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RAS/MAPK gene dysfunction underlies various cancers and neurocognitive disorders. Although the roles of RAS/MAPK genes have been well studied in cancer, less is known about their function during neurodevelopment. There are many genes that work in concert to regulate RAS/MAPK signaling, suggesting that if common brain phenotypes could be discovered they could have a broad impact on the many other disorders caused by distinct RAS/MAPK genes. We assessed the cellular and molecular consequences of hyperactivating the RAS/MAPK pathway using two distinct genes in a cell type previously implicated in RAS/MAPK-mediated cognitive changes, cortical GABAergic interneurons. We uncovered some GABAergic core programs that are commonly altered in each of the mutants. Notably, hyperactive RAS/MAPK mutants bias developing cortical interneurons towards those that are somatostatin positive. The increase in somatostatin-positive interneurons could also be prevented by pharmacological inhibition of the core RAS/MAPK signaling pathway. Overall, these findings present new insights into how different RAS/MAPK mutations can converge on GABAergic interneurons, which may be important for other RAS/MAPK genes and related disorders.

Recurrent mutations in the gene encoding the Speckle Type POZ Protein, SPOP, the substrate-binding components of E3-ubiquitin ligase, have been identified as common alterations in prostate cancer (PCa) and endometrial cancer (EC). SPOP mutations are associated with PCa and EC growth and progression. Here, we characterized SPOP mutations in PCa and EC, and their association with molecular and immune signatures. 7,398 PCa and 19,188 EC samples were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Real world overall survival (rwOS) was extracted from insurance claims and depicted using Kaplan-Meier survival curves for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U tests, with p-values adjusted for multiple comparisons (q Citation Format: Ludimila Cavalcante, Sachin K. Desmukh, Benedito A. Carneiro, Kartik Angara, Tyler E. Mattox, Sharon Wu, Joanne Xiu, Philip Walker, Matthew Oberley, Chadi Nabhan, Emmanuel S. Antonarakis. Opposing roles of SPOP mutations in human prostate and endometrial cancers [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 937.

Therapy resistance in glioblastoma (GBM), a hypervascular, hyperproliferative, and hypoxic neoplasia of the central nervous system with an extremely high mortality rate has always been an attractive topic of investigation. The transient benefits of adjuvant antiangiogenic therapies (AAT) in normalizing blood vessels, controlling abnormal vasculatures, and preventing recurrence coupled with higher rates of relapse are attributed to the AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms such as vascular mimicry (VM). An overactive IL-8-CXCR2 axis-driven VM contributed to AAT therapy resistance in preclinical models of GBM. Intervening the IL-8-CXCR2 axis with SB225002 (a CXCR2 antagonist) reduced (i) the GBM tumor burden, (ii) CXCR2 + and endothelial-like GBM subpopulations, and (iii) VM structures in the tumors. This chapter throws light on these novel findings and corroborates the therapeutic potential of a CXCR2 antagonist in combination with other antitumor agents in preclinical and clinical trials to reverse GBM therapy-induced resistance.

Glioblastoma (GBM) is the most prevalent malignant tumor in the central nervous system. Owing to its hypervascular state and inevitable recurrence following standard therapies of surgical resection, radiation, and chemotherapies, clinicians as well as scientists have instituted antiangiogenic agents to counter the development of blood vessels in the recurrent and rapidly growing GBM. However, investigations from our group and from other laboratories, as well as from clinical data, indicate the development of therapy resistance and activation of alternate neovascularization in GBM following antiangiogenic therapies (AATs). Alternate neovascularization occurs in three distinct pathways, such as angiogenesis, vasculogenesis, and vascular mimicry. Beside angiogenesis, different mechanisms are postulated for the development of alternative neovascularization and therapy resistance in GBM following AAT including bone marrow-derived cell-mediated vasculogenesis, transdifferentiation of glioma stem cells to form vascular mimicry, and vasculogenic as well as angiogenic myeloid cell-mediated neovascularization. Specific agents that target bone marrow-derived cells, myeloid cells, and transdifferentiation of glioma stem cells could be added to current therapeutic strategies in GBM management to overcome the resistance.

