Precision Medicine and Imaging

Tracking Cell Transplants in FemoralOsteonecrosis with Magnetic ResonanceImaging: A Proof-of-Concept Study in PatientsAshok J. Theruvath1,2,3, Hossein Nejadnik1,3, Anne M. Muehe1,3, Felix Gassert1,3,Norman J. Lacayo4, Stuart B. Goodman5, and Heike E. Daldrup-Link1,3,4

Abstract

Purpose: Osteonecrosis is a devastating complication ofhigh-dose corticosteroid therapy in patients with cancer. Coredecompression for prevention of bone collapse has beenrecently combined with the delivery of autologous concen-trated bonemarrow aspirates. The purpose of our study was todevelop an imaging test for the detection of transplanted bonemarrow cells in osteonecrosis lesions.

Experimental Design: In a prospective proof-of-conceptclinical trial (NCT02893293),weperformed serialMRI studiesof nine hip joints of 7 patients with osteonecrosis before andafter core decompression. Twenty-four to 48 hours prior to thesurgery, we injected ferumoxytol nanoparticles intravenouslyto label cells in normal bonemarrowwith iron oxides. Duringthe surgery, iron-labeled bone marrow cells were aspiratedfrom the iliac crest, concentrated, and then injected into thedecompression track. Following surgery, patients received

follow-up MRI up to 6 months after bone marrow celltransplantation.

Results: Iron-labeled cells could be detected in the accesscanal by a dark (negative) signal on T2-weighted MR images.T2� relaxation times of iron-labeled cell transplants weresignificantly lower compared with unlabeled cell transplantsof control patients who were not injected with ferumoxytol(P ¼ 0.02). Clinical outcomes of patients who received fer-umoxytol-labeled or unlabeled cell transplants were not sig-nificantly different (P ¼ 1), suggesting that the added feru-moxytol administration did not negatively affect bone repair.

Conclusions: This immediately clinically applicableimaging test could become a powerful new tool to monitorthe effect of therapeutic cells on bone repair outcomesafter corticosteroid-induced osteonecrosis. Clin Cancer Res;24(24); 6223–9. �2018 AACR.

IntroductionOsteonecrosis is a debilitating and devastating complication of

high-dose corticosteroid therapy: 15%–47% of patients withleukemia and 3%–44% of patients with systemic lupus erythe-matosus (SLE) develop an osteonecrosis as a result of high-dosecorticosteroid therapy (1–4). Seventeen to 22% of these patientsprogress to hip joint collapse (5, 6), which leads to major long-term morbidities, such as severe joint pain requiring narcoticanalgesia, impaired joint function and, severely limited ambula-tion, ultimately requiring total joint replacement.

The pathogenesis of osteonecrosis is multifactorial andinvolves corticosteroid-induced ischemia, necrosis of cells of themesenchymal and hematopoietic lineages, as well as hypertrophyof fat cells that further compress microvessels and thereby per-petuate ischemia (7). Progressive death of cells that providestructural support for the underlying bone ultimately leads tobone collapse. Interventions to save the affected bone are effectiveonly if performed in a preventive manner (8). Various noninva-sive and surgical procedures have been used to prevent progres-sion to bone collapse (9). Core decompression has been estab-lished inmany academic centers and involves drilling a track to anosteonecrosis segment to release the presumed increased pressurein osteonecrosis and facilitate revascularization. Recently, thiscore decompression procedure has been combined with thedelivery of concentrated bone marrow aspirates containing mes-enchymal stromal cells (MSC) and enriched osteoprogenitor cells(10–12). These cell transplants are expected to facilitate theregeneration of normal bone marrow in osteonecrosis throughdirect or indirect mechanisms. However, success or failure of thisnew treatment canonly be diagnosed after severalmonths to years(11, 12). Our group has previously shown in a rat model thatintravenously injected iron oxide nanoparticles are taken up byMSCs in the bone marrow and could be tracked with MRI aftertransplantation into osteochondral defects (13, 14). Others havealso noted in vivo labeling capacity of immune cells in the bonemarrow (15, 16).

