Immunohistochemistry study of tumor vascular normalization and anti-angiogenic effects of sunitinib versus bevacizumab prior to dose-dense doxorubicin/cyclophosphamide chemotherapy in HER2-negative breast cancer

Purpose Tumor angiogenesis controlled predominantly by vascular endothelial growth factor and its receptor (VEGF-VEGFR) interaction plays a key role in the growth and propagation of cancer cells. However, the newly formed network of blood vessels is disorganized and leaky. Pre-treatment with anti-angiogenic agents can “normalize” the tumor vasculature allowing effective intra-tumoral delivery of standard chemotherapy. Immunohistochemistry (IHC) analysis was applied to investigate and compare the vascular normalization and anti-angiogenic effects of two commonly used anti-angiogenic agents, Sunitinib and Bevacizumab, administered prior to chemotherapy in HER2-negative breast cancer patients. Methods This prospective clinical trial enrolled 38 patients into a sunitinib cohort and 24 into a bevacizumab cohort. All received 4 cycles of doxorubicin/cyclophosphamide chemotherapy and pre-treatment with either sunitinib or bevacizumab. Tumor biopsies were obtained at baseline, after cycle 1 (C1) and cycle 4 (C4) of chemotherapy. IHC was performed to assess the tumor vascular normalization index (VNI), lymphatic vessel density (LVD), Ki67 proliferation index and expression of tumor VEGFR2. Results In comparison to Bevacizumab, Sunitinib led to a significant increase in VNI post-C1 and C4 (p < 0.001 and 0.001) along with decrease in LVD post-C1 (p = 0.017). Both drugs when combined with chemotherapy resulted in significant decline in tumor proliferation after C1 and C4 (baseline vs post-C4 Ki67 index p = 0.006 for Sunitinib vs p = 0.021 for Bevacizumab). Bevacizumab resulted in a significant decrease in VEGFR2 expression post-C1 (p = 0.004). Conclusion Sunitinib, in comparison to Bevacizumab showed a greater effect on tumor vessel modulation and lymphangiogenesis suggesting that its administration prior to chemotherapy might result in improved drug delivery. Trial registry ClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish

Mycobacterial granuloma formation involves significant stromal remodeling and the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as anti-angiogenic or vascular normalizing therapies are beneficial host-directed therapies in pre-clinical models of tuberculosis. Here we demonstrate that vascular normalization through vascular endothelial-protein tyrosine phosphatase inhibition decreases granuloma hypoxia, the opposite effect of hypoxia-inducing anti-angiogenic therapy. Vascular normalization leads to increased T cell and decreased neutrophil recruitment to granulomas, correlates of a protective immune response against mycobacterial infection.

Hypoxia Regulated Inhibin Promotes Tumor Growth and Vascular Permeability By ACVRL1 and CD105 Dependent VE-Cadherin Internalization

Hypoxia, a driver of tumor growth and metastasis, regulates angiogenic pathways that are targets for vessel normalization and ovarian cancer management. However, toxicities and resistance to anti-angiogenics limits their use making identification of new targets vital. Inhibin, a heteromeric TGFb ligand, is a contextual regulator of tumor progression acting as an early tumor suppressor, yet also an established biomarker for ovarian cancers. Here, we demonstrate a previously unknown role for inhibins and find that hypoxia increases inhibin levels in ovarian cancer cell lines, xenograft tumors, and patients. Inhibin is regulated specifically through HIF-1, shifting the balance from activins to inhibins. Hypoxia regulated inhibin promotes tumor growth, endothelial cell invasion and permeability. Targeting inhibin in vivo through knockdown and anti-inhibin strategies robustly reduces permeability in vivo and alters the balance of pro and anti-angiogenic mechanisms resulting in vascular normalization. Mechanistically, inhibin regulates permeability by increasing VE-cadherin internalization via ACVRL1 and CD105, a receptor complex that we find stabilized directly by inhibin. Our findings are the first to demonstrate direct roles for inhibins in vascular normalization via TGF-b receptors providing new insights into the therapeutic significance of inhibins as a strategy to normalize the tumor vasculature in ovarian cancer.

Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization

SHP2 mediates the activities of multiple receptor tyrosine kinase signaling and its function in endothelial processes has been explored extensively. However, genetic studies on the role of SHP2 in tumor angiogenesis have not been conducted. Here, we show that SHP2 is activated in tumor endothelia. Shp2 deletion and pharmacological inhibition reduce tumor growth and microvascular density in multiple mouse tumor models. Shp2 deletion also leads to tumor vascular normalization, indicated by increased pericyte coverage and vessel perfusion. SHP2 inefficiency impairs endothelial cell proliferation, migration, and tubulogenesis through downregulating the expression of proangiogenic SRY-Box transcription factor 7 (SOX7), whose re-expression restores endothelial function in SHP2-knockdown cells and tumor growth, angiogenesis, and vascular abnormalization in Shp2-deleted mice. SHP2 stabilizes apoptosis signal-regulating kinase 1 (ASK1), which regulates SOX7 expression mediated by c-Jun. Our studies suggest SHP2 in tumor associated endothelial cells is a promising anti-angiogenic target for cancer therapy.

