Bhagelu R. Achyut

Bone marrow derived myeloid cells orchestrate antiangiogenic resistance in glioblastoma through coordinated molecular networks

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 Our unpublished data and published studies have indicated the involvement of immunosuppressive myeloid cells in therapeutic resistance in glioma.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.

CCL9 Induced by TGFβ Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1(+)CD11b(+) immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFβ signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFβ-targeted therapy.

Application of Umbilical Cord Blood Derived Stem Cells in Diseases of the Nervous System

Umbilical cord blood (UCB) derived multipotent stem cells are capable of giving rise hematopoietic, epithelial, endothelial and neural progenitor cells. Thus suggested to significantly improve graft-versus-host disease and represent the distinctive therapeutic option for several malignant and non-malignant diseases. Recent advances in strategies to isolate, expand and shorten the timing of UCB stem cells engraftment have tremendously improved the efficacy of transplantations. Nervous system has limited regenerative potential in disease conditions such as cancer, neurodegeneration, stroke, and several neural injuries. This review focuses on application of UCB derived stem/progenitor cells in aforementioned pathological conditions. We have discussed the possible attempts to make use of UCB therapies to generate neural cells and tissues with developmental and functional similarities to neuronal cells. In addition, emerging applications of UCB derived AC133+ (CD133+) endothelial progenitor cells (EPCs) as imaging probe, regenerative agent, and gene delivery vehicle are mentioned that will further improve the understanding of use of UCB cells in therapeutic modalities. However, safe and effective protocols for cell transplantations are still required for therapeutic efficacy.

Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments

ABSTRACT 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 × 106 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.

Vascular mimicry in glioblastoma following anti-angiogenic and anti-20-HETE therapies

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.

Anti-Jagged Immunotherapy Inhibits MDSCs and Overcomes Tumor-Induced Tolerance

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.

When Seed and Soil Theory Meets Chicken or Egg Theory in Cancer Metastasis.

Cancer metastasis has been a serious problem since decades. Seed and soil hypothesis of metastasis remains true and all the metastatic tumors follow this nature’s law [1,2]. Advance metastasis or multi-organ metastasis is difficult to treat due to multi-organ dysfunction. One of the major issues in metastasis is that diagnosis occurs at the advanced stages. Secondly, we have not understood the complete mechanisms so well so far due to intricate nature of metastasis. In metastasis, seed (tumor cell) migrates to the soil (distant organs, e.g. lung, liver, brain, and bone). Several experimental studies have been done that suggested the role of bone marrow derived progenitor cells [3] (e.g. CD11b+ [4] and VEGFR1+ [5] cells) in the initiation of metastasis. Several chemokines, such as SDF-1, TNF-α, TGF-β and VEGF-A influence the recruitment of different cell types to pre-metastatic sites resulting into increased expression of specific molecules in the niche like S100A8, S100A9, lysyl oxidase (LOX), fibronectin, MMP9 and MMP2 in the initiation of premetastatic niche [6,7], which are bonafide candidates of therapeutic targeting [8]. In addition, tumor induced hypoxia has been shown to promote the premetastatic niche formation by recruiting CD11b+/Ly6Cmed/Ly6G+ cells [9] and producing LOX [10]. Recently, much attention has been given to the tumor-derived exosomes or micro vesicles that carry almost every essential cellular macromolecule and has signals to polarize cells in the tumor microenvironment and create premetastatic niche in the distant organs, before the seed (tumor cell) arrives [11,12]. Exosomes derived from melanomas were shown to educate pro-metastatic progenitor cells in the bone marrow [13]. Renal-carcinoma-derived exosomes were found to promote angiogenesis in lung tumor metastases [14]. In addition, using murine mammary carcinoma demonstrated that, tumor-derived microvesicles use osteopontin to mobilize pro-angiogenic cells from the bone marrow [15]. Surprisingly, exosomes perform cell independent miRNA biogenesis to promote tumorigenesis and metastasis [16]. Firstly, tumor derived exosomes has pro-angiogenic functions that helps tumor in building required vasculature for tumor growth. For example, Yoon et al. [17] investigated pro-angiogenic role of tumor-secreted exosomes by showing Egr-1 activation in endothelial cells through ERK1/2 and JNK signaling pathways and endothelial cell migration, which was facilitated by the tumor cell derived extracellular vesicles. On the other hand, tumor derived exosomes involved in the destruction of vasculature integrity for metastasis. For example, miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is reported as a potent regulator of tumor cell migration through targeting the tight junction protein ZO-1 via exosomes. Tumor cell secreted exosomes deliver miR-105 to the site of endothelial monolayers that efficiently destroys tight junctions and hence the integrity of barriers against metastasis [18]. Although exosomes have attracted much attention and are considered as a bonafide targets for cancer therapy, their roles in tumor metastasis is poorly investigated. In addition, technologies and methods to study exosomes are growing day by day. It is possible that tumor cell exosomes are delivered to the distant organs that manipulates host environment before any immune cells or chemokine. However, what initiates tumor cell migration to the distant organs, remains unclear, i.e. chicken comes first or egg and warrants further investigations.

Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

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 < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.

Myeloid cell signatures in tumor microenvironment predicts therapeutic response in cancer

Tumor microenvironment (TME) consists of several immune and nonimmune cell populations including tumor cells. For many decades, experimental studies have depicted profound contribution of TME toward cancer progression and metastasis development. Several therapeutic strategies have been tested against TME through preclinical studies and clinical trials. Unfortunately, most of them have shown transient effect, and have largely failed due to aggressive tumor growth and without improving survival. Solid tumors are known to have a strong myeloid component (eg, tumor-associated macrophages) in tumor development. Recent data suggest that therapeutic responses in tumor are characterized by alterations in immune cell signatures, including tumor-associated myeloid cells. Polarized tumor-associated myeloid cells (M1–M2) are critical in impairing therapeutic effect and promoting tumor growth. The present review is intended to compile all the literatures related to the emerging contribution of different populations of myeloid cells in the development of tumor and therapeutic failures. Finally, we have discussed targeting of myeloid cell populations as a combination therapy with chemo-, targeted-, or radiation therapies.

Myeloid Derived Suppressor Cells: Fuel the Fire

Low oxygen tension, hypoxia, is a characteristic of many tumors and associated with the poor prognosis. Hypoxia invites bone marrow derived cells (BMDCs) from bone marrow to the site of tumor. These recruited CXCR4+ BMDCs provide favorable environment for the tumor growth by acquiring pro-angiogenic phenotype such as CD45+VEGFR2+ Endothelial Progenitor Cells (EPC), or CD45+Tie2+ myeloid cells. CD11b+CD13+ myeloid population of the BMDCs modulate tumor progression. These myeloid populations retain immunosuppressive characteristics, for example, myeloid derived suppressor cells (MDSCs), and regulates immune- suppression by inhibiting cytotoxic T cell function. In addition, MDSCs were observed at the premetastatic niche of the distant organs in other tumors. Protumorigenic and prometastatic role of the myeloid cells provides a basis for therapeutic targeting of immunosuppression and thus inhibiting tumor development and metastasis.