microcirculation of secondary bone tumors in vivo: the impact of minor surgery at a distal site

Microcirculation of Secondary Bone Tumors In Vivo: The Impact ofMinor Surgery at a Distal Site

Christian Schaefer,1 Ina Fuhrhop,1 Malte Schroeder,3 Lennart Viezens,1 Jasmin Otten,4 Walter Fiedler,4 Wolfgang Ruther,2 NilsHansen-Algenstaedt1

1Orthopaedic Spine Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany, 2Department ofOrthopaedics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3Department of Trauma-, Hand- and Reconstructive Surgery,University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4Oncology and Hematology, Department of Internal Medicine II, UniversityMedical Center Hamburg-Eppendorf, Hamburg, Germany

Received 24 November 2009; accepted 17 March 2010Published online 29 April 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21166

ABSTRACT: Microcirculatory properties of tumors play a pivotal role in tumor progression and inefficacy of therapies. It has beenhypothesized that surgical interventions result in an accelerated tumor growth by increasing the level of pro-angiogenic cytokines withsubsequent amplification of tumor angiogenesis. To characterize the microvascular properties of secondary bone tumors in vivo anddetermine the impact of minor surgery on the microcirculation, we performed intravital microscopy over 25 days using a xenograft modelof breast cancer tumor growth (MCF-7) in bone. After engraftment of tumors the mice were treated with a mastectomy versus no surgery.Tumor growth was accompanied by angiogenic sprouting and decreased vascular diameters while blood flow rate and tumor perfusionremained constant. Mastectomy initially led to a significant reduction of functional vascular density, increased vascular diameter, anddecreased blood flow velocities. However, neither tumor growth nor tissue perfusion was different between the groups. The presentedstudy corroborates the assumption that tumor microcirculation in bone exhibits similar time-dependent alterations compared to softtissue tumors. A minor surgical intervention did not change tumor growth kinetics however microcirculatory properties were altered.Whether major surgery has an impact on tumor growth in bone should be clarified in further studies. © 2010 Orthopaedic ResearchSociety. Published by Wiley Periodicals, Inc. J. Orthop. Res. 28: 1515–1521, 2010

Keywords: microcirculation; angiogenesis; intravital microscopy; tumor; bone; metastasis; surgery

The skeleton is the most common organ to be affected bymetastatic cancer especially by tumors originating frombreast or prostate, which posses a propensity to spreadto bone.1 Primary tumors and skeletal-related complica-tions, as pathologic fracture or spinal cord compression,contribute to the necessity of surgical interventions inclinical treatment of these patients.1,2 In this contextthere is growing evidence that surgical interventionscan induce the switch of micrometastases from a dor-mant state to a rapid growing tumor.3–6 However, theeffect on established tumors remains widely unknown.Release of growth factors such as vascular endothe-lial growth factor (VEGF) plays an important role inmaintenance of angiogenesis during wound healing sub-sequent to surgical trauma7 and has been demonstratedto have systemic effects at distant sites on tumor growthand other tissues.8−10 In this context surgery-relatedmodulation of angiogenic factors has been proposed toinfluence this balance and might therefore contribute toan acceleration of tumor growth at a distal site throughalterations of the tumor microcirculation.3,9

The properties of tumor microcirculation are knownto play a pivotal role in tumor growth, metastasis,and inefficacy of therapies.11−14 It has been shown

Additional Supporting Information may be found in the online ver-sion of this article.The first two authors contributed equally to this study.Correspondence to: Christian Schaefer (T: +49-40-7410-55574; F:+49-40-7410-49338; E-mail: [email protected])© 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

that the angiogenic phenotype of a tumor dependson the site of growth.15 However, little is knownabout the microcirculation of bone metastases com-pared to soft tissue tumors despite the importance oflocal tumor–host interactions.15−17 We recently pub-lished a bone tumor model, which allows continuousobservation of microvascular properties in vivo, anddescribed morphological angiogenic alterations duringtumor growth.16,18 Since functional properties of tumormicrocirculation greatly contribute to tumor perfusionand drug delivery,12,13 the objective of the present studywas to characterize angiogenesis and tumor perfusionof experimental bone tumors. Furthermore, we hypoth-esized a systemic influence of a surgical intervention ata distal site on tumor growth through microcirculatoryalterations.

