Tie2-expressing Monocytes Research Articles

ME-05 * COUNTERATTACKING THE FORCE BEHIND GLIOMA INVASION

Brain tumor dispersal is the major cause behind the extreme difficulty of treatment and the inevitable recurrence of malignant gliomas. Determining the mechanism underlying the invasion of brain tumor cells will suggest rational therapeutic strategies. Importantly, acquisition of a highly aggressive invasive phenotype is being observed in patients with recurrent malignant gliomas treated with antiangiogenesis therapy and is considered to be responsible for the therapy failure. Although the cause for this tumor behavior remains elusive, it has been hypothesized that the tumor microenvironment may be one major influence in the invasive development. In this regard, we have recently described the recruitment of Tie2-expressing monocytes (TEMs) into the tumor/normal brain interface upon anti-VEGF therapies in preclinical models and in human surgical specimens, and reported that this specific myeloid population is responsible for the heightened invasive properties of brain tumor stem cells. Here we investigated changes in the cytokine milieu of these tumors and identified increased expression of Angiopoietin 2 (Ang2), a Tie2 ligand, after therapies targeting either VEGF (bevacizumab, aflibercept) or VEGFR (DC101), but no after standard chemotherapy. In vitro and in vivo models were combined to decipher the role of Ang2, and we observed that expression of Ang2 functioned as a potent chemoattractant of TEMs. Moreover, we observed that Ang2 stimulates the tissue remodeling properties of TEMs, further enhancing the pro-invasive properties of this tumor recruited population. Of interest, modulation of molecules blocking the Ang/Tie2 axes in a preclinical model jeopardizes the tumor recruitment of TEMs and drastically resulted in a complete abolition of the heightened invasion induced by antiangiogenesis therapy. These results might have important implications in the clinical scenario since decrease of the invasive tumor pattern could potentially render recurrent tumors amenable to surgical removal ultimately prolonging the survival of patients with malignant gliomas.

356 Discovery of novel pyrido[2,3-b]pyrazine as fibroblast growth factor receptor (FGFR-1, 2, 3 & 4) kinase inhibitors with nanomolar affinity

Background: Altiratinib is a Type II switch pocket inhibitor of MET kinase as well as TIE2 and VEGFR2 kinases. This profile provides an agent that exhibits anti-tumor activity in cancers driven by MET overexpression or genomic mutation, and also blocks angiogenic and metastatic processes mediated by the tumor microenvironment. Material and Methods: Altiratinib was evaluated in MET, TIE2, and VEGFR2 biochemical studies, including evaluation in a number of MET activation mutants. Cellular activity was evaluated in tumor cell lines exhibiting MET amplification (MKN-45 gastric, EBC-1 NSCLC) or overexpression (B16/F10 melanoma; U87 glioblastoma). In vivo pharmacokinetic/pharmacodynamic studies were performed in an MKN45 xenograft model. Efficacy was demonstrated in a battery of xenograft/ allograft models including: gastric (MKN-45), melanoma (B16/F10, A375), ovarian (SKOV-3), colorectal (COLO-205), lung (EBC-1), breast (PyMT), and glioblastoma (U87). Results: Altiratinib afforded balanced inhibition of MET, TIE2, and VEGFR2 kinases in the low nM range (IC50s 2−9 nM) and blocked HGF-, ANG-, or VEGFA-induced HUVEC activation and capillary tube formation. Altiratinib retained potency versus activation loop mutant forms of MET (D1228X, Y1230X, M1250X), inhibiting all forms with IC50 24 hr after a single 10mg/kg oral dose in an MKN-45 xenograft pharmacodynamic model. Altiratinib exhibited anti-tumor activity in melanoma (B16/F10, A375), gastric (MKN-45), lung (EBC-1), colorectal (COLO-205), breast (PyMT), ovarian (SKOV-3) and GBM (U87) xenograft or allograft models. In these in vivo studies, altiratinib was shown to inhibit tumor growth, angiogenesis, invasion and/or metastasis. Altiratinib also blocked recruitment of TIE2-expressing monocytes in the PyMT breast cancer model. In some models, altiratinib was shown to increase overall survival. In particular, in the i.c.v. orthotopically implanted U87 glioblastoma model, altiratinib extended survival by 1.7-fold vs vehicle (112 days vs 66 days), while the combination of altiratinib + bevacizumab extended survival by 2.5 fold vs vehicle and by 1.9-fold vs bevacizumab single agent (166 days vs. 88 days). Conclusions: Altiratinib is a balanced inhibitor of MET, TIE2, and VEGFR2 kinases. This profile provides robust inhibition of tumors driven by MET amplification or overexpression, and also provides the potential for altiratinib to block tumor microenvironment angiogenic resistance mechanisms and pro-tumoral effects of TIE2-expressing macrophages in the clinical setting. Altiratinib is currently in Phase 1 clinical trials in patients with solid tumors.

