Time-course-dependent microvascular alterations in a model of ...

Leukemia (2008) 22, 59–65 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu

ORIGINAL ARTICLE Time-course-dependent microvascular alterations in a model of myeloid leukemia in vivo C Schaefer1,2, M Krause2, I Fuhrhop2, M Schroeder2, P Algenstaedt3, W Fiedler4, W Ru¨ther2 and N Hansen-Algenstaedt1,2 1

Department of Neurological Surgery, Spine Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Orthopaedics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3Endocrinology, Department of Internal Medicine III, University Medical Center Hamburg-Eppendorf, Hamburg, Germany and 4Oncology and Haematology, Department of Internal Medicine II, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2

Functional and morphological properties of tumor microcirculation play a pivotal role in tumor progression, metastasis and inefficiency of tumor therapies. Despite enormous insights into tumor angiogenesis in solid tumors, little is known about the time-course-dependent properties of tumor vascularization in hematologic malignancies. The aim of this study was to establish a model of myeloid leukemia, which allows long-term monitoring of tumor progression and associated microcirculation. Red fluorescent protein-transduced human leukemic cell lines (M-07e) were implanted into cranial windows of severe combined immunodeficient mice. Intravital microscopy was performed over 55 days to measure functional (microvascular permeability, tissue perfusion rate and leukocyte–endothelium interactions) and morphological vascular parameters (vessel density, distribution and diameter). Tumor progression was associated with elevated microvascular permeability and an initial angiogenic wave followed by decreased vessel density combined with reduced tissue perfusion due to loss in small vessels and development of heterogenous tumor vascularization. Following altered geometric resistance of microcirculation, leukocyte–endothelium interactions were more frequent without increased leukocyte extravasation. It was concluded that time-dependent alterations of leukemic tumor vascularization exhibit strong similarities to those found in solid tumors. The potential contribution to the development of barriers to drug delivery in leukemic tumors is discussed. Leukemia (2008) 22, 59–65; doi:10.1038/sj.leu.2404947; published online 27 September 2007 Keywords: angiogenesis; microcirculation; drug delivery; intravital microscopy; myeloid leukemia

about the properties of vasculature in hematologic malignancies. Recently published studies demonstrated the influence of tumor microenvironment on leukemic cell survival without addressing the influence of functional and morphological properties of microcirculation governing the metabolic microenvironment.12,13 The known linkage of tumor angiogenesis to tumor microenvironment and their contribution to cancer progression and drug resistance in solid tumors raise the question whether vascular properties exhibit comparable effects on hematologic malignancies. Pathological morphology of tumor vascularization in bone marrow of patients suffering from leukemia has been described comparable to those found in solid tumors.14 We hypothesized that the functional and morphological alterations in leukemic tumor vascularization might be similar to those found in solid tumors and might contribute to barriers to drug delivery in hematologic malignancies. This study reports long-term results of a newly established model of localized myeloid leukemia in severe combined immunodeficient mice. Therefore, red fluorescent proteintransduced human leukemic cell line (M-07e) and intravital microscopy were used. This technique allows non-invasive and continuous monitoring of microvascular properties, correlating functional parameters with morphological aspects. Tumor growth and alterations of functional and morphological vascular parameters from tumor onset until late-stage tumor disease are reported and their potential contribution to the development of barriers to drug delivery in leukemic tumors is discussed.