BackgroundEnvironmental insults that activate the maternal immune system are potent primers of developmental neuropathology and maternal immune activation (MIA) has emerged as a risk factor for neurodevelopmental and psychiatric disorders. Animal models of MIA provide an opportunity to identify molecular pathways that initiate disease processes and lead to neuropathology and behavioral deficits in offspring. MIA-induced behaviors are accompanied by anatomical and neurochemical alterations in adult offspring that parallel those seen in affected human populations.MethodsWe performed transcriptional profiling and neuroanatomical characterization in a time course across mouse embryonic cortical development, following MIA via single injection of the viral mimic polyinosinic:polycytidylic acid (polyI:C) at E12.5. Transcriptional changes identified in the cortex of MIA offspring at E17.5 were validated and mapped to cortical neuroanatomy and cell types via protein analysis and immunohistochemistry.ResultsMIA induced strong transcriptomic signatures, including induction of genes associated with hypoxia, immune signaling, and angiogenesis. The acute response identified 6h after the MIA insult was followed by changes in proliferation, neuronal and glial differentiation, and cortical lamination that emerged at E14.5 and peaked at E17.5. Decreased numbers of proliferative cell types in germinal zones and alterations in neuronal and glial cell types across cortical lamina were identified in the MIA-exposed cortex.ConclusionsMIA-induced transcriptomic signatures in fetal offspring overlap significantly with perturbations identified in neurodevelopmental disorders (NDDs), and provide novel insights into alterations in molecular and developmental timing processes linking MIA and neuropathology, potentially revealing new targets for development of novel approaches for earlier diagnosis and treatment of these disorders.

Tuberous Sclerosis Complex is a complex syndrome that affects multiple organs and is caused by dysfunction of either the TSC1 or TSC2 genes. One of the least understood features of TSC is the impact of TSC1&2 variants on brain phenotypes, including elevated rates of autism spectrum disorder and seizures. Moreover, while a great deal of work has uncovered how loss of either gene can alter various neural cell types, the impact of many variants in TSC and on these cell types is poorly understood. In particular, missense variants that cause minor changes in the proteins are expected to cause functional changes that differ from a complete loss of the protein. Herein, we examined how some missense variants in TSC1 impacted the development of cortical inhibitory interneurons, a cell type whose molecular, cellular and physiological properties are known to be altered after loss of mouse Tsc1. Importantly, we found that most missense variants complemented phenotypes caused by loss of Tsc1 and resulting in elevated MTOR activity as well as several cell intrinsic physiological properties. However, distinct variants showed deficits in complementing an increase in parvalbumin levels, which is observed after loss of Tsc1 and demonstrated smaller amplitudes of after hyperpolarizations. These data suggest subtle but sensitive phenotypes can be detected by some TSC1 missense variants and provide an in vivo system in which to better assess TSC variants.

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.

Neurofibromatosis 1 (NF1) is caused by mutations in the NF1 gene, which encodes the protein, neurofibromin, an inhibitor of Ras activity. Cortical GABAergic interneurons (CINs) are implicated in NF1 pathology, but the cellular and molecular changes to CINs are unknown. We deleted mouse Nf1 from the medial ganglionic eminence, which gives rise to both oligodendrocytes and CINs that express somatostatin and parvalbumin. Nf1 loss led to a persistence of immature oligodendrocytes that prevented later-generated oligodendrocytes from occupying the cortex. Moreover, molecular and cellular properties of parvalbumin (PV)-positive CINs were altered by the loss of Nf1, without changes in somatostatin (SST)-positive CINs. We discovered that loss of Nf1 results in a dose-dependent decrease in Lhx6 expression, the transcription factor necessary to establish SST(+) and PV(+) CINs, which was rescued by the MEK inhibitor SL327, revealing a mechanism whereby a neurofibromin/Ras/MEK pathway regulates a critical CIN developmental milestone.

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.

SummaryNeurofibromatosis-1 (NF-1) is a monogenic disorder caused by mutations in theNF1gene, which encodes the protein, Neurofibromin, an inhibitor of Ras GTPase activity. While NF-1 is often characterized by café-au-lait skin spots and benign tumors, the mechanisms underlying cognitive changes in NF-1 are poorly understood. Cortical GABAergic interneurons (CINs) are implicated in NF-1 pathology but cellular and molecular changes to CINs are poorly understood. We deletedNf1from the medial ganglionic eminence (MGE), which gives rise to both oligodendrocytes and CINs that express somatostatin and parvalbumin. Loss ofNf1led to a persistence of immature oligodendrocytes that prevented later born oligodendrocytes from occupying the cortex. Moreover, PV+ CINs were uniquely lost, without changes in SST+ CINs. We discovered that loss ofNf1results in a graded decrease inLhx6expression, the transcription factor necessary to establish SST+ and PV+ CINs, revealing a mechanism wherebyNf1regulates a critical CIN developmental milestone.