An imaging test, which could directly track transplantedbone marrow cells in vivo, could help us better understand thecontribution of these cells to bone repair processes, diagnose

1Department of Radiology, Pediatric Radiology, Lucile Packard Children's Hos-pital, Stanford University, Stanford, California. 2Department of Diagnostic andInterventional Radiology, University Medical Center Mainz, Mainz, Germany.3Pediatric Molecular Imaging Program, Molecular Imaging Program at Stanford(MIPS), Stanford University, Stanford, California. 4Department of Pediatrics,Pediatric Hematology/Oncology, Lucile Packard Children's Hospital andStanford Cancer Center, Stanford University, Stanford, California. 5Departmentof Orthopaedic Surgery and Bioengineering, Stanford Hospital, StanfordUniversity, Stanford, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A.J. Theruvath, H. Nejadnik, and A.M. Muehe are co-first authors of this article.

Corresponding Author: Heike E. Daldrup-Link, Pediatric Radiology Section andMolecular Imaging Program at Stanford (MIPS), Department of Radiology,Stanford School of Medicine, 725 Welch Rd, Stanford, CA 94305-5654. Phone:650-497-8601; Fax: 650-723-8402; E-mail: heiked@stanford.edu

doi: 10.1158/1078-0432.CCR-18-1687

�2018 American Association for Cancer Research.

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complications earlier, and facilitate the development of moresuccessful cell therapies that canprevent bone collapse. The abilityto track therapeutic cells noninvasively in vivo could have directimpact on patient management, for example, by stratifyingpatients with unsuccessful or lost cell transplants to revisionsurgeries or alternative treatment options. To address this unmetclinical need, we developed an imaging test for the detection ofbone marrow cell transplants in osteonecrosis in a "first in-patient" proof-of-concept clinical trial.

Materials and MethodsStudy design

This prospective, nonrandomized, HIPAA-compliant proof-of-concept clinical trial was approved by our institutional reviewboard and performed under an investigator-initiated IND (111154). The study was conducted in accordance with the BelmontReport. We invited pediatric and young adult patients from May2015until December 2017 to participate if theymet the followinginclusion criteria: (i) age 8–40 years, (ii) avascular necrosis of theproximal femur, (iii) planned core decompression with trans-plantation of autologous bone marrow aspirates, and (iv) will-ingness to give written informed consent. Patients were excludedif they had: (i) active leukemia, (ii) contraindications toMRI, (iii)hemosiderosis or hemochromatosis, or (iv) if theywere pregnant.

We recruited 7 patients (mean age 30� 8.4 years; range: 17–38years) with history of high-dose corticosteroid treatment forleukemia (n ¼ 3), Hodgkin lymphoma (n ¼ 1), asthma (n ¼1), SLE (n¼ 1), or inflammation of unknown origin (n¼ 1). Thepatients included4 female (mean age 30�9.5 years; range: 17–38years) and 3male patients (mean age 31� 8.7 years; range: 21–36years). The patients had nine early-stage (ARCO stage II) epiph-yseal osteonecrosis in their femoral heads: 3 patients had osteo-necrosis of the right femoral head, 2 patients of the left femoralhead, and 2 patients had bilateral osteonecrosis.

All patients received core decompression with transplantationof either iron-labeled or unlabeled bone marrow aspirates. Toachieve iron labeling of bone marrow cells in vivo, we injected

ferumoxytol (Feraheme) intravenously as published previously(13). Four patients with six core decompression proceduresreceived an intravenous injection of ferumoxytol at a dose of5 mg Fe/kg at 24 to 48 hours before surgery. Three patients withthree core decompressionprocedures didnot receive ferumoxytol.Ferumoxytol-labeled or unlabeled bone marrow cells were har-vested by an iliac crest aspiration using an autologous cell aspi-ration and concentration system (Zimmer-Biomet) and mixedwith demineralized bone matrix from DePuySynthes. Follow-ing core decompression, the graft matrix enriched with labeledor unlabeled bone marrow cells was injected into the accesscanal by an experienced orthopedic surgeon (S.B. Goodman).Follow-up imaging was performed by MRI.

To compare the clinical outcome of core decompression withandwithout labeled cells,weenrolled5 additional control patientswith seven femoral osteonecrosis, which were treated with coredecompression and unlabeled bone marrow cell transplants andwho did not receive serial MRI. These comprised 1 female and 4male patients (mean age 30� 6.8 years; range: 21–38 years) withhistory of status post high-dose corticosteroid treatment.