Introduction Functional and morphological properties of neoplastic microcirculation in solid tumors play a pivotal role in tumor progression, metastasis and inefficiency of tumor therapies.1–4 Attention has recently been directed to the role of bone marrow vascularity and angiogenic factors in hematologic disorders.5 Bone marrow vascular density is increased in 80% of patients suffering from leukemia.6 Leukemia cells exhibit an autocrine and paracrine secretion of angiogenic substances, for example, vascular endothelial growth factor and fibroblast growth factor, and expression of corresponding receptors.7–10 While angiogenesis in solid tumors and its influence on tumor microenvironment has been intensively investigated,2,4,11 little is known Correspondence: Dr N Hansen-Algenstaedt, Spine Center and Department of Orthopedics, University Medical Center HamburgEppendorf, Martinistrasse 52, Hamburg 20246, Germany. E-mail: [email protected] Received 4 July 2007; revised 6 August 2007; accepted 13 August 2007; published online 27 September 2007

Materials and methods

Tumor model A human myeloid leukemic cell line (M-07e, red fluorescent protein transduced)15,16 was implanted in cranial windows17 of 12-week-old, male, severe combined immunodeficient mice from our gnotobiotic colony. A suspension of 0.5 106 M-07e cells in 4 ml of nutrition media was used to implant the tumor cells on the cranial hemispheres before closing with a coverslip for non-invasive and continuous intravital microscopy. Tumor growth, functional and morphological vascular parameters were monitored on days 6, 13, 17, 25, 37, 43, 49 and 55 using fluorescence microscopy (n ¼ 6–34). Tumor area was measured using an image-processing system (NIH Image 1.62), when leukemic cells formed a tumor mass. Due to technical limitations of area determination at early tumor progression with disseminated cells, some of the tumors were omitted at day 6. All animal procedures were performed according to the German Animal Welfare Committee (assurance no. 57/00,

Microvascular alterations in myeloid leukemia C Schaefer et al

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Microvascular parameters Vessel density, vessel diameter, tissue perfusion rate, blood flow velocity and blood flow rate. To measure vessel density, vessel diameter, tissue perfusion rate, blood flow rate and blood flow velocity, 100 ml of fluorescein isothiocyanate-Dextran (MW ¼ 2 000 000 kDa, 10 mg ml-1; Sigma, St Louis, MO, USA) was injected through the tail vein to visualize functional vessels. Fluorescence images were recorded digitally and non-compressed for 20 s in five distinct regions and analyzed off-line. Using an image-processing system (NIH Image 1.62), vessel diameter and length were measured to calculate vessel density, defined as total length of vessels per unit area (cm cm2). Blood flow velocity (VRBC) was measured optoelectronically using a two-slit method (Exbem 3.0, Pixlock Muenster, Germany), corrected by a taking into account the decreasing blood flow velocity from the middle of the vessel to its wall. Corrected blood flow velocity (Vmean) was calculated as follows: Vmean ¼ VRBCa (a ¼ 1.3 for blood vessels o10 mm; linear extrapolation 1.3 oao1.6 for blood vessels 410 and o15 mm; and a ¼ 1.6 for blood vessels 415 mm).18 Blood flow rate (BFR), representing the perfusion of single vessel was calculated using Vmean and the diameter (D) of the vessel: BFR ¼ VmeanD2p/4. The tissue perfusion rate was obtained using vascular density, blood flow rate of single vessel and surface area as described previously.17

Microvascular permeability. Effective microvascular per-

meability was measured as described previously.17 Briefly, after the injection of tetramethylrhodamine isothiocyanate-labeled bovine serum albumin (MW ¼ 67 000 kDa, excitation wavelength 525–555 nm, emission wavelength 580–635 nm; Molecular Probes, Eugene, OR, USA) (10 mg ml1, 0.1 ml 25 g1 body weight), the fluorescence intensity was measured intermittently for 25 min and recorded digitally (PowerLab/200; ADInstruments Pty Ltd, Unit 6, Castle Hill, NSW 2154, Australia). The P-value was calculated as P ¼ (1–HT)V/S(1/(I0– Ib)dI/dt þ 1/K), where I is the average fluorescence intensity of the whole image, I0 is the value of I immediately after filling of all vessels by tetramethylrhodamine isothiocyanate-bovine serum albumin and Ib is the background fluorescence intensity. The average hematocrit (HT) of vessels is assumed to be equal to 19%.19 V and S are the total volume and surface area of vessels within the tissue volume covered by the surface image. The time constant of bovine serum albumin plasma clearance (K) is 9.1 103 s.20