Can TIE-2 expressing monocytes represent a novel marker for hepatocellular carcinoma?

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is a global health problem representing the sixth most common cancer and the third cause of cancer related death worldwide. The number of deaths per year in HCC is comparable to the incidence number, underlying the aggressive behavior of HCC and the modest efficacy of available curative treatments. Effective HCC treatment is problematic also due to the lack of early and specific diagnostic markers. In this regard, particular interest has been put on the tyrosine kinase with Ig and endothelial growth factor (EGF) homology domains 2 (TIE2), a receptor of angiopoietins, predominantly present on endothelial cells but also observed on monocytes [TIE-2-expressing monocytes (TEMs)]. Recently, a work by Matsubara et al. showed that the amount of circulating TEMs is higher in hepatitis virus C (HCV)/HCC patients compared to HCV patients or healthy subjects. Additionally the authors showed that TEMs have a diagnostic potential for HCC. Whereas the molecular mechanisms responsible for this observation remain elusive and further studies are necessary to confirm this finding, the work of Matsubara et al. may contribute to the identification of a novel HCC prognostic and diagnostic marker.

TIE2-expressing monocytes: a possible cellular diagnostic and prognostic biomarker for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and the third leading cause of cancer-related deaths worldwide (1). Major risk factors for HCC include chronic infection by hepatitis B virus (HBV) or hepatitis C virus (HCV) and alcoholic liver cirrhosis. Advanced and symptomatic HCC disease is not amenable to curative surgery, therefore screening programs for high-risk populations are implemented to increase early detection and effective surgical treatment of HCC (2). However, a definitive diagnosis of HCC requires concordant findings from several invasive and non-invasive diagnostic parameters, including liver biopsy, serum alpha-fetoprotein (AFP) levels, computed tomography, or magnetic resonance imaging (2). Nevertheless, noninvasive imaging is not sufficiently sensible for detecting an early-stage HCC, similarly AFP has low sensitivity and specificity to be used as a screening biomarker (3). In this framework, effective biomarkers for the early detection as well the progression of HCC are greatly needed. Matsubara et al. in Hepatology (4) described the possible significance of circulating CD14+CD16+ monocytes expressing Tyrosine kinase with Ig and EGF homology domains 2 (TIE2) [TIE2-expressing monocytes (TEMs)] as biomarkers for the detection of both early- and late-stage HCC. Indeed, TIE2 is mainly expressed on endothelial cells and binds all the known angiopoietins (ANGs) and TEMs are peripheral as well as tumor-infiltrating myeloid cells presumed to be characterized by a profound pro-angiogenic activity (5-7). In particular, TEMs have been reported in tumors of the kidney, colon, pancreas and lung, as well as in soft tissue sarcomas (7) where angiogenesis is known to be important for tumor progression. In this framework, HCC is a highly vascularized tumor, the progression of early lesions to overt HCC is strongly associated with increase of vasculature (8,9) and the antiangiogenic drug, sorafenib prolongs to a certain extent survival of patients with advanced HCC. Matsubara et al. (4) report on the frequency of circulating TEMs in a cohort of 168 HCV-infected patients, of which 89 were with HCC. The investigators found that the frequency of TEMs was significantly higher in patients with HCC than HCV-infected patients without HCC or healthy subjects. No difference was found among HCC patients at different stages of the disease, thus establishing TEMs as a stage-independent, diagnostic biomarker for HCC. In addition, higher blood TEM levels correlate to a worse recurrence-free survival after HCC resection or radiofrequency ablation (RFA) therapy, suggesting a role for TEMs also as prognostic biomarker. Furthermore, they also identified TEMs in HCC tumor lesions preferentially localized in perivascular tumor areas correlating with increased microvessel density in the tumors. Therefore, it’s possibly suggesting that HCC-infiltrating TEMs are proangiogenic. Moreover, high circulating and intratumoral TEM levels correlate with a more advanced Child-Pugh stage, suggesting that TEM frequency correlates positively with the degree of liver inflammation/stage of cirrhosis and negatively with liver function. The biology underlying TEM’s involvement in human tumor angiogenesis and progression is essentially unknown and several questions need to be addressed in order to possibly improve HCC anti-angiogenetic therapy. Indeed, the proangiogenic activity exerted by TEMs in HCC may represent one of the reasons for the limited efficacy of sorafenib or other antiangiogenic treatments. Furthermore, elevated circulating levels of the TIE2 ligand, ANG2, correlate with a worse prognosis in untreated HCC patients and predict shorter survival in HCC patients treated with Sorafenib (10). The study by Matsubara et al. (4) is the first to present evidence suggesting that circulating TEMs may be a diagnostic biomarker for both early- and late-stage HCC, although they are not strictly peculiar of HCC but detectable also in other tumors (5,7). These very interesting findings need to be validated on a larger scale of HCC patients to confirm the relevance of TEMs as diagnostic/prognostic marker for HCC as well as therapeutic target for improving the efficacy of current treatments for HCC.

TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. In the past few years, the mechanisms of hepato-carcinogenesis have been elucidated and the involvement of a number of pathways, including angiogenesis, aberrant signal transduction, and dysregulated cell cycle control have been demonstrated. Myeloid lineage cells, such as macrophages and monocytes, have been reported to regulate angiogenesis in mouse models. TIE2, a receptor of angiopoietins, conveys pro-angiogenic signals and identifies a monocyte/macrophage subset with pro-angiogenic activity. Recently, one study suggests that TIE2-expressing monocyte/macrophage (TEMs) frequency can be used as a diagnostic marker for HCC.

PRESENCE OF A DISTINCTIVE MYELOID POPULATION IS ASSOCIATED WITH THE INVASIVE TUMOR PHENOTYPE OBSERVED AFTER ANTI-ANGIOGENESIS THERAPIES

BACKGROUND: The addition of anti-angiogenic therapy to the few treatments that are available to patients with malignant gliomas was based on the fact that these tumors are highly vascularized and on findings from initial preclinical and clinical studies which showed encouraging results. However, tumors that initially respond to this therapy invariably recur with the acquisition of highly aggressive and invasive phenotype. Although several myeloid populations have been associated to the recurrence of these tumors, a specific targetable population has not been yet identified as responsible for the heightened invasion observed upon antiangiogenesis therapies. METHODS: Immunocompromised mice and immunocompetent GFP macrophage Fas-induced apoptosis (MAFIA) transgenic mice were used to model the response of malignant gliomas to several antiangiogenesis therapies, such as bevacizumab, VEGF Trap, DC101, and to temozolomide. Analysis of tumors and monocytic populations were performed by immunohistochemistry and double immunofluorescence. In vitro studies encompass the use of isolated monocytes from donors and a novel experimental model based on a THP1 human monocytic cell line. Data was corroborated in surgical specimens from human malignant gliomas RESULTS: Here we present evidence for the accumulation of Tie2-expressing cells at the tumor/normal brain interface of immunocompromised mice treated with anti-VEGF therapies in regions with heightened tumoral invasion. This cell population was further characterized as having markers of M2 polarized monocytes/macrophages compatible with Tie2-expressing monocytes (TEMs). We validated these results using the immunocompetent MAFIA transgenic mice treated with an anti-VEGFR compound. In contrast, TEMs were almost undetectable in the brains of glioma-bearing mice treated with temozolomide or anti-VEGF therapies that did not result in an invasive tumoral pattern. In vitro studies showed that TEMs enhanced the invasive properties of glioma cells compared with monocytes that did not express Tie2 (non-TEMs). Moreover, TEMs displayed a higher gelatinase enzymatic activity and, specifically, secreted higher levels of MMP2 and MMP9 than non-TEMs did. Consistently Tie2/MMP9 monocytic cells were detected in the invasive tumoral edge of mice treated with anti-VEGF therapies. Of clinical relevance, we detected the presence of TEMs in human surgical specimens after anti-VEGF therapy. CONCLUSIONS: Collectively, our results described the association of a specific myeloid/monocytic subpopulation characterized as Tie2-expressing monocytes with the heightened invasion observed after antiangiogenic therapy. Our results suggest that his distinctive population might explain the mechanism of escape of malignant gliomas to anti-angiogenesis therapies. Therefore, combination of antiangiogenesis therapies with approaches to decrease the tumor recruitment of TEMs might be required to obtain a therapeutic effect. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Anti-vascular endothelial growth factor therapy-induced glioma invasion is associated with accumulation of Tie2-expressing monocytes.

