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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.

Diabetes doubles the risk of vascular cognitive impairment and dementia (VCID), a disease is associated with small vessel disease (SVD) of the brain. Given that diabetes mediates early cerebrovascular dysfunction, and microemboli are common in the cerebral circulation and can penetrate into the brain parenchymal arterioles without causing an infarction, we tested the hypothesis that entrapment of microemboli in dysfunctional vessel walls accelerates the development of SVD ultimately resulting in VCID in diabetes. Diabetes was induced by a high fat diet and low dose streptozotocin (35 mg/kg, IP) injection in male Wistar rats. Cholesterol crystal microemboli [40-70 μm, 3000 (Group A) or 6000 (Group B) per 200 μl saline] were injected 8 weeks later through internal carotid artery. Cognitive function was monitored by the novel object recognition (NOR) test. White matter injury was assessed by Luxol fast blue (LFB) and hematoxylin/eosin (HE) staining in Group A. Cerebral blood flow (CBF), white matter hyperintensities (T2 map), microbleeds (T2 asterisk), and microinfarcts (T2 W) were assessed by MRI in Group B. In Group A, there was increased white matter degeneration in diabetic animals compared to controls. This degeneration was aggravated by microembolic injection. We found that cognitive function was exacerbated in Group B diabetes animals. MRI results showed these animals also had decreased CBF but no change on microinfarcts or microbleed. These results suggest that white matter degeneration starts in the early stages of diabetes, and that microemboli exacerbate these pathological changes even before the appearance of any neurobehavioral deficits. Additional studies are needed to examine the precise time course for development of cerebral neurovascular changes relative to appearance of behavioral deficits and explore potential therapeutic targets for VCI in diabetes.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2−/− mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression., Dissemination of tumor cells from the primary site is an early event. Here, the authors show that the early disseminated tumor cells are actively cleared by the host cytotoxic T lymphocytes induced by the primary tumor and that infiltration of granulocytic myeloid-derived suppressor cells counteracts such immune protection and allow metastasis development.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope (131)I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

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 resistance and recurrence. Previous investigations showed that there were changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and rethinking is needed how to treat GBM. It is hypothesized 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 better to target TME-associated cells.

Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer.

Glioblastoma (GBM) is a hypervascular and malignant form of brain tumors. Anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in clinical and preclinical studies, which resulted into marked hypoxia and recruited bone marrow derived cells (BMDCs) to the tumor microenvironment (TME). In vivo animal models to track BMDCs and investigate molecular mechanisms in AAT resistance are rare. We exploited recently established chimeric mouse to develop orthotopic U251 tumor, which uses as low as 5 × 10(6) GFP+ BM cells in athymic nude mice and engrafted >70% GFP+ cells within 14 days. Our unpublished data and published studies have indicated the involvement of immunosuppressive myeloid cells in therapeutic resistance in glioma. Similarly, in the present study, vatalanib significantly increased CD68+ myeloid cells, and CD133+, CD34+ and Tie2+ endothelial cell signatures. Therefore, we tested inhibition of CSF1R+ myeloid cells using GW2580 that reduced tumor growth by decreasing myeloid (Gr1+ CD11b+ and F4/80+) and angiogenic (CD202b+ and VEGFR2+) cell signatures in TME. CSF1R blockade significantly decreased inflammatory, proangiogenic and immunosuppressive molecular signatures compared to vehicle, vatalanib or combination. TCK1 or CXCL7, a potent chemoattractant and activator of neutrophils, was observed as most significantly decreased cytokine in CSF1R blockade. ERK MAPK pathway was involved in cytokine network regulation. In conclusion, present study confirmed the contribution of myeloid cells in GBM development and therapeutic resistance using chimeric mouse model. We identified novel molecular networks including CXCL7 chemokine as a promising target for future studies. Nonetheless, survival studies are required to assess the beneficial effect of CSF1R blockade.

Recent evidence suggests that myeloid cells are critical in cancer development and therapy resistance processes. Pharmacological targeting of tumor-associated myeloid cells is an emerging approach among upcoming immune therapies. Surprisingly, myeloid cells are heterogeneous, including a subset of the myeloid cell displaying angiogenic properties in solid tumors. There is an urgent need to delineate angiogenic myeloid cell populations in order to facilitate specific targeting of protumor myeloid cells among heterogeneous pool. This review article is intended to compile all the relevant information in the literature for improved understanding of angiogenic myeloid cells and their role in tumor refractoriness to cancer therapy.