Leukocyte–endothelium interaction. Leukocyte–endothelium interactions in vessels were monitored as described previously.17 Briefly, mice were injected intravenously with a bolus (20 ml) of 0.1% rhodamine-6G in 0.9% saline and leukocytes were visualized via an intensified charge-coupled device camera and recorded non-compressed digitally on a computer (Apple Power Macintosh, G4, Dual 500 MHz Power PC, 1 GB SDRAM). The numbers of rolling (Nr) and adhering (Na) leukocytes were counted for 30 s along a 100-mm segment of a vessel. Rolling was defined as a slow movement along the vessel wall (o50% of VRBC), whereas adhering was defined as a stable position at the vessel wall for 30 s. The total flux of cells for 30 s was also measured (Nt). The total leukocyte flux was normalized by the cross-sectional area of the individual vessel: leukocyte flux (cells mm2 s1) ¼ 106 Nt/(p(D/2)2 30 s). Leukemia

Rolling count, the ratio of rolling leukocytes (Nr) to total cell flux (Nt) was calculated as follows: rolling count (%) ¼ 100 Nr/ Nt. The mean leukocyte adhesion at the vessel wall was calculated by normalizing the vessel surface area: adhesion density (cells mm2) ¼ 106 Na/(pD 100 mm). Shear rate of individual blood vessel was measured to determine the forces of blood flow influencing the interactions between leukocytes and endothelium. Shear rate was calculated according to the following equation: shear rate (s1) ¼ 8 Vmean/D.

Statistical analysis The results are presented as mean7s.e.m. Statistical analysis was performed with StatView (Abacus, Berkeley, CA, USA) using the Mann–Whitney U-test for comparison of vascular parameters between different observation points after testing for outliers using the extreme Studentized deviate. Correlation of parameters was tested by Spearman rank correlation test. Statistical significance was based on P-values smaller than 5%.

Discussion Despite the growing evidence of involvement of the vasculature in hematologic malignancies,5,10,21,22 little is known about the functional and morphological alterations of microcirculation in these disorders. It is known that temporal and spatial varying, functional and morphological properties of tumorvasculature determine tumor progression and influence the drug delivery to neoplastic tissues.4,23 To address this multistep process subsequent to leukemic tumor growth, we established a long-term model of localized human myeloid leukemia in severe combined immunodeficient mice using intravital microscopy at an ectopic site. Leukemic tumor progression was associated with elevated microvascular permeability and an initial angiogenic wave followed by decreased vessel density combined with reduced tissue perfusion due to loss in small vessels and development of heterogenous tumor vascularization. Following altered geometric resistance of microcirculation, leukocyte– endothelium interactions were more frequent without increased leukocyte extravasation.

Biphasic growth of leukemic M-07e tumors Following tumor cell implantation in the cranial window, the leukemic M-07e cells formed a tumor mass on the brain surface (Figure 1), which showed an exponential growth initially (Figure 2a; Table 1). The increase in tumor area per time reached a maximum between days 17 and 25 (1.02 mm2 day1), with a decrease thereafter. The phenomenon of decreasing tumor growth during tumor progression is known for solid tumors and could be explained by focal necrosis due to imbalances in angiogenic/antiangiogenic substances with resulting spatially reduced tumor perfusion.24,25

Microvascular permeability is accompanied by angiogenic sprouting and might contribute to elevated interstitial pressure and impaired distribution of therapeutic agents Increased permeability is a prerequisite for angiogenesis allowing the extravasation of plasmaproteins and migration of endothelial cells2 and is a common phenomenon in solid tumors.26 Elevated vascular leakage results from the release of several cytokines through tumor cells and surrounding tumor stroma, for example vascular endothelial growth factor, leading