The addition of anti-angiogenic therapy to the few treatments available to patients with malignant gliomas was based on the fact that these tumors are highly vascularized and on encouraging results from preclinical and clinical studies. However, tumors that initially respond to this therapy invariably recur with the acquisition of a highly aggressive and invasive phenotype. Although several myeloid populations have been associated to this pattern of recurrence, a specific targetable population has not been yet identified. Here, we present evidence for the accumulation of Tie2-expressing monocytes/macrophages (TEMs) at the tumor/normal brain interface of mice treated with anti-VEGF therapies in regions with heightened tumoral invasion. Furthermore, we describe the presence of TEMs in malignant glioma surgical specimens that recurred after bevacizumab treatment. Our studies showed that TEMs enhanced the invasive properties of glioma cells and secreted high levels of gelatinase enzymatic proteins. Accordingly, Tie2⁺MMP9⁺ monocytic cells were consistently detected in the invasive tumor edge upon anti-VEGF therapies. Our results suggest the presence of a specific myeloid/monocytic subpopulation that plays a pivotal role in the mechanism of escape of malignant gliomas from anti-VEGF therapies and therefore constitutes a new cellular target for combination therapies in patients selected for anti-angiogenesis treatment.

Abstract P4-15-13: Rebastinib in combination with paclitaxel ablates TIE2-expressing macrophages, reduces metastasis, and increases survival in the PyMT metastatic breast cancer model

Abstract In cancer models, TIE2 kinase plays an important role in angiogenesis, vasculogenesis, and tumor metastasis. TIE2 expression is largely restricted to vascular endothelial cells, tissue macrophages, and bone marrow derived TIE2-expressing monocytes (TEMs), which are proangiogenic, provasculogenic and enhance invasiveness. The hypoxic tumor environment engendered by damaging the vasculature with chemotherapy, radiation, or anti-angiogenic treatments leads to rebound tumor vascularization by an angiogenic switch from the VEGF pathway to the angiopoietin/TIE2 pathway. This leads to recruitment of provasculogenic TEMs from the bone marrow, leading to the growth of residual tumor cells and disease progression. Significantly, a subset of TIE2-expressing macrophages are located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs). Recent observations have linked TIE2-expressing macrophages within TMEM structures to intravasation of cancer cells into circulation and subsequent dissemination to metastatic sites. We hypothesized that TIE2 inhibition should decrease migration and association of TEMs with blood vessels in the tumor stroma, therefore blocking their proangiogenic activity and leading to reduced tumor growth. TIE2 inhibition may also alter TMEM function, leading directly to a blockade of metastasis. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined multiple dosing schedules of rebastinib in combination with paclitaxel. Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases (Table 1). Furthermore, the combination of rebastinib and paclitaxel led to a significant further decrease in lung metastases compared to treatment with paclitaxel or rebastinib alone. Rebastinib also enhanced the activity of paclitaxel in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition with targeted therapy represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development for treatment-refractory metastatic breast cancer, with a Phase 1b trial being planned for late 2013. Rebastinib reduces lung metastases in the PyMT breast cancer modelStudynTreatmentLung Metastases (% of Control)110Vehicle100%110Paclitaxel 10 mg/kg Q5D36%110Rebastinib 10 mg/kg BID28%110Rebastinib 10 mg/kg BID + Paclitaxel7%210Vehicle100%210Paclitaxel 10 mg/kg Q5D51%210Rebastinib 10 mg/kg QD + Paclitaxel21%33Vehicle100%33Paclitaxel 10 mg/kg Q5D58%33Rebastinib 10 mg/kg twice/week + Paclitaxel28% Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-13.

Abstract P4-15-12: Rebastinib in combination with eribulin ablates TIE2-expressing macrophages, reduces metastasis, and increases survival in the PyMT metastatic breast cancer model