Therefore, we implanted breast cancer cells trans-duced with red-fluorescent protein (MCF-7 pDsRed) inthe femoral diaphysis of severe combined immunodefi-cient (SCID) mice and performed intravital microscopyusing the “femur window” preparation to analyze micro-circulatory alterations subsequent to tumor growth anddetermine the systemic effect of a minor surgical inter-vention.

METHODSModel of Secondary Bone TumorThe Breast Cancer Cell line MCF-7 (Cell Line Service,Eppelheim, Germany)19,20 was transduced with red fluores-cent protein (pDsRed-Express, excitation/emission wavelength

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557/579 nm, Clontech Laboratories Inc., Mountain View, CA)using commercial lipofectamine reagent (Lipofectamine, Invit-rogen, Kahlsruhe, Germany). Cells were grown in D-MEM/F12media containing 10% of fetal bovine serum. The cells werecultured at 37◦C and 5% CO2 in a humidified incubator.

For the experiments 12-week-old, female, SCID micewere used (Taconic, Lille Skensved, Denmark). All ani-mal procedures were performed according to the GermanAnimal Welfare Committee (No. 31/04, Behorde fur Wis-senschaft und Gesundheit, Hansestadt, Hamburg, Germany).All surgical procedures were performed at aseptic condi-tions while maintaining body temperature at physiologicallevels using a heating pad. Prior to surgical proceduresmice were anesthetized (7.5 mg ketamine hydrochloride and2.5 mg xylazine/100 g body weight), the skin was shavedand depilated. For noninvasive and continuous intravitalmicroscopy a “femur window” (Machine Shop, University Ham-burg, Germany) was implanted to the right femur diaphysis asdescribed more detailed previously.18 Prior to femur windowfixation and wound closure, a suspension of 1 × 106 pDsRed-MCF-7 cells in 4 �l of nutrition media was implanted into thecancellous bone of the diaphysis. Thereafter, the mice wereallowed to recover for 5 days and the window quality and thegrafting of the tumor were evaluated. Only mice with signifi-cant tumor growth, adequate window quality, and no signs ofinfection were included into the measurements and randomlydivided into two groups (“surgery: n = 8; “control”: n = 7). The“Surgery” group received a mastectomy 5 days after implanta-tion of breast cancer cells and femur window (day 0). Therefore,a 1.5 cm skin incision was made behind the foreleg and theunderlying tissue of the breast was excised. Thereafter the skinwas closed with a nonabsorbable suture (5.0 Ethilon, Ethicon,Johnson & Johnson Medical, GmbH, Norderstedt, Germany).Animals were kept in a 12-h-day-and-night-rhythm at 24◦Cand 50% humidity. Mice were caged individually and fed withstandard laboratory chow (Chow number S5714S040, Ssniff,Soest, Germany) and water ad libitum.

Intravital MicroscopyEpi-illumination techniques were employed to monitor tumorgrowth and vascular parameters on days 0, 3, 5, 9, 15, and20 using fluorescence microscopy (Fig. 1). The measurement ofday 0 was performed prior to mastectomy in the surgery group.Only animals with intact microcirculation and establishedtumor microcirculation on day 0 were included into furthermeasurements. To obtain microcirculatory parameters threelocations within the tumor were investigated using an intrav-ital fluorescence microscope (Axioplan, Zeiss, Oberkochen,Germany) and a 20× long working distance objective (LDAchroplan 20×/0.40, Zeiss). The microscope was equipped withfluorescence filter sets for fluorescein isothiocyanate (FITC)and red fluorescence protein (RFP), an intensified charge-coupled device (CCD) video camera (C2400-97, HamamatsuPhotonics, Hamamatsu, Germany), a Photomultiplier Tube(R4632, Hamamatsu Photonics), and a Computer (Apple PowerMacIntosh, G4, Dual 500 MHz Power PC, 1 GB SDRAM) fordigital signal recording and off-line analysis. During measure-ments the body temperature was maintained at physiologicallevels using a heating pad. To eliminate movements of thefemur chamber due to breathing the chamber was fixed to themicroscope using a special clamp (Machine shop, UniversityHamburg, Germany).18 Analysis of tumor area was performedusing the Axiovision software (Axiovision 4.6, Carl Zeiss JenaGmbH, Jena, Germany).

