Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep 2;116(9):1539-47.
doi: 10.1182/blood-2009-06-230474. Epub 2010 May 14.

Leukemia Regression by Vascular Disruption and Antiangiogenic Therapy

Affiliations
Free PMC article

Leukemia Regression by Vascular Disruption and Antiangiogenic Therapy

Gerard J Madlambayan et al. Blood. .
Free PMC article

Abstract

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

Figures

Figure 1
Figure 1
Effects of OXi4503 and bevacizumab on subcutaneous leukemic chloromas. NOD/scid/IL2Rγ−/− (NOG) mice were subcutaneously inoculated with KG-1 human acute myelogenous leukemia (AML) cells. After chloromas were palpable mice were treated with intraperitoneal injections of bevacizumab, OXi4503, combination (OXi+Bev), or controls. Leukemia growth was measured every other day. (A) OXi4503 alone decreased leukemia growth in comparison to controls. Combination Oxi4503+Bev treatment resulted in regression of cancer size. Bevacizumab alone had no effect on tumor growth. (B) Comprehensive staining of chloromas was performed for: H&E (scale bar: 200 μm), TUNEL/MECA-32 (scale bar: 100 μm), (TUNEL/CD45 (scale bar: 50 μm), and CD45/VEGF-A (scale bar: 100 μm). Sections showed that OXi4503 monotherapy led to chloromas with central cores made up mainly of nonvascularized, TUNEL+ apoptotic cells and viable rims (outlined by dotted lines) containing MECA-32+ blood vessels at the periphery of leukemias. VEGF-A expression was also observed in viable rims. Combination therapy eliminated the viable rim resulting in widespread apoptosis and a lack of intact blood vessels throughout the tumor mass. Bevacizumab-treated tumors showed no difference in staining compared with controls. (C) Quantification of microvessels based on MECA-32+ blood vessels revealed decreased density within leukemic cores of mice treated with OXi4503 and combination therapy in comparison to bulk control tumors. Blood vessels within viable leukemia rims are increased after OXi4503 treatment but significantly decreased with additional bevacizumab treatment. Values represent mean ± SEM. *P < .05.
Figure 2
Figure 2
OXi4503 induced regression of systemic AML in bone marrow. Irradiated NOG mice were transplanted with human AML of differing subtypes (M1/2, M4, and M5) including a primary human AML specimen that harbored a high-risk FLT3 ITD mutation and after verification of leukemia engraftment were randomly assigned to 1 of 4 treatment cohorts. (A) Six weeks after AML transplant, NOG mice were treated with bevacizumab, OXi4503, combination (OXi4503+Bev), or controls. After 2 weeks of treatment, bone marrow showed persistence of AML in control and bevacizumab-treated mice. However, incidences of AML engraftment were significantly decreased with OXi4503 (1/8 positive) and combination treatment (1/11 positive) in comparison to controls. Shown are representative flow cytometric plots showing leukemic engraftment in control and bevacizumab-treated mice and no leukemic engraftment in Oxi4503 and combination-treated mice. (B) Quantification of AML engraftment by flow cytometry showed significantly decreased engraftment in OXi4503 and combination-treated animals versus controls. (C) PCR analysis for FLT3 ITD AML revealed molecular remissions (*) of high-risk FLT3 ITD+ AML in 40% of OXi4503 and combination-treated animals. (D-E) TUNEL staining showed no detectable apoptotic response to bevacizumab (D); however, a hypoxia-mediated reaction was observed upon HIF-1α staining (E). CD45+ cells were observed throughout bone marrow sections (scale bar: 100 μm). Values represent mean ± SEM. Gating was established using appropriate isotype controls.
Figure 3
Figure 3
Effects of OXi4503 treatment on bone marrow blood vessels and leukemia cells. (A) MECA-32 staining demonstrated abundant blood vessels throughout bone marrow sections regardless of treatment type (scale bar: 50 μm). (B) Quantification of microvessel density was performed for all treatment and control groups. An additional control group without leukemia transplantation was added to assess the effects of leukemia on microvessel density. * represents significance in comparison to control (−)leukemia group; **, significance in comparison to control (+)leukemia group; ***, significance in comparison to bevacizumab alone group; and ****, significance in comparison to OXi4503 alone group. (C) Leukemic KG-1 cells were incubated with OXi4500 at differing concentrations for 24 hours and the numbers of viable cells quantified using trypan blue dye exclusion. Under these conditions, concentrations of 50nM or higher showed significant decreases in cell viability versus controls. (D) OXi4500 treatment induces apoptosis of leukemic KG-1 cells. After a 24-hour treatment with OXi4500, cells were stained with annexin V and PI and the percentage of apoptotic cells determined using flow cytometry. (E) The generation of ROS was assessed in KG-1 cells at different OXi4500 concentrations by H2DCFDA staining. After 48 hours, ROS were detected at concentrations of 50nM or higher. Relative fluorescent intensity values were normalized to controls (0nM OXi4500). Values represent mean ± SEM; *P < .05; **P < .05; ***P < .05; and ****P < .05. Gating was established using appropriate isotype controls.

Comment in

Similar articles

See all similar articles

Cited by 23 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback