Jonas Kloepper

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Significance Inhibition of the VEGF/VEGF receptor (VEGFR) pathway has failed to increase overall survival in phase III trials in patients with glioblastoma (GBM). Previously we identified the angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway as a potential driver of resistance to VEGF inhibition in GBM. Here we show that dual inhibition of VEGFRs and Ang-2 inhibits tumor growth and prolongs vessel normalization compared with VEGFR inhibition alone, resulting in improved survival in murine GBM models. Furthermore, by blocking macrophage recruitment, we demonstrate that macrophages contribute to the beneficial effects of dual therapy. Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Next-generation in vivo optical imaging with short-wave infrared quantum dots

For in vivo imaging, the short-wavelength infrared region (SWIR; 1000–2000 nm) provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging by the biomedical research community. Here, we introduce a class of high-quality SWIR-emissive indium-arsenide-based quantum dots (QDs) that are readily modifiable for various functional imaging applications, and that exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. To demonstrate the unprecedented combination of deep penetration, high spatial resolution, multicolor imaging and fast-acquisition-speed afforded by the SWIR QDs, we quantified, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generated detailed three-dimensional quantitative flow maps of the mouse brain vasculature.

Preclinical Efficacy of Ado-trastuzumab Emtansine in the Brain Microenvironment

BACKGROUND Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

The brain microenvironment triggers HER3-dependent de novo resistance to therapies targeting PI3K or HER2 in HER2-positive and/or PIK3CA-mutant breast cancer cells. No safe haven for metastases Although targeted therapies for cancer offer great promise, they are often much less effective against brain metastases than against peripheral tumors. This is generally attributed to the drugs’ difficulty in penetrating the blood-brain barrier, but Kodack et al. now demonstrate that this is not the only reason. The authors discovered that, at least in breast cancer, the brain microenvironment itself plays a role in treatment resistance in metastatic tumors. Using mouse models and human cancer samples, the researchers found increased expression of human epidermal growth factor receptor 3 (HER3) in breast cancer–associated brain lesions and showed that it facilitates the tumors’ survival in the presence of targeted treatment and that inhibiting can help overcome resistance to therapy. Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges

Immunotherapy has emerged as a major therapeutic modality in oncology. Currently, however, the majority of patients with cancer do not derive benefit from these treatments. Vascular abnormalities are a hallmark of most solid tumours and facilitate immune evasion. These abnormalities stem from elevated levels of proangiogenic factors, such as VEGF and angiopoietin 2 (ANG2); judicious use of drugs targeting these molecules can improve therapeutic responsiveness, partially owing to normalization of the abnormal tumour vasculature that can, in turn, increase the infiltration of immune effector cells into tumours and convert the intrinsically immunosuppressive tumour microenvironment (TME) to an immunosupportive one. Immunotherapy relies on the accumulation and activity of immune effector cells within the TME, and immune responses and vascular normalization seem to be reciprocally regulated. Thus, combining antiangiogenic therapies and immunotherapies might increase the effectiveness of immunotherapy and diminish the risk of immune-related adverse effects. In this Perspective, we outline the roles of VEGF and ANG2 in tumour immune evasion and progression, and discuss the evidence indicating that antiangiogenic agents can normalize the TME. We also suggest ways that antiangiogenic agents can be combined with immune-checkpoint inhibitors to potentially improve patient outcomes, and highlight avenues of future research.

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior

The cerebral cortex is essential for integration and processing of information that is required for most behaviors. The exquisitely precise laminar organization of the cerebral cortex arises during embryonic development when neurons migrate successively from ventricular zones to coalesce into specific cortical layers. While radial glia act as guide rails for projection neuron migration, pre-formed vascular networks provide support and guidance cues for GABAergic interneuron migration. This study provides novel conceptual and mechanistic insights into this paradigm of vascular-neuronal interactions, revealing new mechanisms of GABA and its receptor-mediated signaling via embryonic forebrain endothelial cells. With the use of two new endothelial cell specific conditional mouse models of the GABA pathway (Gabrb3ΔTie2-Cre and VgatΔTie2-Cre), we show that partial or complete loss of GABA release from endothelial cells during embryogenesis results in vascular defects and impairs long-distance migration and positioning of cortical interneurons. The downstream effects of perturbed endothelial cell-derived GABA signaling are critical, leading to lasting changes to cortical circuits and persistent behavioral deficits. Furthermore, we illustrate new mechanisms of activation of GABA signaling in forebrain endothelial cells that promotes their migration, angiogenesis and acquisition of blood-brain barrier properties. Our findings uncover and elucidate a novel endothelial GABA signaling pathway in the CNS that is distinct from the classical neuronal GABA signaling pathway and shed new light on the etiology and pathophysiology of neuropsychiatric diseases, such as autism spectrum disorders, epilepsy, anxiety, depression and schizophrenia.