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., Our understanding on how CAFs can be activated to support tumour progression is still limited. Here, the authors demonstrate that adenosine produced in the tumour microenvironment can enhance the expression of CD73 in CAFs ultimately driving CD8 T-Cell suppression and tumour growth.

Intracerebral hemorrhage (ICH) is a fatal stroke subtype with significant public health impact. Although neuroinflammation is a leading cause of neurological deficits after ICH, no imaging tool is currently available to monitor brain inflammation in ICH patients. Given the role of TSPO in neuroinflammation, herein we investigate whether a second-generation TSPO ligand, [125 I]IodoDPA-713 can be used to monitor the changes in TSPO expression in a preclinical model of intracerebral hemorrhage. Male CD1 mice were subjected to ICH/Sham. The brain sections, collected at different time points were incubated with [125 I]IodoDPA-713 and the brain uptake of [125 I]IodoDPA-713 was estimated using autoradiography. The specificity of [125 I]IodoDPA-713 binding was confirmed by a competitive displacement study with an unlabeled TSPO ligand, PK11195. [125 I]IodoDPA-713 binding was higher in the ipsilateral striatum with an enhanced binding observed in the peri-hematomal brain region after ICH, whereas the brain sections from sham as well as contralateral brain areas of ICH exhibited marginal binding of [125 I]IodoDPA-713. PK11195 completely reversed the [125 I] IodoDPA-713 binding to brain sections suggesting a specific TSPO-dependent binding of [125 I]IodoDPA-713 after ICH. This was further confirmed with immunohistochemistry analysis of adjacent sections, which revealed a remarkable expression of TSPO in the areas of high [125 I]IodoDPA-713 binding after ICH. The specific as well as enhanced binding of [125 I]IodoDPA-713 to the ipsilateral brain areas after ICH as assessed by autoradiography analysis provides a strong rationale for testing the applicability of [125 I]IodoDPA-713 for non-invasive neuroimaging in preclinical models of ICH.

The glucocorticoid-induced leucine zipper (GILZ) mediates anti-inflammatory effects of glucocorticoids. Acute kidney injury (AKI) mobilizes immune/inflammatory mechanisms, causing tissue injury, but GILZ9s impact in AKI is not known. Neutrophils play context-specific pro- (N1) and anti-inflammatory (N2) functional roles. Also, regulatory T lymphocytes (Tregs) and regulatory T-17 (Treg17) cells exert counter-inflammatory effects including suppression of effector T lymphocytes, e.g., Th-17 cells. Thus, utilizing cell preparations of mice kidneys subjected to AKI or sham operation, we determined the effects of GILZ on T cells and neutrophil subtypes in the context of its renoprotective effect; these studies utilized the trans activator of transcription (TAT)-GILZ or the TAT peptide. AKI increased N1 and Th-17 cells but reduced N2, Tregs and Treg17 cells in association with increased interleukin (IL)-17+ but reduced IL-10+ cells accompanied with disruption of mitochondrial membrane potential (ψm) and increased apoptosis/necrosis compared to sham kidneys. TAT-GILZ, compared to TAT, treatment reduced N1 and Th-17 cells but increased N2 and Tregs, without affecting Treg17 cells, in association with reduction in IL-17+ but increased IL-10+ cells; TAT-GILZ caused less disruption of ψm and reduced cell death in AKI. Importantly, TAT-GILZ increased perfusion of the ischemic-reperfused kidney but reduced tissue edema compared to TAT. Utilizing splenic T cells and bone marrow-derived neutrophils, we further showed marked reduction in proliferation of Th cells in response to TAT-GILZ than TAT. Collectively, the results indicate that GILZ exerts renoprotection accompanied with upregulation of regulatory/suppressive arm of immunity in AKI likely via regulating crosstalk between T cells and neutrophils.

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.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.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.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.