61

Figure 1 Macroscopic aspects of native (a–e) and fluorescent (red; f–j) tumor growth. Intravenous application of fluorescein isothiocyanatelabeled Dextran (MW ¼ 2 000 000 kDa) was used to visualize functional microcirculation (green; f–j). After tumor cell implantation (day 17), hemispheres and the typical microvascular anatomy, including the midlined sagittal sinus, can be visualized (a). The corresponding fluorescent image shows that M-07e cells (red) formed a tumor mass (f). Further tumor growth showed a subsequent loss of normal vessel architecture, which can be seen even at lower magnification ( 1.25) on days 25 (b and g), 37 (c and h), 43 (d and i) and 55 (e and j). On day 55, cranial surface was completely covered by leukemic tumor (e and j).

Figure 2 (a) Tumor growth (A) and microvascular permeability (P). Implantation of M-07e cells led to exponential increase in tumor area until day 37. Thereafter tumor area per time displayed a slower increase. Initial tumor growth phase was associated with a significant peak of microvascular permeability with a subsequent decrease from day 13 to day 25 reaching a plateau phase from day 25 to the end of the observation period (*Po0.05). (b) Vessel density (VD) and vessel diameter (D). Functional vessel density and diameter were measured after injection of fluorescein isothiocyanate-labeled Dextran. Vessel density increased significantly from day 6 to day 13 with a subsequent reduction from day 17 to day 25 and day 43, respectively. In contrast, vessel diameter showed an inverse slope with initially decreased vessel diameter from day 6 to day 13. Further tumor growth was associated with increased vessel diameters (*Po0.05 for VD and #Po0.05 for D). All values are presented as mean7s.e.m. (c) Vessel distribution. Analysis of vessel distribution, defined as the counts of vessels with defined diameters per observation area, revealed an increase in vessels with small diameter concordant with increased functional capillary density from day 6 to day 13 and day 17, respectively. The initial angiogenic wave was followed by a subsequent reduction in vessels with small diameters from day 17 to end of the observation period.

to loosening of interendothelial cell contacts and transcellular openings.2,26,27 Leukemia cells were reported to secrete autocrine and paracrine angiogenic substances, such as vascular endothelial growth factor and fibroblast growth factor, and express corresponding receptors.7–10 Implantation of leukemic M-07e cells was followed by a 1.5-fold increase in microvascular permeability compared to normal brain tissue on day 6 (Figure 2a; Table 1).17 Since the above-mentioned cytokines induce not only vascular leakage but also act angiogenic, the association between peak vascular permeability on days 13 and 17 and angiogenic sprouting corroborate observations made in solid tumors (Figures 2a and b; Table 1). The maximum permeability showed a subsequent decrease to a plateau phase beginning on day 25, still increased 10-fold compared to normal brain vessels (Figure 2a; Table 1).17 Increased vascular leakage, combined with altered geometric resistance as found in solid tumors, induce elevated interstitial pressure.17,28 This phenomenon leads to impaired drug delivery in neoplastic tissue.11 The stable increase in vascular permeability, found in leukemic tumors subsequent to day 25, might therefore result in impaired

distribution of therapeutic agents in leukemic infiltrations in bone marrow similar to that reported in solid tumors.26 Since the presented data demonstrate ectopic tumor growth at the brain surface and it is known that the same tumor entities develop different angiogenic phenotypes depending on the growth site,29,30 the effect on microvascular permeability should be further investigated at an orthotopic bone marrow model.