Medial ganglionic eminence (MGE)-derived somatostatin (SST)+ and parvalbumin (PV)+ cortical interneurons (CINs), have characteristic molecular, anatomical and physiological properties. However, mechanisms regulating their diversity remain poorly understood. Here, we show that conditional loss of the Tuberous Sclerosis (TS) gene, Tsc1, which inhibits mammalian target of rapamycin (MTOR), causes a subset of SST+ CINs, to express PV and adopt fast-spiking (FS) properties, characteristic of PV+ CINs. These changes also occur when only one allele of Tsc1 is deleted, making these findings relevant to individuals with TS. Notably, treatment with rapamycin, which inhibits MTOR, reverses these changes in adult mice. These data reveal novel functions of MTOR signaling in regulating PV expression and FS properties, which may contribute to some neuropsychiatric symptoms observed in TS. Moreover, they suggest that CINs can exhibit properties intermediate between those classically associated with PV+ or SST+ CINs, which may be dynamically regulated by the MTOR signaling.

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.

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 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.

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.

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.

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.

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.

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

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.

Antiangiogenic agents caused paradoxical increase in pro-growth and pro-angiogenic factors and caused tumor growth in glioblastoma (GBM). It is hypothesized that paradoxical increase in pro-angiogenic factors would mobilize Bone Marrow Derived Cells (BMDCs) to the treated tumor and cause refractory tumor growth. The purposes of the studies were to determine whether whole body irradiation (WBIR) or a CXCR4 antagonist (AMD3100) will potentiate the effect of vatalanib (a VEGFR2 tyrosine kinase inhibitor) and prevent the refractory growth of GBM. Human GBM were grown orthotopically in three groups of rats (control, pretreated with WBIR and AMD3100) and randomly selected for vehicle or vatalanib treatments for 2 weeks. Then all animals underwent Magnetic Resonance Imaging (MRI) followed by euthanasia and histochemical analysis. Tumor volume and different vascular parameters (plasma volume (vp), forward transfer constant (Ktrans), back flow constant (kep), extravascular extracellular space volume (ve) were determined from MRI. In control group, vatalanib treatment increased the tumor growth significantly compared to that of vehicle treatment but by preventing the mobilization of BMDCs and interaction of CXCR4-SDF-1 using WBIR and ADM3100, respectively, paradoxical growth of tumor was controlled. Pretreatment with WBIR or AMD3100 also decreased tumor cell migration, despite the fact that ADM3100 increased the accumulation of M1 and M2 macrophages in the tumors. Vatalanib also increased Ktrans and ve in control animals but both of the vascular parameters were decreased when the animals were pretreated with WBIR and AMD3100. In conclusion, depleting bone marrow cells or CXCR4 interaction can potentiate the effect of vatalanib.

