cxcr4 isapotentialtargetfordiagnosticpet/ct …...ageal dysplasia and cancer highlight the potential...

Personalized Medicine and Imaging

CXCR4 Is a Potential Target forDiagnostic PET/CTImaging in Barrett's Dysplasia and EsophagealAdenocarcinomaHsin-Yu Fang1, Natasha Stephens M€unch1, Margret Schottelius2, Jonas Ingermann1,Haibo Liu3, Michael Schauer1, Stefan Stangl4, Gabriele Multhoff4, Katja Steiger5,Carlos Gerngroß6, Moritz Jesinghaus5,Wilko Weichert5, Anja A. K€uhl7,Antonia R. Sepulveda8, Hans-J€urgen Wester2, Timothy C.Wang3, and Michael Quante1

Abstract

Purpose: Barrett's esophagus represents an early stage in car-cinogenesis leading to esophageal adenocarcinoma. Consider-able evidence supports amajor role for chronic inflammation anddiverse chemokine pathways in the development of Barrett'sesophagus and esophageal adenocarcinoma.

Experimental Design: Here we utilized an IL1b transgenicmouse model of Barrett's esophagus and esophageal adenocar-cinoma and human patient imaging to analyze the importance ofCXCR4-expressing cells during esophageal carcinogenesis.

Results: IL1b overexpression induces chronic esophagealinflammation and recapitulates the progression to Barrett'sesophagus and esophageal adenocarcinoma. CXCR4 expressionis increased in both epithelial and immune cells during diseaseprogression in pL2-IL1b mice and also elevated in esophagealadenocarcinoma patient biopsy samples. Specific recruitment ofCXCR4-positive (CXCR4þ) immune cells correlated with dyspla-

sia progression, suggesting that this immune populationmay be akey contributor to esophageal carcinogenesis. Similarly, withprogression to dysplasia, there were increased numbers ofCXCR4þ columnar epithelial cells at the squamocolumnar junc-tion (SCJ). These findings were supported by stronger CXCR4-related signal intensity in ex vivo fluorescence imaging and auto-radiography with advanced dysplasia. Pilot CXCR4-directed PET/CT imaging studies in patients with esophageal cancer demon-strate the potential utility of CXCR4 imaging for the diagnosis andstaging of esophageal cancer.

Conclusion: In conclusion, the recruitment of CXCR4þ

immune cells and expansion of CXCR4þ epithelial cells in esoph-ageal dysplasia and cancer highlight the potential of CXCR4 asa biomarker and molecular target for diagnostic imaging ofthe tumor microenvironment in esophageal adenocarcinoma.Clin Cancer Res; 24(5); 1048–61. �2017 AACR.

IntroductionThe chemokine receptor 4 (CXCR4) has been shown to be

consistently overexpressed in a variety of solid tumors (1–3). Itsactivation by the only endogenous ligand, CXCL12 (formerlytermed SDF-1), contributes to cancer progression through bothautocrine and paracrine stimulation of cancer growth, as well as

inhibition of apoptosis in neoplastic cells (4). Alternatively, CXCR4signaling may trigger tumor angiogenesis by regulating proangio-genesis factors (5) and attracting endothelial cells to the tumormicroenvironment (6). Furthermore, CXCR4 overexpression pro-motes tumor invasiveness and metastasis by facilitating retentionand homing of tumor cells in cellular niches such as the bonemarrow. CXCR4 is normally expressed bymyeloid cells, B cells, andT cells (7, 8), but is also thought to be expressed by some epithelialcells. In the stomach, CXCR4 also identifies type 2 innate lymphoidcells (ILC2; ref. 9). Expression of CXCR4 by hematopoietic pro-genitor cells (HPCs) and the production of CXCL12 by stromal cellsand osteoblasts are important for homing and retention of thesecells. Together with other cytokines (e.g., SCF and IL7), CXCR4function is required for normal maturation of myeloid and lym-phoid cells, and for the survival and proliferation of B-cell pre-cursors and myeloid progenitor cells (10). While CXCR4 has alsobeen shown to be expressed by columnar epithelial cells in thegastrointestinal tract, where it appears to mark progenitors, the roleof CXCR4 signaling in this setting remains to be elucidated (11).