Growing evidence suggests that renin angiotensin system (RAS) modulators support cognitive function in various animal models. However, little is known about their long-term effects on the brain structure in aged hypertensive animals with chronic cerebral hypoperfusion as well as which specific domains of cognition are most affected. Therefore, in the current study we examined the effects of Candesartan and Compound 21 (C21) (RAS modulators) on aspects of cognition known to diminish with advanced age and accelerate with hypertension and vascular disease. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline and after 4 and 8 weeks of chronic hypoxic hypoperfusion and treatment. Magnetic resonance imaging (MRI) was performed at the end of the 8 week study period followed by animal sacrifice and tissue collection. Both Candesartan and C21 effectively preserved cognitive function and prevented progression of vascular cognitive impairment (VCI) but only candesartan prevented loss of brain volume in aged hypertensive animals. Collectively, our findings demonstrate that delayed administration of RAS modulators effectively preserve cognitive function and prevent the development / progression of VCI in aged hypertensive animals with chronic cerebral hypoperfusion.

Over 700 drugs have failed in stroke clinical trials, an unprecedented rate thought to be attributed in part to limited and isolated testing often solely in "young" rodent models and focusing on a single secondary injury mechanism. Here, extracellular vesicles (EVs), nanometer-sized cell signaling particles, were tested in a mouse thromboembolic (TE) stroke model. Neural stem cell (NSC) and mesenchymal stem cell (MSC) EVs derived from the same pluripotent stem cell (PSC) line were evaluated for changes in infarct volume as well as sensorimotor function. NSC EVs improved cellular, tissue, and functional outcomes in middle-aged rodents, whereas MSC EVs were less effective. Acute differences in lesion volume following NSC EV treatment were corroborated by MRI in 18-month-old aged rodents. NSC EV treatment has a positive effect on motor function in the aged rodent as indicated by beam walk, instances of foot faults, and strength evaluated by hanging wire test. Increased time with a novel object also indicated that NSC EVs improved episodic memory formation in the rodent. The therapeutic effect of NSC EVs appears to be mediated by altering the systemic immune response. These data strongly support further preclinical development of a NSC EV-based stroke therapy and warrant their testing in combination with FDA-approved stroke therapies.

The measurement of extracellular pH (pHe) has significant clinical value for pathological diagnoses and for monitoring the effects of pH-altering therapies. One of the major problems of measuring pHe with a relaxation-based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH-unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation-based dendritic MRI contrast agent in a concentration-independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA-4AmP)44-G5, a G5–PAMAM dendrimer-based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r1) increased from 7.91 to 9.65 mM−1 s−1 (on a per Gd3+ basis) on changing pH from 8.84 to 6.35. At 7 T, r1 relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r2) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA-4AmP)44-G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R2/R1 also showed a linear relationship in a pH-responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA-4AmP)44-G5 agent. Importantly, the nanoprobe (GdDOTA-4AmP)44-G5 shows pH response in the range commonly found in the microenvironment of solid tumors. Copyright © 2015 John Wiley & Sons, Ltd.

The metastasis occurrence, an important prognostic factor, depends on peculiarities such as cellular invasiveness and cell migration, mechanisms controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). An increased expression of this protein promotes tumor growth and metastasis, a mechanism which can be restricted by the use of the effector's inhibitors. Melatonin, a hormone secreted by the pineal gland, has shown oncostatic action and anti-metastatic effects and can modulate the ROCK-1expression. The objective of this study was to investigate the anti-metastatic response mediated by ROCK-1 and through the action of melatonin and its specific inhibitor (Y27632) in vitro and in vivobreast cancer models. Cells from metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer lines were treated with melatonin and Y27632. Cell viability was verified by MTT assay, cell migration/invasion assays in Boyden chamber, ROCK-1 protein and gene expression by western blot and quantitative real time PCR, respectively. In addition, the in vivometastasis study was performed using female athymic nude mice induced by injection of 2x105 MDA-MB-231 viable cells by tail veinfor lung metastasis and byintracardiac for bone metastasis, during 3 weeks. The animals were treated with melatonin and Y27632 for 2 weeks. The metastasis developments were evaluated by single photon emission computed tomography (SPECT). Treatment with melatonin reduced cell viability and migration of both cell lines (p0.05). An statistically significant reduction of ROCK-1 gene expression was observed in all treatment groups (p Support: FAPESP. Citation Format: Debora C Zuccari, Thaiz F Borin, Ali S Arbab, Lívia C Ferreira, Marina G Moschetta, Gustavo R Martins, Larissa B Maschio, Vanessa A Fabri, Verena B Coimbra. Melatonin's inhibitory effect on breast cancer metastasis mediated by ROCK-1 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-20.