Introduction: New theory of “seeds (cancer cells) and soil (distant tissue)” transpired as scientists acquainted exosomes as seeds that cruise to distant organs and release growth factors (fertilizers) to acclimate the “foreign soil” and prepare the metastatic niche. Exosomes are nano-sized (30-150nm) spherical organelles, derived from the endosomal system for intercellular communications. Tumor derived exosomes (TDEs) play an imperative role in tumor growth, stromal reorganization, cancer stemness, neovascularization, and metastatic niche formation by providing proteins, lipids, mRNA, miRNA etc. to tumor microenvironment. Despite intense research in the field, only few studies have investigated exosome biodistribution and thus very little known about their in vivo trafficking, that is also impeding their biomedical application as diagnostic and prognostic marker and as a potential therapeutic delivery vehicle. Also, it would be of interest to investigate the biodistribution of exosomes isolated from tumor cells after different treatment. Application of nuclear imaging techniques, such as single-photon emission computed tomography (SPECT) using radio isotopically labeled exosomes have indisputable advantages over fluorescent imaging regarding tissue penetration and quantitative measurement of the biodistribution. Methods: To determine in vivo trafficking of TDEs and whether exosomes from treated cells are distributed differently, we isolated exosomes from hypoxic (1% O2) 4T1 breast cancer cells without any treatment (control), and treated with GW2580 or HET0016 drug. GW2580, a selective small molecule kinase inhibitor of colony stimulating factor 1-receptor (CSF-1R) and HET0016 that is a highly selective inhibitor of CYP4A in arachidonic acid pathway, have been shown to decrease tumor growth and metastasis. We labeled these three groups of exosomes with radio I-131 and injected them in 4T1 tumor bearing and non-tumor bearing Balb/c mice. Then, we followed up the radio I-131labeled exosome distribution for 3 consecutive days with SPECT. Results: More exosomes were localized in the lungs in tumor bearing mice compared to non-tumor bearing mice. Interestingly, control exosomes were more localized in the metastatic site that is in the lungs than the primary breast tumor. We observed TDEs from untreated cells were localized more in metastatic site than that exosomes from the GW2580 and HET0016 treated cells. Conclusion: This study demonstrates that treatment of tumor cells with drugs that inhibit tumor growth and metastasis potentially yield exosomes with less protumorigenic and less metastatic characteristics and become less homing to metastatic site. TDEs are more efficiently visualized and quantified in vivo by radio-isotope labelling of exosomes. The knowledge attained from this study will open up new horizon in exosome research and biological nanotechnology for diagnosis and prognosis and targeted treatment. Citation Format: Mohammad Harun Rashid, Thaiz Borin, Kartik Angara, Roxan Ara, Achyut Bhagelu, Ali Arbab. Determining and characterizing differential biodistribution of tumor derived exosomes by non-invasive nuclear imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-368.

Glioblastoma (GBM) is a hypervascular and hypoxic 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 to normalize blood vessels and control abnormal vasculature. The benefits of AAT have been transient, and evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance and alternative neovascularization mechanisms 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 serve as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor. In our current studies to understand the tumor-inherent mechanisms of AAT resistance, we identified a crucial pro-migratory and pro-angiogenic chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. AAT-treated groups had significantly higher populations of CXCR2+ stem and endothelial-like subpopulations and these cells lined the VM-like vascular structures carrying functional RBCs in the tumors. These stem cell-like and endothelial-like populations were decreased following treatment with HET0016 or SB225002. Furthermore, knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. Also, HET0016 and SB225002 disrupt the tube-forming capability of U251 GBM cells in an in vitro Matrigel angiogenesis assay. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that tumor cell autonomous IL-8/CXCR2 pathway contributes to VM-mediated AAT resistance in GBM and that HET0016 and SB225002 have a great potential to target therapeutic resistance and can be combined with other chemotherapeutic agents in preclinical and clinical trials. Citation Format: Kartik P. Angara, Thaiz F. Borin, Mohammad H. Rashid, Roxan Ara, Bhagelu R. Achyut, Ali S. Arbab. CXCR2+ tumor cells mediate vascular mimicry driving anti-angiogenic therapy (AAT) resistance in glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2.

Objective: Anti-Angiogenic Therapies (AATs), targeting VEGF-VEGFR pathways, are being used as an adjuvant to normalize Glioblastoma (GBM) vasculature. Unexpectedly, clinical trials have witnessed transient therapeutic effect followed by aggressive tumor recurrence. In pre-clinical studies, targeting VEGFR2 with vatalanib, increased GBM growth under hypoxic microenvironment. There is limited understanding of these unanticipated results. Here, we investigated tumor cell associated phenotypes in response to VEGFR2 blockade. Methods: Human U251 cells were orthotopically implanted in mice (day 0) and were treated with vehicle or vatalanib on day 8. Tumor specimens were collected for immunohistochemistry and protein array. Nuclear translocation of VEGFR2 was analyzed through IHC and western blot. In vitro studies were performed in U251 (p53 and EGFR mutated) and U87 (p53 and EGFR wild type) cells following vehicle or vatalanib treatments under normoxia (21% O2) and hypoxia (1% O2). Proliferation, cell cycle and apoptosis assays were done to analyze tumor cell phenotypes after treatments. Results: Vatalanib treated animals displayed distinct patterns of VEGFR2 translocation into nuclear compartment of U251 tumor cells. In vitro studies suggest that vatalanib significantly induced nuclear translocation of VEGFR2, characterized in chromatin bound fraction, especially in U251 tumor cells grown under normoxia and hypoxia. Anti-VEGFR2 driven nuclear translocation of VEGFR2 was associated with increased cell cycle and proliferation, decreased apoptosis, and displayed increased invasiveness in U251 compared to U87 cells. Conclusion: Study suggests that AAT-induced molecular and phenotypic alterations in tumor cells are associated with mutation status and are responsible for aggressive tumor growth. Therefore, mutation status of the tumor in GBM patients should be taken in to consideration before applying targeted therapy to overcome unwanted effects.