The incidence of esophageal adenocarcinoma, a highly invasivecancer, is rapidly rising in the Western world, and now accountsfor 2% of all cancer-related deaths (12). CXCL12 (SDF-1) andCXCR4 expression in esophageal adenocarcinoma are associatedwith a poor prognosis and metastases to lymph nodes or bonemarrow (13, 14). In vitro, CXCR4-positive (CXCR4þ) esophagealcancer cells show stronger migration ability (15). Knockdown of

1II. Medizinische Klinik, Technische Universitat M€unchen, Munich, Germany.2Pharmazeutische Radiochemie, Technische Universitat M€unchen, Munich, Ger-many. 3Division of Digestive and Liver Diseases, Columbia University MedicalCenter, New York, New York. 4Klinik f€ur RadioOnkologie und Strahlentherapie,Technische Universitat M€unchen, Munich, Germany. 5Institut f€ur Pathologie,Technische Universitat M€unchen, Munich, Germany. 6NuklearmedizinischeKlinik und Poliklinik, Technische Universitat M€unchen, Munich, Germany.7iPATH.Berlin/Medizinische Klinik f€ur Gastroenterologie, Infektiologie undRheumatologie/Research Center ImmunoSciences, Charit�e-Campus BenjaminFranklin, Berlin, Germany. 8Division of Gastrointestinal Pathology, ColumbiaUniversity Medical Center, New York, New York.

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

Corresponding Author: Michael Quante, II. Medizinische Klinik, Klinikum rechtsder Isar, Technische Universit€at M€unchen, Ismaninger Strasse 22, M€unchen81675, Germany. Phone: 4989-4140-7870; Fax: 4989-4140-6796; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-1756

�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 24(5) March 1, 20181048

on June 12, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1756

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-1756&domain=pdf&date_stamp=2018-2-26

http://clincancerres.aacrjournals.org/

CXCR4 expression by siRNA or the use of inhibitors reduces theproliferation and invasion ability of esophageal cancer cells (4,16). In vivo, pharmacologic antagonism of CXCR4 has beenshown to decrease tumor growth and metastasis in xenograftmodels (4, 16, 17). However, these studies have limitations intheir ability to accurately reflect human disease, and do notaddress the role of CXCR4 in the premalignant stage of Barrett'sesophagus. Barrett's esophagus is the predominant precursorlesion for esophageal adenocarcinoma, and is strongly associatedwith gastroesophageal reflux disease (GERD); although it remainsunclear why some patients with GERD develop Barrett's esoph-agus and others do not (18). Furthermore, the overall rate ofcancer progression fromBarrett's esophagus is extremely low, andthus specific biomarkers are needed to identify patients with anelevated risk for developing esophageal adenocarcinoma. Detect-ing cancer at an early andpotentially curable stage in early Barrett'sesophagus progression might considerably improve survival ofpatients with esophageal adenocarcinoma. In this study, weinvestigated the role of CXCR4 expression in esophageal tumorprogression, and explored its relevance for early tumor detectionusing molecular imaging.

We have previously developed the pL2-IL1b transgenic mousemodel of Barrett's esophagus that exhibits chronic inflammation inthe esophagus and recapitulates the histologic progression toesophageal adenocarcinoma (19). To date, this mouse model hasprovided several fundamental insights into the pathogenesis ofBarrett's esophagus, including the emerging paradigm that Barrett'sesophagus and esophageal adenocarcinoma arise from distinctprogenitor cells in the gastric cardia (19). Considerable evidencesupports chronic inflammation as a major factor in the develop-ment of Barrett's esophagus and esophageal adenocarcinoma, as itis able to trigger the expansion of these gastric cardia progenitors.In some patients with GERD, chronic injury to the GE junctionresulting from exposure to gastric contents leads to the upregula-tion of inflammatory cytokines (IL1b, IL6, IL8), which are thoughtto contribute to Barrett's esophagus and esophageal adenocarci-noma. IL1b is highly expressed in Barrett's esophagus (20), and thepL2-IL1b mouse carries a modified human IL1b transgene con-

trolled by the Epstein-Barr virus (L2) promoter, resulting inincreased IL1b gene expression targeting the esophagus and squa-mous forestomach. The pL2-IL1bmice exhibit chronic esophagitisand progress to Barrett's esophagus by 6 months, eventuallydeveloping esophageal adenocarcinoma at an older age.