Vessel Density, Vessel Diameter, Tissue Perfusion Rate, BloodFlow Velocity, Blood Flow RateTo measure vessel density, vessel diameter, tissue perfu-sion rate, and blood flow velocity 100 �l of FITC-Dextran(MW 2,000,000 kDa; 10 mg/ml, Sigma, St. Louis, MO) wasinjected through the tail vein to visualize functional vessels(Fig. 1). Fluorescence images were recorded digitally andnoncompressed for 10 s and analyzed off-line. Using an imageprocessing system (NIH Image 1.63), vessel diameter and

Figure 1. Representative multi-channel images of breast cancer and microcirculation in bone after intravenous application of fluoresceinisothiocyanate-labeled dextran. Red fluorescent protein tumor cells (MCF-7, pDsRed) appear in red and functional vessels in green color.(Day 0) Five days after implantation the tumor was vascularized and an induction of angiogenesis could be seen around the tumor. Onthe left side of the image, the physiologic microcirculation of bone can be seen. (Days 3–20) Significant tumor growth was accompaniedwith angiogenesis response not only at the tumor rim but also on more distant sites as the periosteum. Please note the varying vasculardiameters of vessels and chaotic vascular architecture subsequent to tumor growth in comparison to day 0 (scale bar represents 1 mm).

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Figure 2. Tumor area (A) and microvascular permeability of tumor vessels (B). (A) Tumor area increased significantly from day 0 to day20 in both groups; however, no significant difference could be observed between both groups following mastectomy (control: n = 7; surgery:n = 8). (B) Microvascular permeability of vessels was measured via fluorescence intensity measurements of extravasating fluorochromeafter intravenous application of FITC-BSA (40 mg/kg body weight). A significant reduction of microvascular permeability was observedon days 15 and 20. There was no difference found between both groups (control: n = 6–7; surgery: n = 6–8). Results are presented asmean ± SEM. §p < 0.05 compared to day 0.

length were measured to calculate vessel density, defined astotal length of vessels per unit area, total count of vessels perarea, and mean vascular diameter. Blood flow velocity (VRBC)was measured opto-electronically using a two-slit method(Exbem 3.0, Pixlock e.K., Munster, Germany), corrected by �taking the decreasing blood flow velocity from the middle ofthe vessel to its vessel wall into account. Corrected blood flowvelocity (Vmean) was calculated as follows: Vmean = VRBC × �(� = 1.3, for blood vessels <10 �m; linear extrapolation1.3 < � < 1.6 for blood vessels >10 and <15 �m; and � = 1.6 forblood vessels >15 �m).21 Blood flow rate (BFR), representingthe perfusion of single vessels was calculated using Vmean

and the diameter (D) of the vessel: BFR = Vmean × D2 × �/4.The tissue perfusion rate was obtained using vascular den-sity, BFR of single vessels, and surface area as describedpreviously.22

Microvascular PermeabilityEffective microvascular permeability was measured asdescribed previously.18,23 Briefly, after the injection of FITC-labeled bovine serum albumin (FITC-BSA, MW 67,000;excitation wavelength 494 nm, emission wavelength 520 nm,Molecular Probes, Invitrogen Ltd, Paisley, UK; 40 mg/kgbody weight) the fluorescence intensity was measuredintermittently for 10 min and recorded digitally (Pow-erLab/200 AD Instruments Pty Ltd, Castle Hill, NSW,Australia). The value of permeability was calculated asP = (1 − HT)V/S{1/(I0 − Ib) × dI/dt + 1/K}, where I is the aver-age fluorescence intensity of the whole image, I0 is the value ofI immediately after filling of all vessels by FITC-BSA, and Ib

is the background fluorescence intensity. The average hemat-ocrit (HT) of vessels is assumed to be equal to 19%.24 V andS are the total volume and surface area of vessels within thetissue volume covered by the surface image. The time constantof BSA plasma clearance (K) is 9.1 × 103 s.25

Statistical AnalysisThe results were normalized to day 0 and presented asmean ± standard error of mean (SEM). Statistical analysiswas performed with SPSS (SPSS 16.0, SPSS, Inc., Chicago,IL) using the Mann–Whitney U-test for comparison of dif-ferent groups and the Wilcoxon rank sum test for vascularparameters of one group between different measurementpoints. Statistical significance was based on p-values smallerthan 5%.