Tumor development and therapeutic resistance are linked with tumor-associated macrophage (TAM) and myeloid-derived suppressor cell (MDSC) infiltration in tumors via chemokine axis. Chemokine expression, which determines the pro or anti-inflammatory status of myeloid cells, are partly regulated by the nuclear factor-kappa B (NF-κB) pathway. Here, we identified that conditional deletion of canonical NF-κB signaling (p65) in myeloid cells inhibited syngeneic glioblastoma (GBM) through decreased CD45 infiltration in tumors, as characterized by decreased TAMs (CD206+) and MDSCs (Gr1+ CD11b+), increased dendritic cells (CD86+) and cytotoxic T cells (CD8+) in the p65 knockout (KO) mice. Proinflammatory cytokines (IFNγ, MCP1, MIP1α, and TNFα) and myeloid differentiation factor (Endoglin) were increased in myeloid cells from p65 KO tumor, which demonstrated an influence on CD8+T cell proliferation. In contrast, p65KO athymic chimeric mice with human GBM, failed to inhibit tumor growth, confirming the contribution of T cells in an immune competent model. The analysis of human datasets and GBM tumors revealed higher expression of p65 in GBM-associated CD68+ macrophages compared to neighboring stroma. Thus, canonical NF-κB signaling has an anti-inflammatory role and is required for macrophage polarization, immune suppression, and GBM growth. Combining an NF-κB inhibitor with standard therapy could improve antitumor immunity in GBM.

The WASF3 gene facilitates the metastatic phenotype, and its inactivation leads to suppression of invasion and metastasis regardless of the genetic background of the cancer cell. This reliance on WASF3 to facilitate metastasis suggests that targeting its function could serve as an effective strategy to suppress metastasis. WASF3 stability and function are regulated by the WASF Regulatory Complex (WRC) of proteins, particularly CYFIP1 and NCKAP1. Knockdown of these proteins in vitro leads to disruption of the WRC and suppression of invasion. We have used mouse xenograft models of breast cancer metastasis to assess whether targeting the WRC complex suppresses metastasis in vivo. Stapled peptides targeting the WASF3-CYFIP1 interface (WAHM1) and the CYFIP1-NCKAP1 interface (WANT3) suppress the development of lung and liver metastases. Targeting these critical protein-protein interactions, therefore, could potentially be developed into a therapeutic strategy to control cancer cell invasion and metastasis.

Distant metastasis is the primary cause of death in the majority of the cancer types. Recently, much importance has been given to tumor microenvironment (TME) in the development of invasive malignant tumors, as well as the metastasis potential. The ability of tumor cells to modulate TME and to escape immune-mediated attack by releasing immunosuppressive cytokines has become a hallmark of breast cancer. Our study shows the effect of IV formulation of HET0016 (HPßCD-HET0016) a selective inhibitor of 20-HETE synthesis, administered intravenously in immune-competent in vivo mouse model of murine breast cancer. 4T1 luciferase positive cells were implanted to the mammary fat pad in Balb/c mice. Treatment started on day 15, and was administered for 5 days a week for 3 weeks. The development of metastasis was detected via optical imaging. Blood, spleen, lungs, bone marrow and tumor were collected for flow cytometry, to investigate changes in myeloid-derived suppressive cells (MDSCs) populations and endothelial phenotype. Tumor and lungs were collected for protein analysis. Our results show that HPßCD-HET0016: (1) decreased tumor volume and lung metastasis compared to the vehicle group; (2) reduced migration and invasion of tumor cells and levels of metalloproteinases in the lungs of animals treated with HPßCD-HET0016 via PI3K/AKT pathway; and (3) decreased expression of pro-inflammatory cytokines, growth factors and granulocytic MDSCs population in the lung microenvironment in treated animals. Thus, HPßCD-HET0016 showed potential in treating lung metastasis in a preclinical mouse model and needs further investigations on TME.