Glioblastoma (GBM) is considered one of the most malignant, genetically heterogeneous, and therapy-resistant solid tumor. Therapeutic options are limited in GBM and involve surgical resection followed by chemotherapy and/or radiotherapy. Adjuvant therapies, including antiangiogenic treatments (AATs) targeting the VEGF–VEGFR pathway, have witnessed enhanced infiltration of bone marrow-derived myeloid cells, causing therapy resistance and tumor relapse in clinics and in preclinical models of GBM. This review article is focused on gathering previous clinical and preclinical reports featuring major challenges and lessons in GBM. Potential combination therapies targeting the tumor microenvironment (TME) to overcome the myeloid cell-mediated resistance problem in GBM are discussed. Future directions are focused on the use of TME-directed therapies in combination with standard therapy in clinical trials, and the exploration of novel therapies and GBM models for preclinical studies. We believe this review will guide the future of GBM research and therapy.

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis.

Exosomes are small vesicle cellular products originating from the endocytic pathway abundantly secreted by tumor cells. These exosomes have the ability to alter their immediate microenvironment (ME) through cell-cell interaction by fusion with plasma membrane and subsequent endocytosis or release of their cargo. Exosomes are critical modulators of pre-metastatic niche creation by increasing the recruitment of inflammatory cells. In breast cancer, tumor-derived exosomes recruit myeloid derived suppressor cells in the creation of an immunosuppressive pro-tumorigenic lung niches. Colony stimulating factor 1-receptor (CSF1R), which is a key regulator of myeloid cell proliferation, survival, and differentiation can be blocked by a selective inhibitor GW2580. Overexpression of CSF-1 has been implicated in the creation of increased number of metastatic niches in numerous cancers. In our study, we have demonstrated that the exosomes secreted under hypoxic conditions can initiate early pre-metastatic niche creation in lungs in a metastatic breast cancer model compared to normoxia-secreted exosomes. Exosomes were injected intravenously into Balb/c female mice three days after the implantation of 4T1 breast tumor and continued for a week with injections on alternated days. The animals were pre-treated with GW2580 the day before tumor implantation and continued for a week concomitantly with exosomes on alternate days. Lungs, bone marrow, spleen and brain tissues were collected and analyzed by flow cytometer to detect myeloid and angiogenic cells populations. GW2580 was able to prevent myeloid cell infiltration in lungs and bone marrow. Further, we observed significant increase in anti-tumorigenic M1-macrophage population in the lungs of exosome treated animals pre-treated with GW2580. However, these findings were not observed in the bone marrows of the same group. Vasculogenic leukocyte and angiogenic myeloid cell populations were significantly decreased in the lung of exosome treated animals pre-treated with GW2580. A similar decrease in these populations of cells was not seen in the lungs of animals pre-treated with GW2580 only. These surprising results have led us to hypothesize that GW2580 treatment can prevent the effects of exosomes, which causes infiltration of myeloid cells in the lung to create metastatic niche. These observations indicate a role of CSF1R inhibitor in preventing the distant metastatic niche formation. Citation Format: Thaiz F. Borin, Kartik Angara, Mohammad Rashid, Adarsh Shankar, Asm Iskander, Roxan Ara, Meenu Jain, Bhagelu R. Achyut, Ali S. Arbab. CSF-1R inhibitor prevented pre-metastatic lung niches in metastatic mammary tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2017-1043

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 a selective CYP450 inhibitor 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. Tumor growth and vascular parameters (tumor blood volume, permeability and, extravascular and extracellular space volume) were determined by in vivo dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI). The pharmacokinetics of HPßCD-HET0016 were evaluated in plasma and tumor tissues using IV and IP routes of administration. IV treatment with HPßCD-HET0016 decreased tumor growth, tumor blood volume, permeability and, extravascular and extracellular space volume, when compared with the vehicle group (p Citation Format: Ali S. Arbab, Meenu Jain, Bhagelu Achyut, Kartik Angara, Mohammad H. Rashid, Asm Iskander, Thaiz F. Borin, Wenbo Zhi, Roxan Ara, Irina Lebedyeva, Hassan Bagher-Ebadian. Magnetic resonance imaging based analysis of tumor growth and vascular parameters in animal model of GBM following IV formulated of HET0016 treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2017-2863