Here, we show that CXCR4 expression increases in bothimmune and epithelial cells during progression of Barrett's esoph-agus to low-grade (LGD) and high-grade dysplasia (HGD) andultimately to esophageal adenocarcinoma in the pL2-IL1bmousemodel.Wefind thatCXCR4þ immune cell recruitment specificallycorrelates with increased dysplasia progression, suggesting thatimmune cells are key contributors to esophageal carcinogenesis.We also find that CXCR4 marks epithelial cells near the baseof gastric cardia glands, which expand during the growth of thesecells into the esophagus. EnhancedCXCR4-related signal intensityin ex vivofluorescence imagingandautoradiographywere found tocorrelate with increased CXCR4 expression during disease pro-gression in this model. A novel technique for CXCR4-directed[68Ga]pentixafor-PET/CT imaging in patients with esophagealadenocarcinoma demonstrates focal tracer accumulation inesophageal cancer and in metastatic sites. Our results support thenotionof exploitingCXCR4þ immunecells togetherwithCXCR4þ

epithelial cells for molecular imaging of the tumormicroenviron-ment, thus identifying CXCR4 as a promising biomarker formonitoring and staging of esophageal tumor progression.

Materials and MethodsAnimals

Ageneticmousemodel (pL2-IL1b) that overexpresses IL1b in themouse esophagus and stomach was prepared as described previ-ously (19).Micewere alloweda standard chowdiet frombirthuntilweaning, and water ad libitum. Following weaning and genotyping,between 6 and 8 weeks of age, mice were assigned high-fat diet orremained on the chow diet: Ssniff, V1124-000, Metabolizableenergy (ME):61 kJ%% energy from carbohydrate, 27 kJ% fromprotein and 12 kJ% from fat). High-fat diet: (Ssniff, S5745-E712,ME: 34 kJ% energy from carbohydrate, 18 kJ% from protein and48 kJ%% from fat). All animal experiments were approved by theDistrict Government of Upper Bavaria and performed in accor-dance with the German Animal Welfare and Ethical Guidelines ofthe Klinikum rechts der Isar, TUM (Munich, Germany).

IHC and immunofluorescenceFor mouse samples, after sacrifice, stomach and esophagus

tissues were fixed in formalin and paraffin embedded. Tissueswere cut and put on superfrost slides for drying overnight or at the60�C oven for >1 hour. Standard IHC procedures were performedusing following antibodies: rat anti-mouse CD184 (CXCR4) anti-body (eBioscience, 1:250 4�C overnight) and rabbit anti-a-SMA(Abcam, 1:400, 2 hours room temperature) for mouse tissue.Quantification of CXCR4was assessed on three serial sections (2–3mm) taken every>100mmfor eachmouse. Fivehigh-powerfields[field of vision under maximum magnification (�400)] weretaken by microscope (Zeiss) in each section. The images weretaken in a manner to achieve high power fields with the mostpositively stained cells. Then CXCR4þ cells (brown) and hema-toxylin-counterstained nuclei of the cells (blue) were counted.CD31 immunofluorescence staining was done as described pre-viously (9). For human biopsy samples, Barrett's esophagus,HGD, and adenocarcinoma tissues from esophagectomy or

