therapy will be better to decrease recurrence and improve survival. Therefore, the purpose of this study is to determine the changes in the TME in respect of T-cell population, M1 and M2 macrophage polarization status, and MDSC population following different treatments in a syngeneic model of GBM. In addition to these parameters, tumor growth and survival were also studied following different treatments.The results showed that changes in the TME-associated cells were dependent on the therapeutic agents and the TME-targeting therapy improved the survival of the GBM bearing animals.The current GBM therapies should be revisited to add agents to prevent the accumulation of bone marrow-derived cells in the TME or to prevent the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence. Instead of concurrent therapy, a sequential strategy would be best to target TME-associated cells.

Inflammation occurs in all tissues in response to injury or stress and is the key process underlying hepatic fibrogenesis. Targeting chronic and uncontrolled inflammation is one strategy to prevent liver injury and fibrosis progression. Here, we demonstrate that triggering receptor expressed on myeloid cells 1 (TREM-1), an amplifier of inflammation, promotes liver disease by intensifying hepatic inflammation and fibrosis. In the liver, TREM-1 expression was limited to liver macrophages and monocytes and was highly upregulated on Kupffer cells, circulating monocytes, and monocyte-derived macrophages in a mouse model of chronic liver injury and fibrosis induced by carbon tetrachloride (CCl4) administration. TREM-1 signaling promoted proinflammatory cytokine production and mobilization of inflammatory cells to the site of injury. Deletion of Trem1 reduced liver injury, inflammatory cell infiltration, and fibrogenesis. Reconstitution of Trem1-deficient mice with Trem1-sufficient Kupffer cells restored the recruitment of inflammatory monocytes and the severity of liver injury. Markedly increased infiltration of liver fibrotic areas with TREM-1–positive Kupffer cells and monocytes/macrophages was found in patients with hepatic fibrosis. Our data support a role of TREM-1 in liver injury and hepatic fibrogenesis and suggest that TREM-1 is a master regulator of Kupffer cell activation, which escalates chronic liver inflammatory responses, activates hepatic stellate cells, and reveals a mechanism of promotion of liver fibrosis.

BACKGROUND: Glioblastoma (GBM) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). We identified highly upregulated interleukin 8 (IL-8)-CXCR2 axis in tumor cells in high-grade human glioma and AAT-treated orthotopic GBM tumors. METHODS: Human GBM tissue sections and tissue array were used to ascertain the clinical relevance of CXCR2-positive tumor cells in the formation of VM. We utilized U251 and U87 human tumor cells to understand VM in an orthotopic GBM model and AAT-mediated enhancement in VM was modeled using vatalanib (anti-VEGFR2) and avastin (anti-VEGF). Later, VM was inhibited by SB225002 (CXCR2 inhibitor) in a preclinical study. RESULTS: Overexpression of IL8 and CXCR2 in human datasets and histological analysis was identified as a bonafide candidate to validate VM through in vitro and animal model studies. AAT-treated tumors displayed a higher number of CXCR2-positive GBM-stem cells with endothelial-like phenotypes. Stable knockdown of CXCR2 expression in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. Similar data were obtained following SB225002 treatment. CONCLUSIONS: The present study suggests that tumor cell autonomous IL-8-CXCR2 pathway is instrumental in AAT-mediated resistance and VM formation in GBM. Therefore, CXCR2 can be targeted through SB225002 and can be combined with standard therapies to improve the therapeutic outcomes in clinical trials.

Intracerebral hemorrhage is a devastating neurological injury that produces poor patient outcomes. In this report, Vaibhav et al. demonstrate that remote ischemic post-conditioning noninvasively accelerates hematoma resolution by enhancing AMPK-dependent alternative macrophage activation., Spontaneous intracerebral hemorrhage (ICH) produces the highest acute mortality and worst outcomes of all stroke subtypes. Hematoma volume is an independent determinant of ICH patient outcomes, making clot resolution a primary goal of clinical management. Herein, remote-limb ischemic post-conditioning (RIC), the repetitive inflation–deflation of a blood pressure cuff on a limb, accelerated hematoma resolution and improved neurological outcomes after ICH in mice. Parabiosis studies revealed RIC accelerated clot resolution via a humoral-mediated mechanism. Whereas RIC increased anti-inflammatory macrophage activation, myeloid cell depletion eliminated the beneficial effects of RIC after ICH. Myeloid-specific inactivation of the metabolic regulator, AMPKα1, attenuated RIC-induced anti-inflammatory macrophage polarization and delayed hematoma resolution, providing a molecular link between RIC and immune activation. Finally, chimera studies implicated myeloid CD36 expression in RIC-mediated neurological recovery after ICH. Thus, RIC, a clinically well-tolerated therapy, noninvasively modulates innate immune responses to improve ICH outcomes. Moreover, immunometabolic changes may provide pharmacodynamic blood biomarkers to clinically monitor the therapeutic efficacy of RIC., Graphical Abstract