Therapy resistance in solid tumors is of growing concern due to the failure of multiple therapeutic approaches. Targeting tumor cells alone with chemotherapy, tumor vasculature with antiangiogenic therapies (AAT) and tumor-infiltrating myeloid cells with CSF1R inhibitor have all lead to the development of refractory tumors with greater relapse rates. There is an urgent need to understand molecular mechanisms of therapy resistance in cancer. There could be tumor cell extrinsic and intrinsic mechanisms in the tumor microenvironment. We focused our study to investigate tumor cell intrinsic pathways using glioblastoma (GBM) as a model tumor and AAT (antiVEGF-VEGFR) as a model therapy. The benefits of AAT are transient with increased relapse owing to adaptive responses by the GBM. Our preclinical study and in vitro data, for the very first time, identified that AAT induces transdifferentiation of tumor cells into endothelial-like cells, capable of forming functional blood vessels in the growing tumors, termed as vasculogenic mimicry (VM). We observed that anti-VEGFR2 (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. In addition to this, vatalanib treated tumors show significantly increased Laminin positive loops characteristic of VM in tumor center as well as at the periphery. A positive correlation was observed between the VM-like structures and the tumor size. We also performed in vitro tube formation assays with AAT treated GBM cells alone and HUVEC cells (co-culture) to confirm the role of GBM cells in the formation of mosaic vessels in normoxic conditions. Interestingly, tumor cells are incorporated into the tubes formed by HUVEC cells. We found a higher number of complete tube like structures with AAT treated tumor cells as compared to control. Cytokine array with the condition media from tumor cells treated with AAT showed a significant upregulation in the levels of IL8. We observed a significant increase in the CXCR1+ and CXCR2+ endothelial-like GBM cells following treatment with AAT. Ongoing investigations are focused on study of IL8-CXCR1/2 pathway in VM regulation using loss or gain of function approaches. The study will identify critical mediators of VM in GBM. In clinics, discovering novel targets causing VM associated therapy resistance is essential for identifying subset of patients that could be treated with alternate regimens. Citation Format: Kartik Angara, Mohammad Rashid, Thaiz Borin, Bhagelu Achyut, Meenu Jain, ASM Iskander, Roxan Ara, Ali Arbab. Vascular mimicry mediated mechanisms drive therapy resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2017-787

Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity. Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3968. doi:10.1158/1538-7445.AM2017-3968

Glioblastoma (GBM), a grade IV glioma, is considered highly vascular and invasive subtype. GBM is most lethal during first year after initial diagnosis despite surgical resection, radiotherapy and/or chemotherapy. Antiangiogenic treatments (AATs) against VEGF-VEGFR pathways resulted into increased GBM tumor growth with marked hypoxia and increased recruitment of bone marrow derived cells (BMDCs) to the tumor microenvironment (TME), without any survival benefits. Preclinical studies have found that AAT failures were associated with increased bone marrow-derived tumor-associated myeloid cells (TAMCs) in GBM. Depletion of CSF1R+ myeloid cells using GW2580 decreased recruitment of TAMCs in the tumor, and reduced GBM growth. Interestingly, AAT increased the expression of CXCL7 chemokine, and CSF1R blockade decreased CXCL7 in GBM microenvironment. In addition, CXCL7 expression was correlated with number of tumor infiltrating bone marrow derived cells, phospho-ERK MAPK expression, and proliferation of bone marrow cells in GBM. Here, we looked at the prognostic significance of CXCL7 and its receptor CXCR2 in human GBM. We performed systematic human data analysis (www.oncomine.com), which revealed that CXCL7 is highly expressed (43 to 150 fold) in normal BM compared to other normal organs. This suggests that CXCL7 is required for BM cell survival and migration both in normal and tumor conditions. In GBM datasets, CXCL7 mRNA expression in tumor was increased (> 4.5 fold) in GBM patients (n = 27) compared to normal brain (n = 4). CXCL7 mRNA expression was seen more in stroma (n = 4) than tumor cells (n = 5). Overall survival status analysis showed that patients with GBM who died early (n = 28) had significantly higher CXCL7 mRNA expression compared to living patients (n = 10). In addition, increased CXCL7 mRNA expression was associated with the GBM tumor recurrence (n = 8) compared to primary occurrence (n = 8). Interestingly, CXCL7 mRNA expression was correlated with the CXCR2 mRNA expression (CXCL7 receptor) both in normal (0.7, n = 71) and brain tumor tissues (0.6, n = 68). Similarly, CXCR2 mRNA expression was increased in GBM (n = 78) compared to other brain tumors. CXCR2 mRNA expression was seen more in stroma (n = 4) than tumor cells (n = 5). Patients with GBM, who died early (n = 19) had significantly higher CXCR2 mRNA expression compared to living patients (n = 10). In addition, increased CXCR2 mRNA expression was associated with the GBM tumor recurrence (n = 8) compared to primary occurrence (n = 8). However, data regarding the CXCL7 and CXCR2 expression in response to AATs is lacking in human cancer datasets. In summary, we identified CXCL7-CXCR2 axis as a novel prognostic factor in GBM. However, studies are required to investigate CXCL7-CXCR2 regulation in myeloid cell survival/ polarization in TME. Citation Format: Bhagelu R. Achyut, Adarsh Shankar, Meenu Jain, Kartik Angara, ASM Iskander, Roxan Ara, Ali S. Arbab. CXCL7-CXCR2 axis as a novel prognostic factor in myeloid cell associated glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 252.