MR imagingPatients included in our study received a preoperative MRI and

a follow-up MR scan at 1 week, 4–7 weeks, and 6 months afterbone marrow cell transplantation. MRI has been established as ahighly sensitive and specific test for diagnosing early epiphysealosteonecrosis, before joint damage is apparent on bone scans orradiographs (17). MRI scans were obtained with a 3 Tesla MRIscanner (Discovery 750 MR, GE Healthcare), using a 32-channeltorso phased array coil and the following pulse sequences: T1-weighted fast spin echo (FSE) sequence [TR¼ 600ms (517–721),TE ¼ 15 ms (5.8–19.7), flip angle (FA) ¼ 90� (90�-160�), slicethickness (SL) ¼ 3 mm (3–4.5)], T2-weighted fat saturated FSEsequence [TR¼4450ms (2399–4450), TE¼61ms (57–69), FA¼125�, SL ¼ 4 mm (3–4)], short TI inversion recovery sequences[TR¼5200 ms (5131–5282), TE¼50 ms (47–54), inversiontime¼170 ms, FA ¼ 111�, SL ¼ 3 mm], and a flow-compensated2D fast spoiled gradient recalled (FSPGR) sequence (TR ¼ 21.2ms, TE¼2.2ms, inter-echo interval 2.2ms, FA¼ 25�, SL¼3mm).

MRI analysesImages were analyzed usingOsirix (Pixmeo SARL). The decom-

pression track was equally divided on coronal T2-weighted MRimages in three parts: the proximal, mid, and distal track. Each ofthese areas was manually outlined by one observer, who mea-sured the signal-to-noise ratio (SNR) as the mean signal intensityof the outlined area, divided by the SD of the background noise,which was measured in phase encoding direction within the fieldof view and outside of the patient. In addition, the iron signal inthe same areas was quantified by measuring T2� relaxation timeson corresponding T2� maps, which were generated from FSPGRsequences using the T2 fit map plugin of Osirix. Each of the areaswas considered as an independent observation for MRI analyses.

Standard of referenceThe extent of osseous necrosis on imaging studies was graded

according to the Association Research Circulation Osseous(ARCO) classification (18, 19). All patients had an ARCO stageII at baseline. Stable disease at 6 months after surgery was definedas equal or improvedARCO stage. Progressive diseasewas definedas progression to stage III or IV.

Translational Relevance

Osteonecrosis due to corticosteroid therapy is a devastatingcomplication in patients with cancer. Core decompressionwith transplantation of bone marrow cells has shown prom-ising results to prevent joint collapse. We developed an imag-ing test to track transplanted bone marrow cells in a "first in-patient" clinical trial. We labeled bone marrow cells with asimple intravenous injection of an iron supplement. Iron-labeled bone marrow cells were transplanted into osteone-crosis and could be tracked with MRI. Tracking therapeuticcells in osteonecrosis can improve our understanding of therole of cell transplants in bone regeneration processes, and ourability to develop successful cell therapies for joint repair. Onthe basis of our imaging results, patients could be stratified torevision surgeries, alternative treatment options, or close fol-low-up examinations. By exploiting cell tracking techniquesfor monitoring engraftment outcomes, we anticipate alleviat-ing long-term disabilities of patients with cancer and relatedcosts to our society.

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Statistical analysesAll experiments were analyzed using R version 3.4.4. SNR and

T2� relaxation timeswerepairwise comparedbetween labeled andunlabeled cell transplants, and between decompression trackareas with and without visible iron-labeled cells, using amixed-effects model including a random effect term accountingfor correlation among the measures within a same patient. AFisher exact test was applied for comparison of clinical outcomesof labeled and unlabeled cell transplants. In addition, differencesin time to progression of osteonecrosis from surgery betweenlabeled and unlabeled cell transplants were assessed by log-ranktests. Because of the small sample size and exploring purpose ofthis study, a P < 0.05 without adjustment for multiple compar-isons was considered to indicate significant differences betweenexperimental groups.