Translational Relevance

Barrett's esophagus can be diagnosed and biopsied byesophagogastroduodenoscopy (EGD), but current screeningand surveillance strategies are limitedby the absence of specificbiomarkers highly predictive of increased risk for esophagealadenocarcinoma. Moreover, following diagnosis of esophage-al adenocarcinoma, better methods of PET/CT imaging areneeded to detect small metastases and tumors with lower ratesof glucose metabolism. This translational study, comprising aBarrett's esophagus mouse model and esophageal adenocar-cinoma patient data, clearly demonstrates that the chemokinereceptor 4 (CXCR4) is upregulated in the immune tumormicroenvironment and esophageal progenitor cells, thus pre-senting a highly attractive target for molecular imaging usingdedicated CXCR4-targeted probes. Our findings provide newinsights into the kinetics and pathogenesis of esophagealcancer, and introduce CXCR4-based imaging of the tumormicroenvironment as a potential approach for diagnosis andsurveillance of esophageal adenocarcinoma.

CXCR4 in Esophageal Adenocarcinoma

www.aacrjournals.org Clin Cancer Res; 24(5) March 1, 2018 1049




endoscopic resection specimens were identified from archivedformalin-fixed and paraffin-embedded (FFPE) cases from theDepartment of Pathology and Cell Biology, Columbia University(New York, NY). Five-mm-thick tissue sections were deparaffi-nazed and rehydrated, followed by antigen retrieval, performedin 100-mL Target Retrieval Solution pH9 (Dako, #S2367) using asteaming approach for 25 minutes followed by cooling at roomtemperature for 20 minutes; then, slides were rinsed three timeswith PBS for 5 minutes. Endogenous peroxidase was blocked in30% hydrogen peroxide for 10 minutes. The slides were rinsedwith distilled water once and with PBS three times for 5 minuteseach time. The slideswere thenblocked in10%normal goat serumfor 20 minutes at room temperature. After removal of blockingserum, the sections were incubated with anti-CXRC4 antibody(Abcam, #ab124824) at 1:1,000 dilution for 90 minutes at roomtemperature. Staining of primary antibody was detected usinganti-rabbit biotinylated secondary antibody (Vector Laboratories,# BA-1000) at 1:300 dilution for 30minutes at room temperaturefollowed by avidin-biotinylated peroxidase solution for 30 min-utes at roomtemperature.DABsolution(Dako, #K3468)wasusedas the chromogen; the sections were then counterstained withhematoxylin (Merck Millipore, #105174).

Tissue microarrays from patients with esophageal adenocarci-noma were constructed from formalin-fixed paraffin-embedded(FFPE) tissue blocks obtained from 47 patients with esophagealadenocarcinoma diagnosed at the Charit�e University Hospital(Berlin, Germany) and 12 patients diagnosed at the University ofHeidelberg (Heidelberg, Germany). Patientmaterial was accessedafter written consent and according to the regulations and ethicalvote of the Technical University of Munich (503/16s). IHC forCXCR4was performedwith a Leica BondRxm system (Leica)witha primary CXCR4 antibody (Abcam, #ab124824) and a polymerrefine detection system.

Flow cytometrySingle-cell suspensions of murine esophageal and cardia tissue

along with forestomach regions were generated by choppingtissue with scissors in EDTA solution. Then, the tissue and EDTAsolution was transferred into digestion medium followed byincubation at 150 rpmat 37�C for 30minutes. Digestionmediumfor esophagueal tissue consisted of 5mL Krebs Ringer bufferþ4%(w/v) BSA (0.2 g) þ 2 mg/mL collagenase P (Roche). Digestionbuffer for cardia and forestomach consisted of 5 mL DMEM þ2 mg/mL collagenase P þ 2 mg/mL Pronase (Roche). The fol-lowing antibodies were used for FACS analysis: e450-CD45, APCe780-cd11b, APC- F480, Alexo700-Ly6G, FITC- cd3, and PE-anti-CXCR4, APC e780-Epcam, APC e780-NK1.1, PECy5.5-B220, andPECy7-CD90.2 (all antibodieswere purchased fromeBioscience).7-AAD (eBioscience) was used to quantify live cells. FACS datawere acquired on aGalliosflowcytometer (BeckmanCoulter) andanalyzed using FlowJo software (TreeStar).