It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced ‘metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression., Myeloid-derived suppressive cells (MDSCs) promote metastasis. Here, the authors show that the monocytic MDSCs subset promotes epithelial to mesenchymal transition at the primary site while the granulocytic subset promotes the reverse transition at the metastatic site enabling dynamic tumour cells plasticity.

We report a continuous nanoscale encapsulation of cancer drugs 5-Fluorouracil (FU) and Paclitaxel into biocompatible polycaprolactone (PCL) nanofibers (NFs) using core-sheath electrospinning process. A high potential electric field of 19-23.2 kV was used to draw a compound solution jet from a specialized coaxial spinneret. Using of DMF in both core and Sheath resulted in NFs within 50-160 nm along with large beaded structures. Addition of Trichloromethane (TCM) or Trifluoroethanol (TFE) in sheath turned NFs in more uniform and thin fiber structure. The diameter range for paclitaxel encapsulated fibers was 22-90 nm with encapsulation efficiency of 77.5% and the amount of drug was only 4 to 5% of sheath polymer. Addition of PVA within core resulted drug nanocrystal formation outside of sheath and poor encapsulation efficiency (52%) with rapid initial release (52-53%) in first 3 days. Drug release test of NFs in different pH exhibited increase of release rate with the decrease of media pH. In-vitro cell viability test with FU encapsulated NFs in human prostatic cancer PC3 cells exhibited 38% alive cells at 5 μM concentration while in pristine FU 43% cells were alive. Paclitaxel encapsulated NFs with breast cancer cells also exhibited increased efficacy in comparison to pristine anticancer drugs. Continuous decrease of cell density indicated the slow release of cancer drugs from the NFs. Both PCL+Paclitaxel and PCL+5FU treated conditions caused breast cancer cell death between 40% to 50%.

Myeloid-derived suppressor cells (MDSCs) are immune regulatory myeloid cells, which have emerged as critical regulators of tumor progression and therapy resistance through promoting immune suppression and contributing to tumor vasculature development mechanisms (1-6). Human and mice MDSCs differ in surface, cytoplasmic and nuclear markers (2,7,8). MDSCs characterization and functions have been updated time-to-time (9). After decades of MDSC discovery, we have gained ample knowledge that how MDSC works to promote cancer associated immune suppression. However, in the translational era, scientist started exploiting old knowledge to develop next generation therapeutic modalities. In the same line of thought, Apolloni et al . developed a MDSC cell line using mouse MDSCs (CD11b + /Gr-1 + ) isolated from the spleens of immunosuppressed mice (10). MDSCs were immortalized using a retrovirus encoding the v- myc and v- raf oncogenes, which expressed monocyte/macrophage markers (10). Establishing MDSC cell line has opened a new door for its tremendous applications in cancer therapy.

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p

Dynamic contrast-enhanced T1-weighted magnetic resonance imaging (DCE-MRI) is being increasingly used in various clinical trials involving brain tumors. It allows characterization of the vascular microenvironment in tumors by measurement of a range of parameters, such as Ktrans (forward transfer constant), kep (reverse transfer constant), ve (volume of the extravascular extracellular space), and vp (blood plasma volume) (1,2). These parameters reflect specific physiologic characteristics and hence relate to various aspects of tumor biology. One of the hurdles in obtaining these quantitative metrics is a lack of robust methods for approaching the problem of parametric estimation using multicompartmental pharmacokinetic models (3). Another issue is that current Food and Drug Administration (FDA)-approved extravascular contrast agents (CAs) used for the assessment of vascular parameters in solid tumors are relatively small (molecular weight

There is no available targeted therapy or imaging agent for triple negative breast cancer (TNBC). We developed a small-sized dendrimer-based nanoparticle containing a clinical relevant MRI contrast agent, GdDOTA and a NIR fluorescent dye, DL680. Systemic delivery of dual-modal nanoparticles led to accumulation of the agents in a flank mouse model of TNBC that were detected by both optical and MR imaging. In-vivo fluorescence images, as well as ex-vivo fluorescence images of individual organs, demonstrated that nanoparticles accumulated into tumor selectively. A dual modal strategy resulted in a selective delivery of a small-sized (GdDOTA)42-G4-DL680 dendrimeric agent to TNBC tumors, avoiding other major organs.