Initial angiogenic wave was followed by loss in small vessels and reduced tissue perfusionFpossible contribution to heterogenous microenvironment and barriers to drug delivery The vascular density of tumors has evolved to be a useful prognostic indicator of tumor progression in some tumor entities31,32 and is increased in bone marrow of patients with acute myeloid leukemia.21,33 Morphology of bone marrow vasculature in hematologic malignancies exhibits similar alterations as described for solid tumors.14 Despite the known impact of functional and morphological properties on tumor Leukemia


62 Table 1

Tumor growth and microvascular parameters Day 6

Day 13

Day 17

Day 25

Tumor area (mm ) Permeability ( 108 cm s1) Vessel density (cm cm2) Vessel diameter (mm) Tissue perfusion rate ( 104 ml cm2 s1) Blood flow rate ( 104 m3 s1) Blood flow velocity (mm s1) Shear rate (s1) Leukocyte density (cells mm2) Rolling count (%) Leukocyte flux (cells s1 mm2)

2.5670.38 (n ¼ 24) 4.7270.77 (n ¼ 28) 186.6875.78 (n ¼ 34) 11.7270.32 (n ¼ 34) 10.4470.72 (n ¼ 31) 5.4570.40 (n ¼ 30) 298.79714.88 (n ¼ 31) 137.01712.23 (n ¼ 33) 178.65721.61 (n ¼ 32) 3.7670.49 (n ¼ 32) 81367785 (n ¼ 33)

3.1870.94 (n ¼ 9) 40.1179.42 (n ¼ 10) 236.50714.70 (n ¼ 11) 9.5370.29 (n ¼ 10) 10.9271.28 (n ¼ 11) 4.8070.53 (n ¼ 11) 363.65727.65 (n ¼ 11) 230.57724.39 (n ¼ 11) 157.84732.23 (n ¼ 11) 3.6570.77 (n ¼ 11) 82697846 (n ¼ 10)

5.7770.90 (n ¼ 31) 28.0575.21 (n ¼ 32) 219.0671.21 (n ¼ 33) 9.7670.24 (n ¼ 32) 9.8770.96 (n ¼ 33) 4.5270.29 (n ¼ 32) 314.39718.06 (n ¼ 33) 145.3779.33 (n ¼ 29) 142.86729.85 (n ¼ 28) 5.9970.67 (n ¼ 28) 79797618 (n ¼ 28)

12.7471.43 (n ¼ 29) 17.0572.72 (n ¼ 29) 153.1379.28 (n ¼ 30) 9.9370.26 (n ¼ 29) 5.1970.60 (n ¼ 27) 3.5670.29 (n ¼ 28) 250.49713.62 (n ¼ 28) 136.3378.50 (n ¼ 29) 162.82725.06 (n ¼ 29) 5.7170.78 (n ¼ 30) 87477983 (n ¼ 30)

Tumor area (mm2) Permeability ( 108 cm s1) Vessel density (cm cm2) Vessel diameter (mm) Tissue perfusion rate ( 104 ml cm2 s1) Blood flow rate ( 104 m3 s1) Blood flow velocity (mm s1) Shear rate (s1) Leukocyte density (cells mm2) Rolling count (%) Leukocyte flux (cells s1 mm2)

Day 37 24.2071.78 (n ¼ 25) 17.5574.53 (n ¼ 25) 120.1976.15 (n ¼ 25) 12.5270.48 (n ¼ 25) 5.6670.55 (n ¼ 25) 4.3770.44 (n ¼ 24) 213.55718.25 (n ¼ 25) 111.4377.84 (n ¼ 21) 169.45717.59 (n ¼ 21) 9.1571.32 (n ¼ 22) 66787917 (n ¼ 22)

Day 43 25.9671.98 (n ¼ 11) 19.0674.10 (n ¼ 11) 116.3577.65 (n ¼ 11) 12.6170.69 (n ¼ 10) 4.9270.38 (n ¼ 11) 4.1770.37 (n ¼ 11) 213.85716.76 (n ¼ 11) 138.19715.01 (n ¼ 11) 154.99719.05 (n ¼ 10) 7.2970.99 (n ¼ 10) 63797795 (n ¼ 11)