Immunohistochemistry Study of Tumor Vascular Normalization and Anti-angiogenic Effects of Sunitinib Versus Bevacizumab Prior to Dose Dense Doxorubicin/cyclophosphamide Chemotherapy in HER2 Negative Breast Cancer

PurposeTumor angiogenesis controlled predominantly by vascular endothelial growth factor and its receptor (VEGF-VEGFR) interaction plays a key role in the growth and propagation of cancer cells. However, the newly formed network of blood vessels is disorganized and leaky. Pre-treatment with anti-angiogenic agents can “normalize” the tumor vasculature allowing effective intra-tumoral delivery of standard chemotherapy.Immunohistochemistry (IHC) analysis was applied to investigate and compare the vascular normalization and anti-angiogenic effects of two commonly used anti-angiogenic agents, Sunitinib and Bevacizumab, administered prior to chemotherapy in HER2 negative breast cancer patients.MethodsThis prospective clinical trial enrolled 38 patients into a sunitinib cohort and 24 into a bevacizumab cohort. All received 4 cycles of doxorubicin/cyclophosphamide chemotherapy and pre-treatment with either sunitinib or bevacizumab. Tumor biopsies were obtained at baseline, after cycle 1 (C1) and cycle 4 (C4) of chemotherapy. IHC was performed to assess the tumor vascular normalization index (VNI), lymphatic vessel density (LVD), Ki67 proliferation index and expression of tumor VEGFR2. ResultsIn comparison to Bevacizumab, Sunitinib led to a significant increase in VNI post C1 and C4 (p <0.001 and 0.001) along with decrease in LVD post C1 (p= 0.017). Both drugs when combined with chemotherapy resulted in significant decline in tumor proliferation after C1 and C4 (baseline vs post C4 Ki67 index p=0.006 for Sunitinib vs p=0.021 for Bevacizumab). Bevacizumab resulted in a significant decrease in VEGFR2 expression post C1 (p=0.004). ConclusionSunitinib, in comparison to Bevacizumab showed a greater effect on tumor vessel modulation and lymphangiogenesis suggesting that its administration prior to chemotherapy might result in improved drug delivery.Clinical trial registrationClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

Combined Treatment with Acalabrutinib and Rapamycin Inhibits Glioma Stem Cells and Promotes Vascular Normalization by Downregulating BTK/mTOR/VEGF Signaling

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with a median duration of survival of approximately 14 months after diagnosis. High resistance to chemotherapy remains a major problem. Previously, BTK has been shown to be involved in the intracellular signal transduction including Akt/mTOR signaling and be critical for tumorigenesis. Thus, we aim to evaluate the effect of BTK and mTOR inhibition in GBM. We evaluated the viability of GBM cell lines after treatment with acalabrutinib and/or rapamycin through a SRB staining assay. We then evaluated the effect of both drugs on GBM stem cell-like phenotypes through various in vitro assay. Furthermore, we incubated HUVEC cells with tumorsphere conditioned media and observed their angiogenesis potential, with or without treatment. Finally, we conducted an in vivo study to confirm our in vitro findings and analyzed the effect of this combination on xenograft mice models. Drug combination assay demonstrated a synergistic relationship between acalabrutinib and rapamycin. CSCs phenotypes, including tumorsphere and colony formation with the associated expression of markers of pluripotency are inhibited by either acalabrutinib or rapamycin singly and these effects are enhanced upon combining acalabrutinib and rapamycin. We showed that the angiogenesis capabilities of HUVEC cells are significantly reduced after treatment with acalabrutinib and/or rapamycin. Xenograft tumors treated with both drugs showed significant volume reduction with minimal toxicity. Samples taken from the combined treatment group demonstrated an increased Desmin/CD31 and col IV/vessel ratio, suggesting an increased rate of vascular normalization. Our results demonstrate that BTK-mTOR inhibition disrupts the population of GBM-CSCs and contributes to normalizing GBM vascularization and thus, may serve as a basis for developing therapeutic strategies for chemoresistant/radioresistant GBM.

Vascular Normalization: A New Window Opened for Cancer Therapies

Preclinical and clinical antiangiogenic approaches, with multiple side effects such as resistance, have not been proved to be very successful in treating tumor blood vessels which are important targets for tumor therapy. Meanwhile, restoring aberrant tumor blood vessels, known as tumor vascular normalization, has been shown not only capable of reducing tumor invasion and metastasis but also of enhancing the effectiveness of chemotherapy, radiation therapy, and immunotherapy. In addition to the introduction of such methods of promoting tumor vascular normalization such as maintaining the balance between proangiogenic and antiangiogenic factors and targeting endothelial cell metabolism, microRNAs, and the extracellular matrix, the latest molecular mechanisms and the potential connections between them were primarily explored. In particular, the immunotherapy-induced normalization of blood vessels further promotes infiltration of immune effector cells, which in turn improves immunotherapy, thus forming an enhanced loop. Thus, immunotherapy in combination with antiangiogenic agents is recommended. Finally, we introduce the imaging technologies and serum markers, which can be used to determine the window for tumor vascular normalization.

Recent Advances in Glioma Therapy: Combining Vascular Normalization and Immune Checkpoint Blockade

Glioblastoma (GBM) accounts for more than 50% of all primary malignancies of the brain. Current standard treatment regimen for GBM includes maximal surgical resection followed by radiation and adjuvant chemotherapy. However, due to the heterogeneity of the tumor cells, tumor recurrence is often inevitable. The prognosis of patients with glioma is, thus, dismal. Glioma is a highly angiogenic tumor yet immunologically cold. As such, evolving studies have focused on designing strategies that specifically target the tyrosine kinase receptors of angiokines and encourage immune infiltration. Recent promising results from immunotherapies on other cancer types have prompted further investigations of this therapy in GBM. In this article, we reviewed the pathological angiogenesis and immune reactivity in glioma, as well as its target for drug development, and we discussed future directions in glioma therapy.