ResultsIron-labeled bone marrow cells can be detected with MRI aftertheir transplantation into osteonecrosis

The overall concept of our study is shown in Fig. 1. Patientswith osteonecrosis received an intravenous injection of the ironsupplement ferumoxytol prior to a scheduled core decompres-sion to label bone marrow cells with iron, which can bedetected by a dark signal on MRI. One to 2 days later, thepatients underwent a core decompression, bone marrow aspi-ration from the iliac crest, and transplantation of concentratediron–labeled bone marrow cells through the decompressiontrack into the osteonecrosis in the femoral head. MRIs wereperformed before and within 1 week after the surgery, as well asat 4–7 weeks, and 6 months to track transplanted iron-labeledbone marrow cells in osteonecrosis.

MR images before ferumoxytol administration showed a focalosteonecrosis lesion in the proximal femoral epiphysis with atypical serpiginous border on T1- and T2-weighted MR images(Fig. 2A). All osteonecrosis were consistent with stage II lesionsaccording to the ARCO classification: the joint surfaces were intactand there was no evidence for subchondral fractures. This isimportant, because only joints without signs of bone collapsecanbe rescuedby a core decompression.Next, patients received anintravenous injection of ferumoxytol. Postcontrast MR images

showed a significant hypointense (dark) enhancement of thenormal bone marrow on T2-weighted MR images (Fig. 2B).

The patients underwent core decompression, harvest, andconcentration of iron-labeled bone marrow cells from the iliaccrest and transplantation of iron-labeled bone marrow cells intothe decompression track. T2-weighted MR images after injectionof iron-labeled marrow cells demonstrated hypointense (dark)signal in the decompression track and osteonecrosis (Fig. 2C, 3Aand B). In comparison, control patients who had received unla-beled bone marrow cell transplants did not show hypointensesignal changes in the access canal (Fig. 3C and D). SNR forferumoxytol-labeled cell transplants were significantly lowercompared with unlabeled cell transplants (33.82 � 12.43 vs.129.56 � 10.93; P ¼ 0.002; Fig. 3E). Likewise, T2� relaxationtimes, which represent more robust measures of tissue ironconcentrations, were significantly lower for ferumoxytol-labeledcell transplants than for unlabeled cell transplants (9.04� 0.7 vs.13.7 � 2.50; P ¼ 0.02; Fig. 3F)

Within the decompression track of patients who had receivediron-labeled cell transplants, we noted areas that showed strongiron signal, presumably representing areas where iron-labeledcells were delivered and areas that showed no iron signal, pre-sumably representing areas where iron-labeled cells were notdelivered. We divided each decompression canal in three areas(proximal, mid, and distal decompression canal) and comparedSNR and T2� relaxation times of areaswhere cell transplants couldbe visually detected or not detected. SNR and T2� relaxation timeswere significantly lower for areas where cell transplants couldbe visually detected or not detected (68.45 � 32.41, P ¼ 0.002;14.2 � 2.18, P ¼ 0.007; respectively; Fig. 3E and F).

Iron supplement administration before decompression doesnot affect bone repair outcomes

To evaluate the long-term implications of ferumoxytol admin-istrations on a decompression surgery, we investigated the MRIsignal of transplanted cells over time and found a slow decline ofthe iron signal (Fig. 2DandE): comparedwithunlabeled controls,SNR and T2� relaxation times of labeled cell transplants were notsignificantly different at 4–7 weeks (P > 0.05; Fig. 3G andH). Thisimplies either metabolization of the iron label or disappearanceof the cell transplant or a combination of both.

Figure 1.

Study concept. A, Twenty-four to 48 hours prior to a planned core decompression for osteonecrosis treatment, patients received an intravenous injection ofthe FDA-approved iron supplement ferumoxytol. B, Ferumoxytol is taken up by cells in normal bone marrow, leading to hypointense (dark) signal on MRI.C, 24–48 hours after iron supplement administration, iron-labeled bone marrow cells were harvested from the iliac crest during core decompression. D, Theosteonecrotic bone was decompressed by drilling a track to the osteonecrosis through a minimally invasive procedure. E, Iron-labeled bone marrowcells were injected through the decompression track.

