Giorgio Seano

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

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.

Role of vascular density and normalization in response to neoadjuvant bevacizumab and chemotherapy in breast cancer patients.

Significance Emerging evidence indicates patients who benefit from antiangiogenic therapies have improved vessel function. To determine how bevacizumab modulates vessel morphology to improve vessel function we conducted a phase II trial of preoperative bevacizumab followed by bevacizumab combined with chemotherapy in HER2-negative breast cancer patients. Our results suggest that the clinical response to bevacizumab may occur through an increase in the extent of vascular normalization primarily in patients with a high baseline tumor microvessel density. If validated, these observations suggest approaches to improve antiangiogenic therapy and to identify patients likely to benefit. Preoperative bevacizumab and chemotherapy may benefit a subset of breast cancer (BC) patients. To explore potential mechanisms of this benefit, we conducted a phase II study of neoadjuvant bevacizumab (single dose) followed by combined bevacizumab and adriamycin/cyclophosphamide/paclitaxel chemotherapy in HER2-negative BC. The regimen was well-tolerated and showed a higher rate of pathologic complete response (pCR) in triple-negative (TN)BC (11/21 patients or 52%, [95% confidence interval (CI): 30,74]) than in hormone receptor-positive (HR)BC [5/78 patients or 6% (95%CI: 2,14)]. Within the HRBCs, basal-like subtype was significantly associated with pCR (P = 0.007; Fisher exact test). We assessed interstitial fluid pressure (IFP) and tissue biopsies before and after bevacizumab monotherapy and circulating plasma biomarkers at baseline and before and after combination therapy. Bevacizumab alone lowered IFP, but to a smaller extent than previously observed in other tumor types. Pathologic response to therapy correlated with sVEGFR1 postbevacizumab alone in TNBC (Spearman correlation 0.610, P = 0.0033) and pretreatment microvascular density (MVD) in all patients (Spearman correlation 0.465, P = 0.0005). Moreover, increased pericyte-covered MVD, a marker of extent of vascular normalization, after bevacizumab monotherapy was associated with improved pathologic response to treatment, especially in patients with a high pretreatment MVD. These data suggest that bevacizumab prunes vessels while normalizing those remaining, and thus is beneficial only when sufficient numbers of vessels are initially present. This study implicates pretreatment MVD as a potential predictive biomarker of response to bevacizumab in BC and suggests that new therapies are needed to normalize vessels without pruning.

Modeling human tumor angiogenesis in a three-dimensional culture system

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.

The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85β.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

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.

Blockade of MMP14 Activity in Murine Breast Carcinomas: Implications for Macrophages, Vessels, and Radiotherapy

BACKGROUND Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.

Increased Expression of α6 Integrin in Endothelial Cells Unveils a Proangiogenic Role for Basement Membrane

The integrin alpha6 subunit is part of the alpha6beta1 and alpha6beta4 integrin complexes, which are known to be receptors for laminins and to mediate several biological activities such as embryogenesis, organogenesis, and invasion of carcinoma cells. However, the precise role of alpha6 integrin in angiogenesis has not yet been addressed. We observed that both vascular endothelial growth factor-A and fibroblast growth factor-2 strongly upregulate alpha6 integrin in human endothelial cells. Moreover, alpha6 integrin was positively modulated in angiogenic vessels in pancreatic neuroendocrine carcinoma. In this transgenic mouse model of spontaneous tumorigenesis, alpha6 integrin expression increased in the angiogenic stage, while being expressed at low levels in normal and hyperplastic tissue. We studied the functional role of alpha6 integrin during angiogenesis by lentivirus-mediated gene silencing and blocking antibody. Cell migration and morphogenesis on basement membrane extracts, a laminin-rich matrix, was reduced in endothelial cells expressing low levels of alpha6 integrin. However, we did not observe any differences in collagen matrices. Similar results were obtained in the aortic ring angiogenesis assay. alpha6 integrin was required for vessel sprouting on basement membrane gels but not on collagen gels, as shown by stably silencing this integrin in the murine aorta. Finally, a neutralizing anti-alpha6 integrin antibody inhibited in vivo angiogenesis in chicken chorioallantoic membrane and transgenic tumor mouse model. In summary, we showed that the alpha6 integrin participated in vascular endothelial growth factor-A and fibroblast growth factor-2-driven angiogenesis in vitro and in vivo, suggesting that it might be an attractive target for therapeutic approaches in angiogenesis-dependent diseases such as tumor growth.