Meenal Datta

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.

Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery

Significance Tuberculosis (TB) is the second most lethal pathogen worldwide. Pulmonary granulomas are a hallmark of this disease. By discovering similarities between granulomas and solid cancerous tumors, we identified a novel therapeutic target for TB, the abnormal granuloma-associated vasculature that contributes to the abnormal granuloma microenvironment. We then asked if we could “normalize” granuloma vasculature by blocking VEGF signaling, an approach originally shown to enhance cancer treatment. Our results demonstrate that bevacizumab, a widely prescribed anti-VEGF antibody for cancer and eye diseases, is able to create more structurally and functionally normal granuloma vasculature and improve the delivery of a low-molecular-weight tracer. This effect suggests that vascular normalization in combination with anti-TB drugs has the potential to enhance treatment in patients with TB. Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can “normalize” their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens.

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.

Solid stress and elastic energy as measures of tumour mechanopathology

Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that two-dimensional spatial mappings of solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and wide size ranges can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; (ii) solid stress increases with tumour size; and (iii) mechanical confinement by the surrounding tissue significantly contributes to intratumoural solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies.

Mathematical Model of Oxygen Transport in Tuberculosis Granulomas

Pulmonary granulomas—the hallmark of Mycobacterium tuberculosis (MTB) infection—are dense cellular lesions that often feature regions of hypoxia and necrosis, partially due to limited transport of oxygen. Low oxygen in granulomas can impair the host immune response, while MTB are able to adapt and persist in hypoxic environments. Here, we used a physiologically based mathematical model of oxygen diffusion and consumption to calculate oxygen profiles within the granuloma, assuming Michaelis–Menten kinetics. An approximate analytical solution—using a priori and newly estimated parameters from experimental data in a rabbit model of tuberculosis—was able to predict the size of hypoxic and necrotic regions in agreement with experimental results from the animal model. Such quantitative understanding of transport limitations can inform future tuberculosis therapeutic strategies that may include adjunct host-directed therapies that facilitate oxygen and drug delivery for more effective treatment.

Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green

Significance Imaging in the shortwave IR (SWIR) spectral window allows the observation of processes deep within living animals. Recent studies have shown that SWIR imaging enables unprecedented imaging opportunities, including contact-free monitoring of vital signs, generation of microvasculature blood flow maps, real-time metabolic imaging, and molecularly targeted imaging. Yet, whereas bright SWIR fluorophores have been developed for preclinical research settings, applications in the clinic have been held back by the conventional belief that no clinically approved fluorophore is available. Here, we show that indocyanine green, a clinically approved near-IR dye, exhibits a remarkable amount of SWIR emission, which enables state-of-the-art SWIR imaging with direct translation potential into clinical settings, and even outperforms other commercially available SWIR emitters. Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000–2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

Quantifying solid stress and elastic energy from excised or in situ tumors

Solid stress, distinct from both tissue stiffness and fluid pressure, is a mechanical stress that is often elevated in both murine and human tumors. The importance of solid stress in tumor biology has been recognized in initial studies: solid stress promotes tumor progression and lowers the efficacy of anticancer therapies by compressing blood vessels and contributing to hypoxia. However, robust, reproducible, and objective methods that go beyond demonstration and bulk measurements have not yet been established. We have developed three new techniques to rigorously measure and map solid stress in both human and murine tumors that are able to account for heterogeneity in the tumor microenvironment. We describe here these methods and their independent advantages: 2D spatial mapping of solid stress (planar-cut method), sensitive estimation of solid stress in small tumors (slicing method), and in situ solid-stress quantification (needle-biopsy method). Furthermore, the preservation of tissue morphology and structure allows for subsequent histological analyses in matched tumor sections, facilitating quantitative correlations between solid stress and markers of interest. The three procedures each require ∼2 h of experimental time per tumor. The required skill sets include basic experience in tumor resection and/or biopsy (in mice or humans), as well as in intravital imaging (e.g., ultrasonography).

Anti-VEGF treatment improves neurological function in tumors of the nervous system

Research of various diseases of the nervous system has shown that VEGF has direct neuroprotective effects in the central and peripheral nervous systems, and indirect effects on improving neuronal vessel perfusion which leads to nerve protection. In the tumors of the nervous system, VEGF plays a critical role in tumor angiogenesis and tumor progression. The effect of anti-VEGF treatment on nerve protection and function has been recently reported - by normalizing the tumor vasculature, anti-VEGF treatment is able to relieve nerve edema and deliver oxygen more efficiently into the nerve, thus reducing nerve damage and improving nerve function. This review aims to summarize the divergent roles of VEGF in diseases of the nervous system and the recent findings of anti-VEGF therapy in nerve damage/regeneration and function in tumors, specifically, in Neurofibromatosis type 2 associated schwannomas.

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.