Tracking Cell Transplants in Osteonecrosis Patients with MRI

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To evaluate whether the ferumoxytol administration prior tothe core decompression had any effect on bone repair, we com-pared clinical outcomes of patients who did or did not receiveferumoxytol. Of six femoral heads treated with labeled celltransplants, one (17%) progressed to collapse. Of ten femoralheads treated with unlabeled cells, three (30%) progressed tocollapse (Table 1). This difference was not significant (P ¼ 1,Fisher exact test), suggesting that ferumoxytol administrationbefore a core decompression did not adversely affect clinicaloutcomes. In addition, time to progression of osteonecrosis fromsurgery between labeled and unlabeled cell transplants were alsonot significantly different (Fig. 3I; P ¼ 0.8)

We noticed that one joint that progressed to collapse afteradministration of iron-labeled cells had apparently received lesscells compared with all other joints that did not collapse, asindicated by less iron signal in the treated decompression track(Supplementary Fig. S1). We quantified the hypointense (dark)area in the access canal through operator-defined regions of

interests. In the femur that progressed to collapse, 3.1% of thearea of the decompression track contained labeled cells. In thefemur that did not progress to collapse, 16.6% � 3.5% of thedecompression track were covered by cells.

Overall, the collective successful outcome of osteonecrosistreated with core decompression plus cell transplants was betterthan previously reported for core decompression alone: in ourstudy, 12 of 16 femurs (75%) showed no collapse within 1 yearor more after the intervention, compared with success rates of53%–71% for core decompressiononly, reportedpreviously (20).

DiscussionOur data showed that a simple intravenous injection of the iron

supplement ferumoxytol before a scheduled core decompressionled to iron labeling of the bone marrow in patients. After harvestfrom bone marrow and transplantation into osteonecrosislesions, the iron-labeled bonemarrow cells could then be tracked

Figure 2.

Serial MR images of osteonecrosis before and after core decompression and transplantation of iron-labeled bone marrow cells. A, T1-weighted, T2-weighted,and X-ray images of the right femur show an osteonecrosis (arrows) in the femoral epiphysis with typical fat-equivalent center and serpiginous borders.B, Twenty-four hours after intravenous injection of ferumoxytol, the normal bone marrow in the iliac crest shows hypointense (dark) enhancement (asterisks) onT2-weighted MR images. C, One week after core decompression and injection of iron-labeled bone marrow cells, a hypointense (dark) signal is noted in thedecompression track (arrows), consistent with delivery of iron-labeled cells. MRI follow-up at 4 weeks (D) and 24 weeks (E) after core decompression andtransplantation of labeled cells shows decline in iron signal over time. The femoral epiphysis did not collapse during this 6-month follow-up period.

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withMRI in the early postoperative period. We previously provedin animal models that intravenously injected ferumoxytol nano-particles are phagocytosed by bone marrow cells (13, 14) and areslowly metabolized over time (21). We found that ferumoxytolnanoparticles accumulate in different cell populations in the bonemarrow, which are capable of phagocytosis, including MSCs,macrophages, dendritic cells, and osteoprogenitor cells. In ourprevious work, we could show that ferumoxytol was taken up byMSCs, stored in lysosomes, and had no effect on the viability anddifferentiation potential of the iron-containing cells. Further-more, MSCs could be tracked in rodents with MRI (13).

Other investigators reported the ability to track iron oxidenanoparticle–labeled neural stem cells (22, 23), autologous mes-enchymal stem cells (24), and dendritic cells (25) with MRI inpatients. In these previous studies, autologous cells were firstharvested, then iron-labeled in cell cultures, and then trans-planted. Our approach is different in that we labeled bonemarrow cells in vivo by intravenous injection of an FDA-approvediron supplement. The iron-labeled cells could then be detectedwith MRI. This in vivo labeling approach is more practical in aclinical setting because it does not require any manipulation ofthe therapeutic cells and thereby, enables bone marrow cellharvest and transplantation in one surgery.