RESULTSTumor Growth Was Not Affected by Surgical InterventionAfter implantation of breast cancer cells into the diaph-ysis of mice, significant tumor growth could be observedin both groups (Figs. 1 and 2A, S-Movie 1). Tumor areaincreased from day 0 to day 5 in the control (233 ± 88%;n = 7) and surgery group (259 ± 32%; n = 8) and furtherto day 20 (control: 512 ± 119%; surgery: 711 ± 146%;p < 0.05). In comparison of both groups no significantdifference in tumor area could be found.

Increased Microvascular Permeability Subsequent toTumor OnsetMicrovascular permeability was measured on day 0(control: n = 7; surgery: n = 7), day 3 (control: n = 6;surgery: n = 8), day 5 (control: n = 6; surgery: n = 8),day 9 (control: n = 7; surgery: n = 7), day 15 (con-trol: n = 7; surgery: n = 6), and day 20 (control: n = 7;surgery: n = 7). Subsequent to tumor cell implantationthe microvascular permeability showed a peak on day3 (control: 262 ± 87%; surgery 132 ± 35%) with a subse-quent decline to a steady state in both groups on day 15(control: 56 ± 20%; surgery: 48 ± 26%; p < 0.05). No dif-ferences were observed between both groups (Fig. 2B).

Surgical Intervention Was Accompanied by a DecreasedAngiogenic Response during Tumor GrowthMorphological parameters of microcirculation wereevaluated in a total of 8,914 vessels in both groupsafter intravenous administration of FITC-dextran. Inthe control group seven animals and three locations andin the surgery group eight animals and three locationswere analyzed. Vascular density, defined as total lengthof vessels per observation area, showed an increase inthe control group (n = 7) from day 0 (100%) to day 9(142 ± 16%; p < 0.01) and day 15 (171 ± 32%; p = 0.15)with a subsequent decrease thereafter to 150 ± 27%(Fig. 3A). After mastectomy the vessel density showed adelayed increase of vascular density till day 5 (120 ± 6%;p < 0.01; n = 8) without further alterations thereafter.Analysis of vessel count per area demonstrated thatthe initial increase in vascular density in the controlgroup resulted from angiogenic sprouting until day 9



Figure 3. Vessel density (A), vessel count per area (B), and mean vessel diameter (C) was measured after contrasting plasma withintravenous application of FITC-dextran (control: n = 7; surgery: n = 8). (A) Vessel density, defined as total length of perfused vessels perarea, showed a significant increase from day 0 to day 15 in the control group and from day 0 to day 5 in the surgery group. Followingmastectomy the vascular density was significantly reduced on day 3. (B) Vessel count per area increased in the control group; however,no alteration was found in the surgery group compared to day 0. The vessel count decreased on day 3 significantly subsequent to surgicalintervention. (C) Mean vessel diameter decreased in both groups from day 0 to day 15. The increase was pronounced in the control groupleading to significant lower values on day 15 compared to the surgery group. Results are presented as mean ± SEM. §p < 0.05 for controland $p < 0.05 for surgery group compared to day 0. *p < 0.05 control versus surgery.

(177 ± 32%; p < 0.05; n = 7) (Fig. 3B), which was atten-uated in the surgery group (n = 8). The increase invascular density and vessel counts on day 3 in the con-trol group was significant compared to the surgery group(p < 0.05). Concomitant effect of the tumor angiogenesisin both groups was the decreased mean vascular diame-ter on day 15 (control: 69 ± 7%, n = 7; surgery: 88 ± 5%,n = 8; p < 0.01) due to increasing number of vessels withsmall diameter (Fig. 3C). The pronounced angiogene-sis in control group led to smaller diameters on day 15compared to surgery group (p < 0.05).