Abstract In cancer models, TIE2 kinase plays an important role in angiogenesis, vasculogenesis, and tumor metastasis. TIE2 expression is largely restricted to vascular endothelial cells, tissue macrophages, and bone marrow derived TIE2-expressing monocytes (TEMs), which are proangiogenic, provasculogenic and enhance invasiveness. The hypoxic tumor environment engendered by damaging the vasculature with chemotherapy, radiation, or anti-angiogenic treatments leads to rebound tumor vascularization by an angiogenic switch from the VEGF pathway to the angiopoietin/TIE2 pathway. This leads to recruitment of provasculogenic TEMs from the bone marrow, leading to the growth of residual tumor cells and disease progression. Significantly, a subset of TIE2-expressing macrophages are located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs). Recent observations have linked TIE2-expressing macrophages within TMEM structures to intravasation of cancer cells into circulation and subsequent dissemination to metastatic sites. We hypothesized that TIE2 inhibition should decrease migration and association of TEMs with blood vessels in the tumor stroma, therefore blocking their proangiogenic activity and leading to reduced tumor growth. TIE2 inhibition may also alter TMEM function, leading directly to a blockade of metastasis. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined dosing rebastinib in combination with eribulin, an inhibitor of microtubule dynamics that recently was FDA-approved for treatment-refractory metastatic breast cancer. Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases. Furthermore, the combination of rebastinib and eribulin led to a significant further decrease in lung metastases compared to treatment with eribulin alone (Table 1). Rebastinib also enhanced the activity of eribulin in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development in combination with eribulin for treatment-refractory metastatic breast cancer, with a Phase 1b trial being planned for late 2013. Rebastinib reduces lung metastases in the PyMT breast cancer modelTreatmentLung Metastases (% of Control)Vehicle100%Eribulin 1 mg/kg three times/week71%Rebastinib 10 mg/kg twice/week + Eribulin 1 mg/kg23%Eribulin 0.3 mg/kg three times/week71%Rebastinib 10 mg/kg twice/week + Eribulin 0.3 mg/kg51%Eribulin 0.1 mg/kg three times/week72%Rebastinib 10 mg/kg twice/week + Eribulin 0.1 mg/kg43% Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-12.

The Frequency of Tumor-infiltrating Tie–2-expressing Monocytes in Renal Cell Carcinoma: Its Relationship to Angiogenesis and Progression

To examine the frequency of tumor-infiltrating Tie-2-expressing monocytes (TEMs) in renal cell carcinoma (RCC) and its association with microvessel density (MVD) and other clinical-pathologic features. This study enrolled 65 consecutive patients with RCC treated with radical nephrectomy. The frequency of tumor-infiltrating TEMs, which was defined as CD14(+) Tie-2(+) cells, was assessed using flow cytometry. MVD was measured by immunohistochemistry using anti-CD34 antibody. The association between clinicopathologic parameters, MVD, and the frequency of tumor-infiltrating TEMs in RCC was assessed. High frequency of tumor-infiltrating TEMs was significantly associated with advanced stage (P= .018), positive lymph nodes (P= .013), high grade (P= .019), and metastases (P= .006). Correlation analysis revealed that the frequency of TEMs was positively correlated with MVD. Our findings revealed a significant association between prognostic tumor features, MVD, and the frequency of tumor-infiltrating TEMs in RCC and indicated that TEMs may play an important role in angiogenesis and progression of RCC.

Tie2 expressing monocytes in cancer research

Tie2 expressing monocytes (TEMs) only present in human circulating blood and tumor organs,and have an important role in tumor angiogenesis and progression.TEMs are effectively recruited to tumors by angiopoietin 2 (Ang2) and hypoxia and then differentiate into macrophages,which promote the angiogenesis in experimental tumor models by providing paracrine support to nascent blood vessels.Studies show that TEMs are up-regulated in hepatocellular carcinoma,colorectal cancer,breast cancer,malignant glioma and other human cancers,which suggests that TEMs are conducive to diagnosis and prognosis of tumors.With the further research,TEMs are applied to deliver drugs which can obtain significant anti-tumor responses and inhibit metastasis with the ability of tumor-homing.Meanwhile,TEMs may also be a potential target for the anticancer drugs.However,the present researches indicate that the effects of TEMs in tumor microvessel density,clinical stage and prognosis are still questionable.Current works that aim at describing and predicting the concrete function of TEMs have attracted significant attention from researchers. Key words: Angiogenesis; Neoplasms ; Tie2 expressing monocytes; Angiopoietin 2

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

NGR-TNF, a novel vascular-targeting agent, does not induce cytokine recruitment of proangiogenic bone marrow-derived cells