Corticosteroid-induced osteonecrosis leads to bone collapse inabout 26% of patients (6). Core decompression can prevent ordelay bone collapse in at least 50% of these patients (20). Theaddition of bone marrow cell transplants to classical decompres-sion surgeries has improved outcomes compared with coredecompression alone (11, 26, 27). It is discussed controversially,

whether bone marrow cell transplants support osteonecrosisrepair directly or indirectly: Wang and colleagues and Lee andcolleagues suggest that mesenchymal stromal cells and osteopro-genitor cells can directly regenerate bone injuries (28, 29), Mur-phy and colleagues and Linero and colleagues suggest that MSCssupport tissue repair through indirect paracrine mechanisms (30,31), and Lim and colleagues and Pepke and colleagues questionany therapeutic effect of bone marrow cells (32, 33). However,Lim and colleagues and Pepke and colleagues also mention thatnumerous factors such as number of cells, area of transplantation,disease stage, and follow-up period play an important role inclinical outcome. Our imaging test could be used to correlate thenumber and distribution of transplanted bone marrow cell trans-plants with outcomes. This information could be used to under-stand and optimize the effect of bone marrow cell transplants onbone repair outcomes. In addition, our imaging test could dis-criminate between successfully engrafting or lost cell transplantsin the early postoperative period. This information is importantfor the treating physician, who could stratify patients with failedcell transplants to alternative operative and nonoperative treat-ment options.

Our data showed that iron labeled and unlabeled celltransplants showed similar success rates in preventing bonecollapse, confirming that our in vivo labeling approach did notaffect the efficacy of the therapeutic cells. Previous studiesshowed that high intracellular iron concentrations and ironoverload can impair chondrogenic differentiation (34, 35) aswell as osteogenic differentiation of MSCs and inhibit osteo-blast activity, while facilitating osteoclast function (36). Ourgroup has shown previously that this effect is dose-dependentand that the proliferation and function of MSCs is notimpaired when the cells are loaded with less than 10 pg Feper cell (37). Our investigations in preclinical models showedthat intravenous injection of ferumoxytol (28 mg Fe/kg) leadsto uptake of approximately 4.3 pg Fe per cell, which is belowthis threshold (13).

Figure 3.

Ferumoxytol-labeled cell transplants can be detected in the decompression track after core decompression. A, Coronal T2-weighted MR image of the left femurof a patient who was treated with core decompression and injection of iron-labeled cells into the decompression track. Areas of hypointense (dark) signal(arrow) are noted in the decompression track, consistent with delivery of iron-labeled cells. B, Superimposed color-coded signal intensities show areasof iron-labeled cells (arrow) as displayed by blue color. C, Coronal T2-weighted MR of the left femur of a patient, who was treated with core decompression andinjection of unlabeled cells into the decompression track. Unlabeled cells are noted by an intermediate signal in the decompression track. D, Superimposedcolor-coded signal intensities show medium ranged signal intensities (green/yellow). Signal-to-noise ratios (SNR; E) and T2� relaxation times (F) during the firstweek after surgery for areas that showed iron signal compared with areas where iron-labeled cells were not delivered and unlabeled controls show significantlylower SNR (P¼ 0.002, n¼ 18; P¼ 0.002, n¼ 10; respectively) and T2� relaxation times (P¼ 0.007, n¼ 9; P¼ 0.02, n¼ 6; respectively). SNR (G) and T2� relaxationtimes (H) at 4–7 weeks reveal no significant differences between the groups, suggesting interval iron metabolization. Data are mean � SEM. P values weredetermined by mixed-effects model including a random effect term accounting for correlation among the measures within a same patient. I, Time to progression ofosteonecrosis between labeled and unlabeled cell transplants are not significantly different (P ¼ 0.8, n ¼ 16). P value was determined by log-rank test.

Table 1. Absolute and relative (%) number of osteonecrosis with labeled andunlabeled cells, which progressed to bone collapse

Labeled Unlabeled Total

Progression 1 (17%) 3 (30%) 4 (25%)No progression 5 (83%) 7 (70%) 12 (75%)Total 6 (100%) 10 (100%) 16 (100%)

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It has been reported previously, thatMSCs and osteoprogenitorcells bear the potential tomigrate to the site of osteonecrotic bonelesions (38, 39). In principle, our imaging test should be alsosuitable to investigate this process. Because of the avascular center,a newly developed osteonecrotic lesion takes up less ferumoxytolthan surrounding healthy bone marrow. In case of an intrinsicmigration of ferumoxytol-labeled bone marrow cells from nor-mal marrow to the site of injury, osteonecrotic lesions couldbecome hypointense (dark) over time in MRI. However, trackingintrinsic in vivo migrations of ferumoxytol labeled cells to osteo-necrosis might involve fewer cells and therefore, require moresensitive pulse sequences.