Glioblastoma (GBM) is one hypervascular and hypoxic tumor known among solid tumors. Antiangiogenic therapeutics (AATs) have been tested as an adjuvant to normalize blood vessels and control abnormal vasculature. Evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance to AATs. Here, we identified that GBM following AAT (Vatalanib) acquired an alternate mechanism to support tumor growth, called vascular mimicry (VM). We observed that Vatalanib induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer-side. The PAS+ matrix is positive for basal laminae (laminin) indicating vascular structures. Vatalanib treated GBM displayed various stages of VM such as initiation (mosaic), sustenance, and full-blown VM. Mature VM structures contain red blood cells (RBC) and bear semblance to the functional blood vessel-like structures, which provide all growth factors to favor tumor growth. Vatalanib treatment significantly increased VM especially in the core of the tumor, where HIF-1α was highly expressed in tumor cells. VM vessels correlate with hypoxia and are characterized by co-localized MHC-1+ tumor and HIF-1α expression. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at periphery of the tumors. In summary, AAT induced resistance characterized by VM is an alternative mechanism adopted by tumors to make functional vessels by transdifferentiation of tumor cells into endothelial-like cells to supply nutrients in the event of hypoxia. AAT induced VM is a potential therapeutic target of the novel formulation of HET0016. Our present study suggests that HET0016 has a potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.

There has been a trend of use of conventional or non-conventional therapies against cancer such as targeting “king pin” tumor cells itself, targeting tumor vasculature lined by endothelial cells, targeting tumor associated macrophages and recently, targeting inhibitory signals on T cells through immunotherapies in tumor microenvironment (TME). However, most of the cases have witnessed therapy resistance flowing a short-term or transient benefit. Despite of several failures in clinical trials targeting tumors and their microenvironments, our understanding is improving every day. It is evident that mutations in tumor cell compartment play a critical role in cancer development as well as therapy resistance. Here, we have discussed studies representing therapy resistance, through p53 as a model mutation and glioblastoma (GBM) as a model tumor.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100 k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

Tumor initiation and evolution is driven by the reciprocal actions between stromal and immune cells within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) specifically M2-macrophages, a critical component of TME; participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their prevalence in most of the human tumor tissue and correlation of their higher infiltration with lower survival, M2-macrophages represent promising targets for diagnosis, prognosis and anticancer therapy. Hence, early in vivo detection and intervention of M2-macrophages in the TME may boost the clinical outcome. Exosomes are biological nanoparticles sizing 30-150 nm, recently drawing huge attention for their potential application as therapeutic and diagnostic tool because they are more biocompatible and biodegradable with lesser toxicity than other synthetic nanoparticles and can easily percolate through the body’s barrier systems or through abnormally formed blood vessels in tumor tissue. We have generated engineered exosomes from HEK293 cells by lentiviral transfection system that carry specific peptide sequence on their external surface for targeting CD206 positive M2-macrophages. To determine the in vivo distribution of M2-macrophages, we adopted 111In-oxine based radio-labeling of the targeted exosomes and single-photon emission computed tomography (SPECT). Up to the time, among the used techniques, nuclear imaging is the foremost method to trace exosomes in vivo for better tissue penetration, sensitivity and quantitative analysis. After the labeling with 111In-oxine, we analyzed the binding efficacy and serum stability of the targeted exosome by thin layer paper chromatography (TLPC) using 100% methanol and 2M Sodium acetate solution (1:1) as eluent. More than 98% of 111In-oxine was bound to exosomes and only 5% of free 111In-oxine was dissociated from the exosomes even 24 hours after the incubation with 20% serum. When we injected these radio-labeled targeted exosomes in to 4T1 breast tumor-bearing mice, they went mostly to the periphery of the primary tumor, metastatic area in the lungs, spleen and to the liver. We performed ex vivo quantification of radioactivity from individual organ by gamma counter after final SPECT scan and observed similar distribution of the radio-labeled targeted exosomes. We also labeled the exosomes with DiI dye and injected into the same mice followed by euthanasia after 3 hours and organ collection. Fluorescent imaging from the tumor and spleen showed the adherence of exosomes to the CD206 positive macrophages confirming the targeting efficacy of the exosomes. Henceforth, we intend to utilize these exosomes as a therapeutic probe for carrying chemotherapeutic or antibody to intervene the actions of M2-macrophages in primary and distal TME. Citation Format: Mohammad H. Rashid, Thaiz F. Borin, Roxan Ara, Kartik Angara, Achyut Bhagelu, Jingwen Cai, Yutao Liu, Ali S. Arbab. CD206 positive M2-macrophage targeting engineered exosomes as a potential diagnostic and therapeutic tool [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 1139.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. Using the syngeneic mouse models, we demonstrated that although both orthotopically-implanted murine 4T1 and EMT6 tumors are capable of disseminating into secondary organs, only 4T1 tumors develop overt metastasis. However, EMT6 tumors induce an anti-tumor immunity in syngeneic mice and that eradicates disseminated tumor cells (DTC) in distant organs. Following the complete removal of primary EMT6 tumors, mice do not develop detectable metastasis and generate an immunological memory that leads to complete elimination of repeatedly injected tumor cells via tail vein. Conversely, these cells readily grow and metastasize in immuno-deficient athymic or Rag2- mice, and when g-MDSCs from 4T1 tumor-bearing mice were co-injected into immunocompetent EMT6 primed mice. In contrast to complete resection, mice with residual tumors following surgery exhibited an enhanced growth of local and concomitant growth of DTCs at metastatic site with increased g-MDSCs accumulation in lung and spleen. Together, our results suggest that some tumors are capable of inducing anti-tumor immunity against the DTCs when complete resection of primary tumor cures animals. However, in the presence of residual tumors, inflammation induced by surgical procedure promote the growth of DTCs. Citation Format: Raziye Piranlioglu, Eunmi Lee, Maria Ouzuonova, Riley Rodier, Adam Greer, Feyzanur Bayraktar, Omer Can Durmus, Ali S. Arbab, Muthushamy Thangaraju, Max S. Wicha, Esteban Celis, Hasan Korkaya. Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model [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 4580.