Inflammation is an important mediator of secondary neurological injury after traumatic brain injury (TBI). Endocannabinoids, endogenously produced arachidonate based lipids, have recently emerged as powerful anti-inflammatory compounds, yet the molecular and cellular mechanisms underlying these effects are poorly defined. Endocannabinoids are physiological ligands for two known cannabinoid receptors, CB1R and CB2R. In the present study, we hypothesized that selective activation of CB2R attenuates neuroinflammation and reduces neurovascular injury after TBI. Using a murine controlled cortical impact (CCI) model of TBI, we observed a dramatic upregulation of CB2R within infiltrating myeloid cells beginning at 72 h. Administration of the selective CB2R agonist, GP1a (1–5 mg/kg), attenuated pro-inflammatory M1 macrophage polarization, increased anti-inflammatory M2 polarization, reduced edema development, enhanced cerebral blood flow, and improved neurobehavioral outcomes after TBI. In contrast, the CB2R antagonist, AM630, worsened outcomes. Taken together, our findings support the development of selective CB2R agonists as a therapeutic strategy to improve TBI outcomes while avoiding the psychoactive effects of CB1R activation.

Myeloid-derived suppressor cells (MDSC) are a major obstacle to promising forms of cancer immunotherapy, but tools to broadly limit their immunoregulatory effects remain lacking. In this study, we assessed the therapeutic effect of the humanized anti–Jagged1/2-blocking antibody CTX014 on MDSC-mediated T-cell suppression in tumor-bearing mice. CTX014 decreased tumor growth, affected the accumulation and tolerogenic activity of MDSCs in tumors, and inhibited the expression of immunosuppressive factors arginase I and iNOS. Consequently, anti-Jagged therapy overcame tumor-induced T-cell tolerance, increased the infiltration of reactive CD8+ T cells into tumors, and enhanced the efficacy of T-cell–based immunotherapy. Depletion of MDSC-like cells restored tumor growth in mice treated with anti-Jagged, whereas coinjection of MDSC-like cells from anti–Jagged-treated mice with cancer cells delayed tumor growth. Jagged1/2 was induced in MDSCs by tumor-derived factors via NFkB-p65 signaling, and conditional deletion of NFkB-p65 blocked MDSC function. Collectively, our results offer a preclinical proof of concept for the use of anti-Jagged1/2 to reprogram MDSC-mediated T-cell suppression in tumors, with implications to broadly improve the efficacy of cancer therapy. Cancer Res; 77(20); 5628–38. ©2017 AACR.

Glioblastoma (GBM) is a hypervascular neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT therapy resistance. Alternative neovascularization mechanisms were shown to be at work in these resilient tumors to counter the AAT therapy insult. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor in the event of the ensuing hypoxia resulting from AAT. In our previous studies, we have demonstrated that AAT accelerates VM in GBM. In this review, we will focus on the origins of VM, visualizing VM in AAT-treated tumors and the development of VM as a resistance mechanism to AAT.

// Liyong Zhang 1 , Yan Wang 1 , Mohammad H. Rashid 1 , Min Liu 2 , Kartik Angara 1 , Nahid F. Mivechi 1 , Nita J. Maihle 1, 3 , Ali S. Arbab 1 and Lan Ko 1, 4 1 Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA, USA 2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA 3 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA 4 Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA, USA Correspondence to: Lan Ko, email: LKO@augusta.edu Keywords: angiogenesis, pericytes, oral cancer, glioblastoma Received: October 21, 2016 Accepted: May 01, 2017 Published: May 25, 2017 ABSTRACT Angiogenesis promotes tumor development. Understanding the crucial factors regulating tumor angiogenesis may reveal new therapeutic targets. Human GT198 ( PSMC3IP or Hop2) is an oncoprotein encoded by a DNA repair gene that is overexpressed in tumor stromal vasculature to stimulate the expression of angiogenic factors. Here we show that pericytes expressing GT198 give rise to tumor cells through angiogenesis. GT198 + pericytes and perivascular cells are commonly present in the stromal compartment of various human solid tumors and rodent xenograft tumor models. In human oral cancer, GT198 + pericytes proliferate into GT198 + tumor cells, which migrate into lymph nodes. Increased GT198 expression is associated with increased lymph node metastasis and decreased progression-free survival in oral cancer patients. In rat brain U-251 glioblastoma xenografts, GT198 + pericytes of human tumor origin encase endothelial cells of rat origin to form mosaic angiogenic blood vessels, and differentiate into pericyte-derived tumor cells. The net effect is continued production of glioblastoma tumor cells from malignant pericytes via angiogenesis. In addition, activation of GT198 induces the expression of VEGF and promotes tube formation in cultured U251 cells. Furthermore, vaccination using GT198 protein as an antigen in mouse xenograft of GL261 glioma delayed tumor growth and prolonged mouse survival. Together, these findings suggest that GT198-expressing malignant pericytes can give rise to tumor cells through angiogenesis, and serve as a potential source of cells for distant metastasis. Hence, the oncoprotein GT198 has the potential to be a new target in anti-angiogenic therapies in human cancer.