Background:Administration of certain chemotherapy drugs at the maximum tolerated dose, vascular-disrupting agents (VDAs) and irradiation can induce mobilisation and tumour homing of proangiogenic bone marrow-derived cells (BMDCs). Increases in cytokines and chemokines contribute to such mobilisation that eventually promotes tumour (re)growth. NGR-TNF is a vascular-targeting agent in advanced clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with tumour necrosis factor-alpha (TNF). We investigated whether NGR-TNF mobilises host BMDCs and growth factors.Methods:Blood was obtained from Lewis lung carcinoma and 4T1 tumour-bearing mice at different time points following NGR-TNF, VDA or anti-VEGFR2/flk-1 antibody treatment. Levels of circulating growth factors were assessed by ELISAs. BMDCs were characterised by FACS. Circulating endothelial progenitor cells were defined as CD45−/CD13+/flk-1+/CD117+/7AAD−, Tie2-expressing monocytes as CD45+/CD11b+/Tie2+ and myeloid-derived suppressor cells as CD45+/CD11b+/Gr1+ cells.Results:NGR-TNF decreases tumour blood vessel density-inducing apoptosis of tumour and tumour endothelial cells. Unlike VDAs, or high-dose NGR-TNF, lower doses of NGR-TNF, comparable to those used in clinical trials, neither mobilise nor recruit to the tumour site proangiogenic BMDCs or induce host growth factors.Conclusion:Low-dose NGR-TNF exerts antitumour activity without inducing proangiogenic host responses, conceivably important for preventing/overcoming resistance and designing combination therapeutic strategies.

FRI0021 The rheumatoid arthritis synovial microenvironment promotes differentiation of monocytes into pro-angiogenic macrophages responsive to angiopoietin signaling

BackgroundVascular remodeling promotes immune cell infiltration and provides nutrients to hyperplastic tissue in the synovial tissue of patients with rheumatoid arthritis (RA). In solid cancers, angiopoietin (Ang-1 and Ang-2) signaling...

Phase I trial of trebananib (AMG 386) plus temsirolimus (Tr + T) in patients (pts) with advanced solid tumors (PJC-008/NCI#9041).

2534 Background: Preclinical data suggest that combined Ang1/2 and mTOR blockade has synergistic anti-cancer activity. The combination of Tr (inhibits angiogenesis by preventing interaction of Ang1/2 with Tie2) with the mTOR inhibitor T was evaluated in pts with advanced solid tumors to determine safety, tolerability, maximum tolerated dose (MTD), pharmacodynamics and preliminary antitumor activity. Methods: Pts were enrolled using 3+3 design. Tr and T were dosed on Day 1 (D1), 8, 15 and 22 of a 28-day cycle. Peripheral blood was collected for evaluation of Tie2-expressing monocytes (TEMs) and thymidine phosphorylase (TP) (an angiogenic enzyme increased in TEMs upon Tie2 stimulation) by flow cytometry. Tumor response was assessed every 2 cycles. Results: 13 pts have been enrolled, 6 at dose level (DL) 1 (15mg/kg Tr + 25mg T) and 7 (1 died from disease before DLT assessment) at DL -1 (15mg/kg Tr + 20mg T). Median age was 57yrs, ECOG 0-1, median previous chemotherapy lines 3 (range 1-8). In DL 1, 1/6 pts experienced DLT (Grade (Gr) 2 pneumonitis). In view of frequent Gr2 adverse events (AEs) in DL 1, DL -1 was evaluated with DLTs in 2/6 evaluable pts (Gr3 mucositis and intolerable Gr2 limb edema preventing start of cycle 2 within 14 days). The most common related AEs (all Gr across both DL) were: fatigue (77%), edema (69%), anorexia (62%), and nausea (54%). Common Gr≥3 AEs included lymphopenia (23%) and fatigue (23%). Of 10 evaluable pts, best RECIST responses were: 1 breast cancer pt (ER+/ HER2-/ PIK3CA mutant) with PR (now in cycle 9), 7 pts with SD, and 2 pts with PD. Four pts with ovarian cancer (1 PIK3CAmutant) had SD ≥11weeks with 2/3 pts (1 not evaluable) demonstrating GCIG response (>50% decrease in CA125). In preliminary analyses, TP expression in TEMs was decreased (mean -18%) in 4pts with tumor shrinkage, but increased (+6%) in 1pt with tumor growth, suggesting a trend between reduced TP and tumor response. Conclusions: The MTD was exceeded at 15mg/kg Tr and 20mg T weekly. The safety of 10mg/kg Tr and 20mg T weekly is currently being evaluated. The combination of Tr and T shows early signs of antitumor activity. TP expression in TEMs by flow cytometry as an early marker of treatment benefit warrants further evaluation. Clinical trial information: NCT01548482.

Abstract 4647: Tie2-expressing monocytes (TEMs) as potential biomarkers of angiopoietin-Tie2 (Ang/Tie2) directed therapies: correlative analysis of a phase I study of AMG386 + temsirolimus (T).