Day 49 26.2371.76 (n ¼ 9) 17.7773.20 (n ¼ 8) 104.8572.71 (n ¼ 8) 13.0770.72 (n ¼ 9) 6.1271.17 (n ¼ 8) 5.5871.01 (n ¼ 8) 234.23725.86 (n ¼ 8) 108.42711.73 (n ¼ 9) 282.27737.15 (n ¼ 8) 6.3971.19 (n ¼ 9) 68517612 (n ¼ 9)

Day 55 29.8871.64 (n ¼ 7) 15.5873.27 (n ¼ 7) 108.8276.47 (n ¼ 7) 13.5970.72 (n ¼ 7) 3.8370.44 (n ¼ 6) 3.7370.51 (n ¼ 6) 182.97724.94 (n ¼ 7) 97.83711.48 (n ¼ 7) 320.23791.94 (n ¼ 7) 9.4470.99 (n ¼ 6) 73627512 (n ¼ 7)

2

All values are presented as mean7s.e.m. Number of observed animals (n).

progression, metabolic environment and tumor susceptibility to therapeutic interventions in solid tumors,34–36 the dynamic and subsequent alterations of the vasculature and microcirculatory parameters in hematologic malignancies have not been elucidated. M-07e cell implantation was followed by a significant increase in vascular density on days 13 and 17 compared to day 6, while mean vessel diameter decreased (Figure 2b; Table 1). Analysis of the vessel frequency distribution, a parameter that shows the frequency of vessel width in the observation area, revealed a time-dependent alteration in vessel distribution during tumor growth (Figure 2c). From day 6 to day 13 and day 17, an increase in vessels with a diameter between 4 and 5 mm was observed. From this observation, we concluded that the initial increase in vessel density resulted from newly established tumor vessels with small diameters. Following the initial ‘angiogenic wave’, vascular density showed a progressive decrease after day 17 resulting in reduced vessel density and approached asymptotically to values around 110 cm cm2 from day 37 to day 55 (Figure 2b; Table 1). Associated mean vessel diameter increased from day 25 until day 55. The ongoing tumor growth from day 25 to day 49, associated with decreasing vessel densities beyond the physiologic level of about 140 cm cm2,17 was represented by a blunted peak of vessels with diameters around 4–7 mm mirroring a loss in small vessels (Figure 2c). Furthermore, a shift in vessel frequency distribution toward higher vessel diameters was observed displaying typical dilated, tortuous and disorganized tumorvascularization as described for solid tumors.2,27,37 Although presence of vessels in tumor tissue are not necessarily correlated with increased interstitial pO2,38 one can conclude that reduced microvascular density results in hypoxic areas within the tumor and heterogenous microenvironment, since diffusion of oxygen is limited to 100 mm.39 The difference between increased bone marrow vascular density, found in leukemic patients,33 and decreased functional vascular density during tumor growth in our model is surprising. An explanation Leukemia

might be the influence of site-specific aspects on tumor vascularization. Depending on the anatomical site in which a tumor is grown, the angiogenic phenotype varies.30 Furthermore, this phenomenon might be explained through methodological differences in the way in which the data were obtained. In contrary to immunohistochemistry, the functional vessel density represents perfused vessels and not total endothelial cell count. The solid stress due to proliferating tumor cells might lead to collapse of small tumor vessels lacking stabilizing pericytes.28,40 This further underlines the necessity to acquire functional data along with morphological data to gain insights into the complex interactions of tumor microcirculation. Neoplastic tissue is perfused by blood passively due to the absence of pericytes around tumor vessels and lack of innervation of vessels. Assuming a constant blood pressure, the tumor perfusion can be described by the vascular density, vessel diameter and blood flow velocity since the flow resistance is not measurable. Despite the angiogenic sprouting described above and the increased blood flow velocity, the tissue perfusion rate of the leukemic tumor was unaltered until day 17 (Figure 3a; Table 1). Further analysis revealed that the mean blood flow rate in single vessel showed a trend toward lower values from day 6 to day 13 leading to a similar blood volume per time in total, distributed on more vessels (Figure 3a; Table 1). The actual observation demonstrates that angiogenesis is not necessarily associated with increased tissue perfusion and corroborates previous studies of solid tumors.41 Following the reduction in vessel density, which could be observed from day 17 onwards, the tissue perfusion decreased. The decrease in tissue perfusion was even more pronounced than the reduction in functional vessel density due to an associated reduction in blood flow velocity with a trend toward lowered blood flow rates in single vessel (Figure 3a; Table 1). The increase in vessel diameter from day 25 to day 37 could compensate the effect of lowered blood flow velocity as could be seen by slight increase in blood flow rate despite further loss in vascular density. Tissue perfusion rate remained unaltered from day 25 to the end of the