Ex vivo fluorescence imagingCXCR4 antibody (eBioscience) was conjugated with Cy5.5

(Alexa Fluor 680,AF680)-NHSEster (Life Technologies) accordingto the manufacturer's protocol. Mice were injected intravenouslywith conjugated CXCR4-Cy5.5 antibody and sacrificed 24 hourspostinjection. After sacrificing, the esophagus and stomach wereexcised and imaged. Fluorescence images were acquired by illu-minating the specimens using 670- and 750-nm diode lasers andguiding the emitted fluorescence through appropriate emission

filters before capturing it using a back illuminated EM-CCDcamera (iXonDU888,Andor), as describedpreviously (21). Signalspecificitywasdeterminedby ImageJ for calculating the ratio of themean signal intensity in the exposed esophagus tissue and thesurrounding background. To demonstrate the specificity of thefluorescent probe, mice were coinjected with CXCR4-Cy5.5 andtheCXCR4 inhibitor AMD3100 (2mg/kg). Injection of AMD3100resulted in a near absence of significant signals in early lesionregions at the SCJ and in esophagus, supporting the notion thatthe fluorescent probe had binding specificity for CXCR4 (datanot shown).

Ex vivo autoradiographyMice (12 months treated with chow diet or high-fat diet, n ¼ 2

per group) were injected intravenously with approximately1.85 MBq [125I]TUM4007 in 100-mL PBS (pH 7.4) under isoflur-ane anesthesia. Like pentixafor, [125I]TUM-4007 is not an anti-body, but is a peptidic probe with a molecular weight <2,000 Da.After 1 hour, mice were sacrificed and perfused with 4% PFA. Thetissues of interest were resected and stored at (�80�C). Cryosec-tions (5 mm) of stomach/esophagus were thenmounted on coverslides and exposed on a Fujifilm BAS-IP MS 2025 (20 � 25 cm)imagingplate (GEHealthcare Lifesciences). Theplatewas scannedwith a CR35 Bio Phosphor Imager (Raytest) in sensitive 25-mmresolution mode. Scanning and data export was performed usingAIDA software (Raytest).

[68Ga]Pentixafor PET/CT in patients[68Ga]pentixafor-PET/CT was performed on five patients

(one woman, four men; mean age, 72 � 12.1 years). Overall,nine PET/CT examinations were carried out. The validationof images and SUV values were performed by an experiencednuclearmedicine consultant. [68Ga]pentixaforwas used in six and[18F]FDG in three scans. In three patients, both [68Ga]pentixafor-and [18F]FDG-PET/CT imaging was performed at the sametime. One patient received [68Ga]pentixafor-PET/CT imagingbefore and after chemotherapy. Another patient only received[68Ga]pentixafor-PET/CT. Injected activity for [68Ga]pentixaforwas 163 � 52 MBq and 361 � 61 MBq for [18F]-FDG. Thewaiting time between injection and imaging was 56 � 9 minutesfor [68Ga]pentixafor and 65 � 6 minutes for [18F]FDG. The CTscan protocol included a low-dose CT from the base of the skull tothe mid-thigh for attenuation correction followed by the PETscan and in some cases a diagnostic CT in the portal venous phase.PET scans were taken with five to seven bed positions for 2 to 3minutes each. PET data were reconstructed with 128 � 128 pixelimages. Images were reconstructed by an attenuation-weightedordered subsets expectation maximization algorithm (four itera-tions, eight subsets) followed by a postreconstruction smoothingGaussian filter (5-mm full-width at half-maximum). For semi-quantitative SUVmean evaluation, a 3D volume of interest (VOI)with a growing seeded method of a 50% isocontour of SUVmax

was used.

ResultsExpression of CXCR4 increases during progression todysplasia in pL2-IL1b mice and in patients with esophagealadenocarcinoma

Weutilized our transgenicmousemodel (pL2-IL1b) of Barrett'sesophagus and dysplasia to analyze the pattern of CXCR4 expres-sion during esophageal carcinogenesis (19). pL2-IL1b mice

Fang et al.

