Dynamic contrast-enhanced magnetic resonance imaging quantification of leukemia-induced changes in bone marrow vascular function

Abstract

In this study we show that dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can quantify the impact of human acute myeloid leukemia (AML) on bone marrow (BM) vasculature by optimizing an in vivo DCE-MRI method and assessing the method’s diagnostic and prognostic potential. AML, the most common acute leukemia in adults, is a very heterogeneous disease with regard to both its genetic basis and outcome of treatment. The BM microenvironment is important because leukemia cells are able to change the functions of normal tissues, favoring proliferation of the cells and protecting them from external harm. BM vascular remodeling induced by AML cells favors a more hypoxic microenvironment, and is thought to protect leukemia stem cells from therapy, being a poor prognostic factor in AML. Most preclinical studies use microscopy-based methods to assess environmental changes, namely vascular leakiness and perfusion, leaving little space for direct translation of the optimized methodology. In the clinic, perfusion imaging is an important tool in the diagnosis and management of various diseases. The advantages of using MRI for perfusion imaging are its superiority in terms of soft tissue contrast, absence of ionizing radiation (compared to computerized tomography and nuclear imaging such as positron emission tomography and single photon emission computed tomography) and the possibility of imaging larger body areas or even the whole body. DCE-MRI is a method commonly used to measure perfusion. DCEMRI quantifies signal changes due to the passing of a contrast agent through the imaging area. By modeling the signal kinetics, it is possible to quantify vascular parameters such as perfusion, leakiness, and blood volume. Even though it has been around for over 20 years, DCE-MRI has not yet been fully implemented in many clinical pathways because of the lack of standardization in image acquisition and analysis methods, rendering it mostly a research-based tool. We used DCE-MRI to quantify the impact of human AML on BM vasculature in an animal model. In-house MatLab scripts were used to quantify non-model-based parameters taken directly from the contrast agent kinetic curves (see below). The parameters quantified are illustrated in Figure 1A. Changes in the first part of the DCE curve reflect blood flow and blood volume, while changes in the second part of the DCE curve reflect changes in vascular permeability and extravascular space (Figure 1A). We have previously reported that leukemia induces BM vascular leakiness, using intravital imaging of the calvaria, which was not observed in mice injected with cord blood (Online Supplementary Figure S1A, B), when compared to healthy, age-matched mice that were not injected (NI). To test the sensitivity of DCE-MRI in quantifying leukemia-induced BM vascular dysfunction, three different leukemia cell lines were used (U937, HL60, and ML1), and mice were scanned at different stages of the disease (Online Supplementary Figure S1C). When compared to healthy, age-matched NI controls, leukemia mice showed altered BM DCE kinetics (Figure 1B), with reduced contrast enhancement (CE; BM vascular density), reduced wash-in rate (WiR; blood flow), increased initial wash-out rate and wash-out rate (iWoR and WoR; vascular permeability and extravascular space) (Figure 1C). BM vascular dysfunction was not dependent on high leukemia burden, as even low levels of leukemia engraftment (<20% mCD45hCD33 cells present in the BM) significantly altered BM vascular function (Figure 1C, open circles). Leukemia is known to promote angiogenesis, but with disorganized and leaky vessels. The reduced CE in our leukemia cohort is in agreement with this, as CE relates to the proportion of functional blood vessels per pixel. With deregulated BM vascular parameters correlating with aging in healthy mice (Online Supplementary Figure S1D), we compared younger (12-16 weeks) versus older (19-32 weeks) mice to understand whether age was affecting leukemia-induced BM vascular dysfunction. As shown in Online Supplementary Figure S1D, older, NI mice showed significantly reduced BM vascular density and increased BM vascular permeability, but we observed no effect of age in leukemia-injected mice (Figure 1C). This indicates that aging and leukemia seem to alter BM vascular permeability (WoR) and functional density (CE) to a similar extent. However, decreased BM blood flow/perfusion (WiR) seems to be a leukemia-specific effect, as it was the only parameter unaltered by aging (Online Supplementary Figure S1E). In fact, receiver operating characteristic (ROC) analysis highlighted WiR as the best parameter for distinguishing healthy from leukemic BM (Figure 1D; AUCWiR = 1.000). Differences in vascular density and function between different areas of the bone have been reported. Using intravital two-photon imaging of the calvaria, we confirmed that at early stages of the disease the areas of leukemia engraftment (GFP cells) coincided with areas of reduced perfusion and irregular vessel structure, confirming a heterogeneous vascular dysfunction linked with leukemia burden (Online Supplementary Figure S1F). For this reason, we hypothesize that averaged DCE-MRI parameters could have some limitations in heterogeneous and low-engrafted mice. To obtain a detailed picture of DCE-MRI-measured vascular aberrations and evaluate their spatial distribution along the femur, we performed pixel-by-pixel (pbp, with each pixel corresponding to an area of ~200 mm) analysis for three DCEMRI parameters: CE, WiR, and WoR. NI mice present a homogeneous distribution of functional vessels in the epiphysis and along the endosteum of the diaphysis (Figure 1E, CE); high vascular perfusion in the central marrow of the diaphysis (Figure 1E, WiR), and higher vascular permeability at the epiphysis and in proximity with the endosteum of the diaphysis. In the presence of AML, there is an aberrant, heterogeneous distribution of these parameters along the femur. In groups injected with HL60 and ML1, pbp analysis highlighted areas (pockets) with severe vascular dysfunction (Figure 1E, white rectangles), surrounded by less affected vasculature. Importantly, areas that showed lower functional vascular density (low CE) corresponded to areas in which both the vascular perfusion (WiR) and permeability (WoR) were mostly affected (Figure 1E, white rectangles). In the group injected with U937 cells, there were no obvious pockets of vascular dysfunction, with this being present throughout the femur. Thus, our pbp analysis approach was successful in resolving the spatial distribution of vascular parameters along the femur and unveiling the heterogeneity of vascular dysfunction in AML xenografts. To further test the clinical potential of BM DCE-MRI, we scanned mice engrafted with samples from AML patients (i.e., with patient-derived xenografts, PDX). Different AML samples had different engraftment capacities (Online Supplementary Figure S2A) and DCE kinetics (Figure 2A), echoing the heterogeneous nature of this dis-