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Figure 3 (a) Tissue perfusion rate (TPR), blood flow rate (BFR) and blood flow velocity (Vmean). Despite the initial significant increase in Vmean, the tissue perfusion rate remained unaltered until day 17. Associated with decreasing Vmean and reduction in vessel density from day 13 to day 17, tissue perfusion rate decreased significantly from day 17 to day 25. Vmean showed a further significant decrease from day 25 to day 37 without effect on tissue perfusion rate due to increasing vessel diameter. Blood flow rate, representing the mean blood flow in single vessel, showed a significant decrease from day 6 to day 25 without further significant alterations (*Po0.05 for TPR and #Po0.05 for Vmean). (b) Shear rate (SR), leukocyte density (LD) and rolling count (RC). Initially increased shear rate, representing hydrodynamic force of blood flow at the vessel wall, was not associated with alterations in rolling count or leukocyte density. However, significantly decreased shear rate from day 13 to day 17 and day 37 was associated with augmented rolling count. Rolling count showed a significant correlation with shear rate (Po0.05). Leukocyte density increased at later stages of tumor growth in spite of unaltered shear rate from day 37 to day 55 implicating a higher level of adhesion molecules’ expression on leukocytes and vessel wall (*Po0.05 for shear rate; #Po0.05 for RC and yPo0.05 for LD). All values are presented as mean7s.e.m.

observation period. Interestingly, increase in tumor area per time was maximum between days 17 and 25, demonstrating that in spite of reduction in tissue perfusion rate, tissue perfusion was still sufficient enough to support tumor growth. The lowered blood flow velocity might be caused through an increased geometric resistance of the tumor vasculature due to a pathological branching pattern as occurring in solid tumors.17,27,41 Whether hematologic malignancies in bone marrow demonstrate similar reduction of tissue perfusion, as found in our ectopic tumor model, remains to be further elucidated. One can conclude that reduced vessel density alongside decreased tissue perfusion found in leukemic tumors might lead to an impaired drug delivery to neoplastic cells42 and an increase in hypoxic areas within the tumor with consecutive reduced efficiency of therapies.11,34

Time-dependent differences in leukocyte–endothelium interaction: possible reasons and implications to cellbased immune therapies Immunomediated antitumor strategies, based on cellular or vaccination approaches, have not been as effective in vivo as anticipated from results obtained in vitro. Leukocyte–endothelium interactions, representing a prerequisite for leukocyte extravasation into tumor stroma, are governed by hydrodynamic and adhesive forces. Both factors are known to be altered following tumor growth and associated angiogenesis in solid tumors.11 The shear rate, representing a parameter of hydrodynamic force of blood flow at the vessel wall, is decreased in solid tumors due to altered structure of vascular bed.17 Furthermore, there is growing evidence that tumor endothelium exhibits heterogeneity in adhesion molecules’ expression, for example, ICAM-1, VCAM-1 and E-selectin.43 Generally leukocyte–endothelium interactions are low in solid tumors.43 Since we found alterations to functional and morphological vascular properties in leukemic tumors, we hypothesized that these alterations have an impact on leukocyte–endothelium interactions similar to solid tumors. Therefore, we quantified interactions between leukocytes and vessel wall in vivo and correlated them with microcirculatory properties.