Abstract LB-347: Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

OBJECTIVE: We aimed to enhance the efficacy of anti-VEGF therapy in glioblastoma (GBM) through additional inhibition of Angiopoietin-2 (Ang-2), a potential mediator of resistance to antiangiogenic therapy using VEGF inhibition. INTRODUCTION: Glioblastoma (GBM) is a uniformly lethal primary brain tumor affecting more than 12.000 patients every year in the US alone. The standard therapy regimen for this highly angiogenic tumor entity comprises maximal safe resection and chemoradiation with temozolomide. The addition of antiangiogenic (anti-VEGF) therapy to the standard of care regimen improved progression-free survival, but failed to improve overall survival of GBM patients. Preclinical and clinical data suggest that resistance to anti-VEGF therapy in GBM is mediated by Ang-2, making this pathway a potential target. EXPERIMENTAL DESIGN: We tested the effect of dual Ang-2/VEGF blockade with A2V on mouse survival using a syngeneic (Gl261) model and a human xenograft (MGG8) model, compared to anti-VEGF antibody therapy (B20). In addition, we used blood-based Gaussian Luciferase (GLUC) assays, immunohistochemistry and flow cytometry to measure changes in tumor growth, microvessel density (MVD), and immune microenvironment, respectively. RESULTS: Gl261 tumors have a highly abnormal tumor vasculature. In this model, treatment with A2V reduced MVD compared to B20. The decrease in MVD was due to a reduction in pericyte-low tumor vessels, while pericyte-high vessels were unaffected. These vascular changes were accompanied by reduced tumor burden and enhanced survival. Interestingly, in the MGG8 tumors, which have a vasculature similar to the normal brain, we detected no change in MVD after A2V treatment. Nevertheless, we found a reduced tumor burden and prolonged animal survival in the MGG8 model. Since vascular normalization may impact immune cell infiltration and function in tumors, we next evaluated these cell populations. We found that A2V therapy reduced pro-tumor M2 polarization of macrophages and microglia and reprogrammed these cells toward the M1 phenotype in both the Gl261 and MGG8 models. Collectively, our data indicate that therapy-induced anti-tumor immunity is mediated by M1-type macrophages but not by T-cell infiltration or function. CONCLUSION: Dual Ang-2/VEGF therapy with A2V reprogrammed macrophages and microglia from pro-tumor M2 toward the anti-tumor M1 phenotype in two GBM models, in addition to normalizing vasculature in tumors with abnormal vessels. These data indicate that dual anti-angiogenic therapy has the potential to overcome resistance to anti-VEGF therapy and confer clinical benefits in GBM patients through vascular and immuno-modulatory effects. Citation Format: Jonas Kloepper, Lars Riedemann, Zohreh Amoozgar, Giorgio Seano, Katharina H. Susek, Veronica Yu, Nisha Dalvie, Robin L. Amelung, Meenal Datta, Jonathan W. Song, Vasileios Askoxylakis, Jennie W. Taylor, Christine Lu-Emerson, Ana Batista, Nathaniel D. Kirkpatrick, Keehoon Jung, Matija Snuderl, Alona Muzikansky, Kay G. Stubenrauch, Oliver Krieter, Hiroaki Wakimoto, Lei Xu, Lance L. Munn, Dan G. Duda, Dai Fukumura, Tracy T. Batchelor, Rakesh K. Jain. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival. [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 LB-347.

Mechanisms of enhanced drug delivery in brain metastases with focused ultrasound-induced blood–tumor barrier disruption

Significance Improved penetration along with accurate prediction and mechanistic understanding of anticancer agent delivery across the blood–brain/blood–tumor barrier (BBB/BTB) are essential for the rational development of effective therapeutic strategies in intracranial malignancies. In this study, we provide insights in drug pharmacokinetics in brain metastases after focused ultrasound-induced BBB/BTB disruption by integrating quantitative microscopy with mathematical modeling. We demonstrate that focused ultrasound-induced BBB/BTB disruption contributes to enhanced interstitial convective transport in solid tumors, in addition to alleviating vascular barriers, and provide evidence of improved penetration of nontargeted and antibody-targeted chemotherapies. Together, our work provides a unified framework for prospective, quantitative, and mechanistic investigation of the penetration of anticancer drugs across the BBB/BTB in brain tumors. Blood–brain/blood–tumor barriers (BBB and BTB) and interstitial transport may constitute major obstacles to the transport of therapeutics in brain tumors. In this study, we examined the impact of focused ultrasound (FUS) in combination with microbubbles on the transport of two relevant chemotherapy-based anticancer agents in breast cancer brain metastases at cellular resolution: doxorubicin, a nontargeted chemotherapeutic, and ado-trastuzumab emtansine (T-DM1), an antibody–drug conjugate. Using an orthotopic xenograft model of HER2-positive breast cancer brain metastasis and quantitative microscopy, we demonstrate significant increases in the extravasation of both agents (sevenfold and twofold for doxorubicin and T-DM1, respectively), and we provide evidence of increased drug penetration (>100 vs. <20 µm and 42 ± 7 vs. 12 ± 4 µm for doxorubicin and T-DM1, respectively) after the application of FUS compared with control (non-FUS). Integration of experimental data with physiologically based pharmacokinetic (PBPK) modeling of drug transport reveals that FUS in combination with microbubbles alleviates vascular barriers and enhances interstitial convective transport via an increase in hydraulic conductivity. Experimental data demonstrate that FUS in combination with microbubbles enhances significantly the endothelial cell uptake of the small chemotherapeutic agent. Quantification with PBPK modeling reveals an increase in transmembrane transport by more than two orders of magnitude. PBPK modeling indicates a selective increase in transvascular transport of doxorubicin through small vessel wall pores with a narrow range of sizes (diameter, 10–50 nm). Our work provides a quantitative framework for the optimization of FUS–drug combinations to maximize intratumoral drug delivery and facilitate the development of strategies to treat brain metastases.