Surgical Intervention Did Not Affect Tissue PerfusionTo evaluate whether the different alterations of mor-phological microcirculatory parameters subsequent tothe surgical intervention affected perfusion of tumortissue, determination of blood flow velocity and BFRof each vessel was performed (Fig. 4). In total 8,914vessels in both groups were analyzed during observa-tion period. In the control group seven animals andthree locations and in the surgery group eight animalsand three locations were observed. Blood flow veloc-ity increased in control group from day 0 to day 3(185 ± 34%; p < 0.05; n = 7); day 15 (257 ± 36%; p < 0.01)

and day 20 (253 ± 39%; p < 0.05). Compared to controls,the surgery group demonstrated a significant smallerincrease in blood flow velocity (day 3: 104 ± 14%; day15: 125 ± 9%; day 20: 124 ± 12%; p < 0.05; n = 8) (Fig.4A). Surprisingly neither BFR, defined as mean bloodvolume per vessel and time, nor tissue perfusion rate,defined as blood volume per area and time, was affectedin both groups throughout the observation period (Fig.4B,C).

DISCUSSIONThe functional and morphological properties oftumor microcirculation are known to modulatetumor progression, metastasis, and inefficacy oftumor therapies.11−14,26 Besides the host tissuemicroenvironment,15,27−30 in which a tumor is grown, ithas been hypothesized that also surgical interventionscould modulate the systemic balance of angiogenicand anti-angiogenic factors and thereby influencethe microcirculatory properties and the growth of atumor.4,5,9,31,32 Here we describe functional and mor-phological microvascular alterations associated withbreast cancer growth in bone in vivo using the bonechamber “femur window” and intravital microscopy.

Figure 4. Blood flow velocity (A), blood flow rate (B), and tissue perfusion rate (C) was determined after intravenous injection of FITC-dextran (control: n = 7; surgery: n = 8). (A) Blood flow velocity increased significantly over time in both groups; however, the increase wasmore pronounced in the control group leading to significant differences between groups on days 3, 15, and 20. (B,C) Neither blood flowrate nor tissue perfusion per area was altered during observation period in both groups. Results are presented as mean ± SEM. §p < 0.05for control and $p < 0.05 for surgery group compared to day 0. *p < 0.05 control versus surgery.



Furthermore, the impact of a minor surgical interven-tion at a distal site on bone tumor microcirculation andgrowth progression was analyzed.

Similar Vascular Alterations during Breast Cancer Growthin Bone Compared to Soft Tissue Tumors ImplicateAnalogous Barriers to Drug DeliveryIncreased microvascular permeability, mediated byloosening of intercellular contact and increased tran-scellular openings, is a prerequisite for angiogenicprocesses and facilitates migration of endothelial cellsand extravasation of plasma proteins.11,33 Initial breastcancer growth was accompanied by an increased level ofmicrovascular permeability in the bone microenviron-ment. The microvascular permeability decreased untilday 15 to a steady state showing an analogous coursefound in soft tissue tumors23 and a substantial delayeddecrease compared to bone tissue after bone chamberimplantation.18

As demonstrated by vascular count and density,tumor growth in bone was associated by angiogenicsprouting of vessels until day 15, which resulted in asignificantly decreased mean vascular diameter due toincreased number of small vessels. An initial angiogenicwave subsequent to tumor growth was described in softtissue23,34; however, the dynamics are potentially influ-enced by the angiogenic phenotype of the tumor and thehost microenvironment.15,16,28,35 We recently describedthe morphological alterations of microcirculation asso-ciated with breast cancer growth in bone using the sameanimal model but a different breast cancer cell line(T47D) and observed an increase in vascular diameter.16

This might result from different growth kinetics of bothtumors. The doubling time of T47D-tumors was foundtwice as high as the doubling time of MCF-7 tumors inthis study. It is known that the solid stress of prolif-erating tumor cells can compress small tumor vesselslacking pericytes.36 Therefore, the decreased vasculardensity and increased vascular diameter in the previ-ous study might have been caused by compression ofsmall vessels, which exhibited no blood perfusion andhave been consecutively not detected.