Abstract Background: The Ang/Tie2 pathway plays a critical role in tumor angiogenesis, and several agents targeting this axis are in development. Tie2 is expressed by monocytes (M), and TEMs may be angiogenic mediators. Thymidine phosphorylase (TP), an angiogenic enzyme, is increased in TEMs upon Tie2 stimulation. We used flow cytometry to evaluate TEMs in a phase I study of AMG386 (Ang1/2 peptibody) + T in patients (pts) with solid tumors. Methods: Peripheral blood was collected from healthy volunteers and pts with advanced cancer (n=10 each) for assay development. For phase I study pts (n=5 to date), blood was collected on Cycle 1 Day 1 (D1), D3 and D8. Extracellular Tie2 staining was performed after hypotonic red blood cell lysis. TP staining followed formaldehyde fixation and Triton X-100 permeabilization. M and lymphocytes (L) were gated with CD45 and CD33. Median fluorescence intensities (MFI) were measured and normalized by calculating a M/L ratio. Tumor response was assessed every 2 cycles. Results: Tie2 staining was present on all M (absent in other leukocytes) in healthy controls and cancer pts, with no discrete Tie2+/- populations. Tie2 and TP M/L were similar between these 2 groups. Tie2, TP and response data from the first 5 pts receiving AMG386 + T are presented. Preliminary results reveal an association between change in TP D1 to D3 and tumor response. TP M/L decreased in all pts with tumor shrinkage (mean -18%), but increased (+6%) in the pt with tumor growth. No consistent association between Tie2 M/L and response was observed. Conclusions: Measurement of Tie2 and TP in circulating M is feasible. No discrete Tie2+ M population is identifiable. Tie2 and TP staining are similar in cancer pts and healthy volunteers. Preliminary analyses reveal an association between an initial reduction in TP and tumor shrinkage in pts receiving AMG386+T. Enrolment continues and additional data will be presented. Tie2, TP and tumor response in the first 5 pts receiving AMG386+T Patient 1 2 3 4 5 Tie2 M/L D1 2.63 2.53 6.56 5.07 9.85 D3 2.58 2.41 6.07 4.94 13.87 D8 3.33 2.61 5.11 7.85 7.95 Tie2% Change D1 to D3 −2.1% -4.9% -7.5% -2.5% 40.7% TP M/L D1 7.41 5.49 8.98 6.59 5.43 D3 6.70 5.83 8.11 5.21 3.81 D8 11.07 9.21 9.19 5.70 5.63 TP% Change D1 to D3 -9.6% 6.2% -9.6% -20.8% -30.0% Best response in target lesions (% change) -25% 14% -31% -14% -7% This study is supported by the National Cancer Institute Grant # U01-CA-132123 Citation Format: David W. Cescon, Philippe L. Bedard, Sue Chow, Helen Chen, Albiruni Razak, Monika Wizemann, Lillian L. Siu, David Hedley. Tie2-expressing monocytes (TEMs) as potential biomarkers of angiopoietin-Tie2 (Ang/Tie2) directed therapies: correlative analysis of a phase I study of AMG386 + temsirolimus (T). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2013-4647

TIE2-expressing monocytes: A novel cellular biomarker for hepatocellular carcinoma?

Note: Editorial Reference EPFL-ARTICLE-186208doi:10.1002/hep.26025View record in Web of Science Record created on 2013-04-25, modified on 2017-05-12

Preliminary evidence of sustained expression of angiopoietin‐2 during monocyte differentiation in chronic hepatitis C

Monocytes are essential precursors of antigen-presenting cells, such as macrophages and dendritic cells, and contribute to the pathogenesis of chronic inflammatory diseases and cancer. As Tie2-expressing monocytes (TEMs) are increased in the peripheral blood of patients with chronic hepatitis C (CHC), we aimed to examine the expression of Tie2 and angiopoietins (Ang1 and Ang2) during monocyte differentiation and maturation in CHC. The expression of Tie2, CD11b, CD80, CD83, CD86 and MHC-II was measured by flow cytometry in peripheral blood monocytes and monocytes-derived cells (Mo-DCs) from nine healthy subjects and eight CHC patients whose HCV infection was unresolved after combination therapy. Ang1 and Ang2 levels were measured in cellular supernatants by ELISA. Mo-DCs from CHC patients expressed differential patterns of maturation markers compared with controls--primarily with regard to CD80. Tie2 was downregulated during monocyte differentiation in controls and CHC patients, whereas Ang1 expression was constant. However, Ang2 levels fell significantly during the differentiation of control monocytes, in contrast with those from CHC patients in whom Ang2 expression remained stable throughout differentiation. Altered expression of the Ang/Tie2 system in monocytes and Mo-DCs from CHC patients might account for the inflammatory and angiogenic disorders that are related to CHC. An increased understanding of Ang/Tie2 system regulation might be helpful in designing strategies to prevent CHC progression.

TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis

Angiogenesis is a critical step in the development and progression of hepatocellular carcinoma (HCC). Myeloid lineage cells, such as macrophages and monocytes, have been reported to regulate angiogenesis in mouse tumor models. TIE2, a receptor of angiopoietins, conveys pro-angiogenic signals and identifies a monocyte/macrophage subset with pro-angiogenic activity. Here, we analyzed the occurrence and kinetics of TIE2-expressing monocytes/macrophages (TEMs) in HCC patients. This study enrolled 168 HCV-infected patients including 89 with HCC. We examined the frequency of TEMs, as defined as CD14+CD16+TIE2+ cells, in the peripheral blood and liver. The localization of TEMs in the liver was determined by immunofluorescence staining. Micro-vessel density in the liver was measured by counting CD34+ vascular structures. We found that the frequency of circulating TEMs was significantly higher in HCC than non-HCC patients, while being higher in the liver than in the blood. In patients who underwent local radio-ablation or resection of HCC, the frequency of TEMs dynamically changed in the blood in parallel with HCC recurrence. Most TEMs were identified in the perivascular areas of tumor tissue. A significant positive correlation was observed between micro-vessel density in HCC and frequency of TEMs in the blood or tumors, suggesting that TEMs are involved in HCC angiogenesis. Receiver operating characteristic analyses revealed the superiority of TEM frequency to AFP, PIVKA-II and ANG-2 serum levels as diagnostic marker for HCC. TEMs increase in patients with HCC and their frequency changes with the therapeutic response or recurrence. We thus suggest that TEM frequency can be used as a diagnostic marker for HCC, potentially reflecting angiogenesis in the liver.

TIE2-expressing monocytes regulate revascularisation of the ischaemic limb

BackgroundMonocytes (CD14+ cells) expressing the receptor TIE2 are a highly angiogenic subset that are pivotal to neovascularisation in the tumour environment. We hypothesised that TIE2-expressing monocytes (TEMs) are also important in neovascularisation of ischaemic tissues. MethodsFlow cytometry was used to quantify circulating TEMs in 40 patients with critical limb ischaemia (20 age-matched and 20 healthy controls). RT-PCR was used to confirm TIE2 expression in FACS-sorted TEMs. ELISA was used to measure circulating levels of the TIE2 ligand angiopoietin 2 (ANG2). Mice were subjected to hindlimb ischaemia and TEMs quantified. In an additional study, haemopoietic stem/progenitor cells, isolated from Pgk-rtTA-miR-126T transgenic mice, were transduced ex vivo with a TRE-miR-Tie2-OFP lentiviral vector and used to reconstitute lethally irradiated mice. These mice were treated with alternate daily doses of doxycycline to silence Tie2 expression on TEMs, and hindlimb ischaemia (HLI) was induced. Conversely, bone-marrow-derived macrophages (BMDMs) were enforced to express Tie2 using a Pgk-Tie2 lentivirus and delivered into the ischaemic hindlimb. Recovery of ischaemia was measured with laser Doppler. FindingsFlow cytometry revealed a ten-fold higher number of circulating TEMs in patients with critical limb ischaemia than in matched controls (mean 3·52% [SE 0·28] vs 0·39 [0·09], p<0·0001). Revascularisation or amputation resulted in a fall in TEM numbers to control levels (p<0·005). Analysis by RT-PCR confirmed TIE2 mRNA expression in TEMs. Circulating ANG2 levels were two-fold higher in patients with critical limb ischaemia than in controls (mean 4354 pg/mL [SE 661] vs 1973 [247], p<0·05). Circulating CD115+/Tie2+ monocyte numbers were higher following hindlimb ischaemia in mice than in sham controls (p<0·05) and Tie2 expression was upregulated in CD45+/CD11b+/F4/80+ macrophages isolated from ischaemic hindlimbs (p<0·05). Tie2 gene knockdown in TEMs inhibited neovascularisation of the ischaemic hindlimb (p<0·0001). Delivery of Tie2-expressing BMDMs into the ischaemic limb accelerated the recovery of blood flow compared with treatment with control BMDMs. InterpretationOur studies suggest that TEMs are mobilised following ischaemia and contribute to the revascularisation of ischaemic tissue, and that TIE2 is important for the proangiogenic functios of TEMs. TEMs may represent a promising, novel therapeutic target for cell therapy in critically ischaemic tissues. FundingBritish Heart Foundation and Royal College of Surgeons of England.