Glioblastoma (GBM) is considered as hypervascular form of brain tumors. Anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels. Surprisingly, AAT resulted into marked hypoxia and recruited bone marrow derived cells (BMDCs) to the tumor microenvironment (TME). Nonavailability of in vivo model limits investigation of BMDCs in AAT resistance. We established a novel chimeric mouse model using as low as 5x106 BM cells from GFP+ transgenic mice, administered through i.v route in athymic nude mice following whole body irradiation, which resulted in >70% engraftment within 14 days. We exploited chimeric mouse model to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. Increased infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes in TME, and increased tumor growth were observed following treatments. Inhibition of CSFR1+ myeloid cells using GW2580 reduced tumor growth by decreasing myeloid and angiogenic cells in TME. This was followed by decreased inflammatory, proangiogenic and immune suppressive factors compared to vehicle and other treated groups. In conclusion, we developed preclinical chimeric model of GBM and role of tumor infiltrated BMDCs were investigated in context of resistance of AATs by utilizing state of art multispectral optical and MR imaging. Study suggests that inhibiting immune suppressive myeloid cells could provide a better therapeutic benefit in GBM. Citation Format: B.R Achyut, Adarsh Shankar, Asm Iskander, Roxan Ara, Kartik Angara, Robert Knight, Alfonso Scicli, Ali Arbab. Bone marrow derived myeloid cells orchestrate resistance to antiangiogenic therapy in novel chimeric mouse model of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A23.

Anti-angiogeneic therapies are being used in glioma due to high expression of vascular and angiogenic growth factors but without improved survival and tumor recur. Our study used vatalanib (PTK) in U251 tumor cells which secretes proangiogenic and proinvasive proteins. We found 7 proteins (MMP-2, MMP-9, SDF-1α, Tie-2, MCP-1, IL-8, VE Cadherin, and VEGFA) which were altered greater than 20 fold in both U251 complete medium and lysate. We hypothesized that miRNA regulation may be involved in overexpression of these secreted factors that might be responsible for antiangiogenic resistance in glioma. We performed miRNA profiling (Affymetrix miRNA v3 platform) in U251 glioma cell line treated with or without PTK drug under hypoxia and normoxia conditions. Fold changes were estimated using normoxia group as control against normoxia+PTK, hypoxia, hypoxia+PTK, and changes greater than 5 fold were considered for further analysis to investigate potential targets. We found miR-15a and miR-21 were > 7-fold upregulated in group with hypoxia+PTK treatment. Targetscan analysis showed mir-15a may target TNFAIP3, VEGFA and mir21 can target MMP-2, which were altered in U251 cell lysate. Our present study identified novel microRNAs and gene targets which may lead to antiangiogenic resistance in glioma and provide a basis for future clinical trials. In future, functional assays will confirm the role of candidate miRNAs in tumor vasculature development following antiangiogenic drug treatment. Citation Format: Meenu Jain, Bhagelu Achyut, Adarsh Shankar, Kartik Angara, Asm Iskander, Ali S. Arbab. MicoRNA mediated regulation of antiangiogenic resistance in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B30.