Similar to the observations made in solid tumors, leukemic M-07e tumor exhibited diminished rolling count compared to host tissue throughout the observation period.44 Shear rate showed a significant increase from day 6 to day 13 (Figure 3b; Table 1). This could be expected from increased blood flow velocity and reduced mean vessel diameter. In spite of the elevated shear rate, the rolling count, representing the initial step in leukocyte– endothelium interaction, remained unaltered (Figure 3b; Table 1). This might be mediated by the production of angiogenic factors, for example vascular endothelial growth factor, leading to increased adhesion molecule expression in pre-existing host vessels and attenuation of increased shear rate.45,46 A significant decrease in shear rate from day 13 to day 17 followed by a further trend toward lower values was observed. This trend was associated with a significant increase in leukocyte rolling at the vessel wall, while leukocyte density at the vessel wall remained unaltered from day 6 to day 43 (Figure 3b; Table 1). Further analysis revealed that rolling count, but not leukocyte density, was significantly correlated with shear rate, indicating that the increase in temporary leukocyte–endothelium interactions was due to lowered hydrodynamic force of blood flow at the vessel wall. This is consistent with observations in which leukocyte rolling is dependent on selectin expression, while the leukocyte arrest needs activation of adhesion molecules, for example integrins.47 Unaltered leukocyte density found in leukemic tumors indicates constant expression levels of adhesion molecules from day 5 to day 43. Later stages of tumor growth presented with a significant increase in leukocyte density at the vessel wall (Figure 3b; Table 1). This increase might be due to upregulation of adhesion molecules and reflects a late maturation process of tumor vascularization. We described this phenomenon of late-stage tumor vascularization previously in solid tumors.17 One can conclude that this latestage effect could be beneficial for cell-mediated therapies.48 However, this effect might also contribute to tumor progression by influencing proliferation and survival of neoplastic cells, as well as modulating neoplastic microenvironments.1 The temporal heterogeneity in leukocyte–endothelium interactions of leukemic tumor microcirculation underlines the necessity of long-term measurements and remains to be further investigated. Leukocyte flux remained unaltered during the Leukemia


64 whole observation period, indicating the absence of systemic inflammation (Table 1). In summary, long-term results of localized leukemic tumor growth and associated alterations of microcirculation were demonstrated. The observed microcirculatory alterations exhibited strong similarities to those described for solid tumors. One can conclude that this might lead to comparable barriers to drug delivery. Furthermore, temporally heterogenous vascularization of leukemic tumors, as presented in this study, raises the question whether efficiency of therapies might be dependent on the timing of their initiation. One can conclude that high molecular weight therapies, such as antibodies, are more efficient in small tumors while microvascular permeability is increased and interstitial pressure is low.49,50 The observed elevated leukocyte density might ameliorate cell-based therapies at later stages of tumor growth. The growing evidence of the connection between hematologic malignancies and angiogenesis makes antiangiogenic therapy a promising approach in the treatment of leukemia.7–10 Antivascular therapies were reported to inhibit leukemic tumor progression51,52 and decrease vascular density.22 Whether this effect is solely mediated by inhibition of tumor cell proliferation or partially by disruption of the vascular network remains unclear and should be investigated in further studies. Therefore, the presented model, enabling the long-term analysis of functional and morphological properties of leukemic vasculature, might be a useful tool to understand the complex interactions between tumor microcirculation and tumor microenvironment during tumor therapies. Besides, optimizing the regime of drugs, the time schedule for therapeutic agents addressing the multifaceted temporal and spatial heterogenous vasculature of leukemic tumor growth, might improve therapeutic effectiveness.

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Acknowledgements This work was supported by a Werner-Otto Stiftung research grant to Nils Hansen-Algenstaedt. Christian Schaefer and Nils Hansen-Algenstaedt were members of the DFG Graduate Kolleg [GRK476]. Petra Algenstaedt was a member of the DFG Graduate Kolleg [GRK336].

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