We found that the quantity of bone marrow cells transplantedinto osteonecrosis correlates with clinical outcomes, which is inaccordance with previously published findings (10, 12, 40, 41).Hernigou and colleagues analyzed the number of transplantedprogenitor cells in patients with osteonecrosis and found thatfemoral heads that received a low number of transplanted cellshad higher risks of bone collapse than femoral heads that receiveda high number of cells (10). In another study, Hernigou andcolleagues evaluated the amount of cells needed for treatment ofnonunion of the tibial shaft in 60 patients and found a positivecorrelation between the volume of mineralized callus and thenumber of cells and concentration (40).

Bone marrow cells from patients with osteonecrosis may haveless bone regeneration potential compared with healthy patients(12). Future studies will compare the in vitro bone-formingcapacity of MSCs and osteoprogenitor cells in patients withosteonecrosis and healthy controls. In patients who underwentbone marrow transplantation, MSC from the donor might bemore effective in regenerating bone.Our imaging test can track thelocation, estimate the quantity, and provide information aboutthe phagocytic activity of bone marrow cells. For example, wepreviously noted that normal bonemarrowcells of youngpatientstake up more iron compared with bone marrow cells of olderpatients (42, 43). Our imaging test cannot determine the efficacyof the transplanted cells to regenerate bone. However, in case ourimaging studies suggest low quantities of iron labeled cells in thedelivered bonemarrowaspirates, we could increase thenumber oftransplanted cells and/or add "off the shelf" MSC products orbanked donor MSCs. We previously established ferumoxytollabeling procedures for ex vivo labeling of donor MSCs (21, 44).

In summary, we provide proof-of-concept for a new imagingtest, which allows to track autologous bone marrow–derived celltransplants in patients with MRI, after a simple intravenous

injection of an FDA-approved iron supplement. The ability todirectly detect and track iron-labeled therapeutic cells in vivo, inpatients, could help us to recognize inter-individual differences inthe delivered quantity and location of transplanted cells andcorrelate resultswith tissue repair outcomes. This could ultimatelyimprove our ability to developmore successful cell therapies. Thisnew imaging test could become a powerful new tool to monitorthe delivery and engraftment of bone marrow–derived therapeu-tic cells noninvasively in patients with cancer with the ability todirectly impact patient management. Future studies under thisclinical trial will correlate MRI signal characteristics of-ironlabeled cells with clinical outcomes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.J. Theruvath, H. Nejadnik, A.M. Muehe,S.B. Goodman, H.E. Daldrup-LinkDevelopment of methodology: A.J. Theruvath, H. Nejadnik, S.B. Goodman,H.E. Daldrup-LinkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.J. Theruvath, H. Nejadnik, A.M. Muehe, F. Gassert,N.J. Lacayo, S.B. Goodman, H.E. Daldrup-LinkAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.J. Theruvath, H. Nejadnik, A.M. Muehe, F. Gassert,H.E. Daldrup-LinkWriting, review, and/or revision of themanuscript: A.J. Theruvath, H. Nejadnik,A.M. Muehe, F. Gassert, N.J. Lacayo, S.B. Goodman, H.E. Daldrup-LinkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.J. Theruvath, H. Nejadnik, A.M. MueheStudy supervision:A.J. Theruvath, A.M.Muehe, S.B. Goodman,H.E.Daldrup-Link

AcknowledgmentsWe thank members of the Daldrup-Link lab for their helpful reviews and

discussions of our study design and research results.We thank Jin Long from theQuantitative Sciences Unit at Stanford University and Ketan Yerneni for theirexcellent statistical consulting. Part of this work was performed at the RichardLucas Center for MR Imaging and at the Stanford Nano Shared Facilities(SNSF) at Stanford University. This work was supported by research grant2R01AR054458 from the National Institute of Arthritis and Musculoskeletaland Skin Diseases.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 29, 2018; revised July 23, 2018; accepted August 27, 2018;published first September 17, 2018.

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2018;24:6223-6229. Published OnlineFirst September 17, 2018.Clin Cancer Res   Ashok J. Theruvath, Hossein Nejadnik, Anne M. Muehe, et al.   Resonance Imaging: A Proof-of-Concept Study in PatientsTracking Cell Transplants in Femoral Osteonecrosis with Magnetic

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