Increased vascular permeability and altered geomet-ric resistance of vascular network as well as temporaland spatial heterogeneous blood flow result in impaireddrug delivery to neoplastic tissues.13,37 To address theimpact of functional microcirculatory parameters on tis-sue homeostasis,38,39 we analyzed blood flow velocityand blood flow in single vessels as well as over-all tissue perfusion. Despite the angiogenic sproutingdescribed above and the increased blood flow velocity,the blood flow and the tissue perfusion rate of the mam-mary carcinoma were found unaltered. Further analysisrevealed that the interplay between vascular sprout-ing with decreased vessel diameters and increasedblood flow velocity in single vessels led to an unal-tered blood volume per time, which was distributedon more vessels. The actual observation demonstratesthat angiogenesis is not necessarily associated with

increased tissue perfusion and corroborates previousstudies of leukemic and solid tumors grown in softtissue.23,39 The presented results demonstrate that theunderlying alterations of tumor microcirculation in boneare similar to those found in tumors grown in otherorgans23,34; therefore, analogous barriers to drug deliv-ery can be assumed in tumors growing in bone. Whetherthe bone microenvironment further influences drugdelivery and therapeutic efficacy should be objective offurther studies.

A Minor Surgical Intervention at a Distal Site Lead toMicrovascular Alterations without Affecting TumorGrowth Kinetics of Breast Cancer Growth in BoneSeveral authors reported increased engraftment ofmetastasis4,5,9,40 or accelerated tumor growth follow-ing surgical interventions.3,6,32 These alterations oftumor growth kinetic have been linked to surgery-induced stress with subsequent impairment of immunefunction4−6,40 or changes in the balance of angio-genic/antiangiogenic substances with subsequent induc-tion or acceleration of tumor angiogenesis.3,9,32 Despitethe clinical relevance of bone metastasis and the impactof the microenvironment on tumor growth, the effectof surgical interventions on growth of tumors in bonetissue has not been investigated. To focus on the micro-circulatory properties and eliminate possible effects bysurgery-induced immunodepression,4−6,40 a xenograftmodel of mammary carcinoma cells grown in SCIDmice was chosen in our study. Since the objective ofthe presented investigation was the determination ofthe growth kinetics of an established tumor and theunderlying microcirculatory alterations rather than theengraftment of tumor cells we performed the surgi-cal intervention after vascularization of the tumor.The minor surgical intervention performed in thisstudy led to significant alterations in microcircula-tory properties in our model of skeletal breast cancergrowth. However, the tumor growth kinetics and tumorperfusion were not affected. We found a decreasedangiogenic response to tumor growth after surgicalintervention as demonstrated by vascular density andvessel count with concomitant increased vascular diam-eter. To our knowledge this is the first time thisphenomenon has been reported. However, the under-lying mechanism remains unclear. Whether this effectresulted from a true decrease in angiogenic sprout-ing or from microvessels without perfusion remainsobjective of ongoing studies. Neither in the controlgroup, nor in the surgery group the blood flow and thetissue perfusion were affected by alterations of mor-phological microvascular parameters. This corroboratesprevious studies and underlines the importance of deter-mination of functional microvascular properties.23,39

After major surgery, such as laparotomy or subtotalhepatectomy, several authors described an effect onexperimental metastasis.3,40 Recently, Rupertus et al.3

described an increase in microvessel density and sub-cutaneous growth of colon carcinoma following major



hepatectomy in mice. In this context, the unchangedtissue perfusion and tumor growth kinetics found inthis study might result from an insufficient stimulusthrough the performed surgical intervention and mightrepresent therefore a limitation in the chosen studydesign.3,4,32 In this context it cannot be excluded thatlarger surgery might have an effect on tumor growthin bone, which should be objective in further investiga-tions.

Here we described sequential alterations of func-tional and morphological microvascular propertiesduring breast cancer growth in bone over 25 daysusing intravital microscopy. Following tumor growth inbone we determined similar alterations of microvascularproperties as found in soft tissue tumors, which mightresult in analogous barriers to delivery of therapeu-tic agents. The performed minor surgical interventiondid neither modulate breast cancer growth kinetics nortumor blood flow. However, differences in vascular den-sity, diameter, and blood flow velocity were found. Itremains to be further elucidated, whether major surgeryhas an impact on tumor progression in bone and whetherthe bone tumor microcirculation has an impact on ther-apeutic efficacy.

THE LIST OF ABBREVIATIONS

SCID severe combined immunodeficientVD functional vascular densityD vascular diameterSEM standard error of meanVEGF vascular endothelial growth factorVRBC blood flow velocityBFR blood flow rate

ACKNOWLEDGMENTSThis work was supported by a Deutsche Forschungsgemein-schaft (DFG) research grant to Christian Schaefer and NilsHansen-Algenstaedt (HA2790/4-1).

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