20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N-(20-hydroxyeicosa-6( Z), 15( Z)-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4, Sox2, and Nanog, which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.

Leiomyosarcoma is the most frequent subtype of the deadly uterine sarcoma and shares many common clinical grounds with leiomyoma, which is in turn the most common solid benign uterine neoplasm. With the recent progress in minimally invasive techniques for managing leiomyomas, accurate preoperative diagnosis of uterine masses has become the most important selection criterion for the safest therapeutic option. Therefore, different imaging modalities would be playing a key role in management of uterine masses. Testing for a sarcoma-specific promoter that expresses its downstream reporter gene only in leiomyosarcoma and not in leiomyoma or healthy uterine tissue. Adenoviral vectors were utilized both in vitro and in vivo to test the specificity of the promoters. Quantitative studies of downstream gene expression of these promoters was carried out both in vitro and in vivo. Our data indicated that human leiomyosarcoma cells highly expressed the reporter gene downstream to survivin promoter (Ad-SUR-LUC) when compared with benign leiomyoma or normal cells (p value of 0.05). Our study suggested that survivin is the unique promoter capable of distinguishing between the deadly sarcoma and the benign counterparts.

Exosomes are critical mediators of intercellular crosstalk and regulator of cellular/tumor microenvironment. Exosomes have great prospects for clinical application as theranostic and prognostic probe. Nevertheless, the advancement of the exosomes research has been thwarted by limited knowledge elucidating the most efficient isolation method and theirin vivotrafficking. Here we have showed that combination of two size-based methods using 0.20 µm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation method yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of differentialin vivodistribution of radioisotope131I-labelled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments (HET0016 and GW2580), myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the protein/cytokine contents of the exosomes. The appliedin vivoimaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.AbbreviationsbFGFbasic fibroblast growth factorCSF1Rcolony stimulating factor 1 receptorCTcomputed tomographyCTLA4cytotoxic T-lymphocyte-associated protein 4EGFepidermal growth factorEMTepithelial to mesenchymal transitionEVsextracellular vesiclesEPCsendothelial progenitor cellsFasLFas ligandG-CSFgranulocyte-colony stimulating factorGM-CSFgranulocyte-macrophage colony-stimulating factorHGFhepatocyte growth factorHSPheat shock proteinICAM-1intercellular adhesion molecule 1IFN-gammainterferon gammaIL – 1betainterleukin-1 betaIL – 1rainterleukin-1 receptor antagonistIL – 2interleukin-2IL – 4interleukin-4IL – 6interleukin-6IL – 7interleukin-7IL – 10interleukin-10IL – 12interleukin-12IL – 13interleukin-13IL – 17interleukin-17KCkeratinocyte-derived chemokineLIXlipopolysaccharide-induced CXC chemokineM-CSFmacrophage colony-stimulating factorMCP-1monocyte chemoattractant protein 1MDCmacrophage-derived chemokineMDSCsmyeloid derived suppressor cellsMFPmammary fat padMIP-1αmacrophage-inflammatory protein-1alphaMMP-2matrix metalloproteinase-2MRImagnetic resonance imagingNISsodium iodide symporterNTAnanoparticle tracking analysisPETpositron emission tomographyPF-4platelet factor 4RANTESregulated on activation, normal T cell expressed and secretedROIsregion of interestSDF-1αstromal cell-derived factor-1SEMstandard error of the meanSPECTsingle-photon emission computed tomographySCFstem cell factorTAMstumor-associated macrophagesTEMtransmission electron microscopyTIMP 2tissue inhibitors of metalloproteinases 2TLPCthin layer paper chromatographyTMEtumor microenvironmentTNF-αtumor necrosis factor-αTSLPthymic stromal lymphopoietinUCultracentrifugationVEGF-Avascular endothelial growth factor AVEGFR2vascular endothelial growth factor receptor 2.