Metastatic breast cancer (BC) and glioblastoma (GBM) are two highly invasive forms of cancer that contribute 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 invasion, growth, and neovascularization in tumors by inducing the proliferation and migration of both, the endothelial cells and the tumor cells, therefore promoting BC and GBM tumor survival. N-hydroxy-N’-(4-butyl-2 methyl phenyl) formamidine (HET0016) is a selective inhibitor of 20-HETE synthesis and thus impairs tumor growth and neovascularization. In this study, we investigate the functional and phenotypic changes in BC (MDAMB231 and MCF7) and GBM (U251 and U87) cells overexpressing CYP4A11, a human CYP4 enzyme, with and without HET0016 treatment. 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 and in vivo by ascertaining the Ki67 levels and incidence of lung metastases foci. Tumor growth with and without CYP4A11 overexpression was followed for 21 days in vivo in athymic nude mice for all groups of cells and the acquisition of stem cell and angiogenic phenotypes by the tumor cells were determined by flow cytometry. The results demonstrated that cells overexpressing CYP4A11 showed protein levels 3-6 times higher than the non-modified cells. CYP4A11-overexpressing MDAMB231 and U87 cells demonstrated increased proliferation capacity, motility and significantly increased tumor growth. Interestingly, MCF7 and U251 cells overexpressing CYP4A11 did not show significant alterations in tumor growth compared to non-modified cells. Additionally, all cells treated with HET0016 showed decreased migration after 24 hours. U87 tumors showed increased stem cell and angiogenic phenotypes compared to non-modified U87 tumors. In conclusion, overexpression of CYP4A11 in Triple Negative BC and U87 GBM cells increased tumor aggressiveness proving that this pathway is an important target for therapeutic inhibition to control tumor growth and invasion. Citation Format: Thaiz F. Borin, Sehar Ali, Kartik Angara, Mohammad Rashid, Stephanie Myers, Divya Chawla, Roxan Ara, Iryna Lebedyeva, Bhagelu R. Achyut, Ali S. Arbab. CYP4A11 overexpression increases aggressiveness in glioblastoma and breast cancer [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 127.

The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM.

Background We examined the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium.

In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.