Clin Cancer Res; 24(5) March 1, 2018 Clinical Cancer Research1050




develop low-grade dysplastic lesions in the setting of Barrett-likemetaplasia at the squamocolumnar junction (SCJ) at approxi-mately 12months of age.When pL2-IL1bmice were fed a high-fatdiet (HFD), they develop accelerated disease, with mid-gradedysplastic lesions along the SCJ by 9 months and larger high-grade dysplastic lesions in the esophagus and SCJ at 12 months(Fig. 1A, i and ii), enabling us to study changes associated withstepwise progression of esophageal carcinogenesis. CXCR4þ cellswere detected by IHC only in the inflamed esophagus of pL2-IL1bmice, but not the esophagus of wild-type mice (Fig. 1A, iii).

Notably, pL2-IL1b mice showed increasing CXCR4 expressionlevels in the esophagus and at the SCJ, which correlated well withincreasing numbers of macroscopic and microscopic dysplasticlesions, along with increasing age (Fig. 1A and B). Furthermore,significantly higher CXCR4 expression was found in tissue sam-ples of human esophageal adenocarcinoma compared withhuman Barrett's esophagus tissue with only LGD (Fig. 1C andD). Overall, these data suggest that CXCR4 expression correlateswith esophageal tumor progression from nondysplastic Barrett'sesophagus to LGD to HGD and esophageal adenocarcinoma.

B

0

10

20

30

40

50

n.s.

*

**

*

*

WT12 months

pL2-IL1β chow12 months

pL2-IL1β HFD9 months


No tumor Low-grade Mid-grade High-gradeDysplasiaprogression

% o

f CXC

R4+ /H

emat

oxyl

in

BE EAC0

10

20

30

CXC

R4

%

D

WT 12 months pL2-IL1β chow 12 months pL2-IL1β HFD 9 months pL2-IL1β HFD 12 months

A

forestomach

SCJ

esophagus

Low-gradePre/small lesions

Mid-gradelesions

Dysplasia lesion progression

No tumorHigh-grade

lesions

(i)

(ii)

(iii)

C BE, LGD EACBE, HGD

Figure 1.

CXCR4 expression increases whiledysplasia progresses in HGDmice (high-fat diet, HFD, pL2-IL1bmice) and in patients withesophageal adenocarcinoma(EAC). A, (i) macroscopic pictureof esophagus and stomach (arrow:dysplasia lesions). HFDaccelerates the developmentof dysplasia lesions along squamo-columnar junction (SCJ), cardia,and esophagus in 9 months.(ii) Representative hematoxylin& eosin (H&E) staining of cardiatissue. (iii) RepresentativeCXCR4 IHC images show thatCXCR4 is increasing throughesophageal dysplasia progression.B, Quantification of CXCR4þ IHC.Quantification was assessedon three sections taken every>100 mm in each mouse. Five to10 positive staining fields weretaken by microscope (�40)in each section. Hematoxylin andCXCR4þ cells were counted. Dataare represented as mean � SEM.� , Two-tailed t test P < 0.05. C,CXCR4 IHC shows more CXCR4expression in HGD and patientswith esophageal adenocarcinomathan LGD in patients withBarrett's esophagus (BE). Originalmagnification, 200�. Epithelialcells are strongly positivein HGD, and show membranousand cytoplasmic staining. Inesophageal adenocarcinoma,epithelial cells express CXCR4(solid arrow) as well as manyimmune cells (opened arrow area).D,Quantification of CXCR4þ IHC inpatients with Barrett's esophagusor esophageal adenocarcinoma(n ¼ 3–4).






Immune cells, but not a-SMAþfibroblasts or endothelial cells,

are the main contributors to increased CXCR4 expressionInterestingly, our IHC analysis showed that the majority of

CXCR4 expression was in the stroma, suggesting that esophagealepithelial (Fig. 1C, solid arrows) or cancer cells may not be solely

responsible for the observed increases in CXCR4 expression.Therefore, we next investigated the nature of the cells contributingto the accelerated expression of CXCR4 in dysplastic esophagealtissue. IHC showed that a-SMAþ

fibroblast cells in the esophagusdid not express CXCR4 (Fig. 2A), indicating that the higher

D CD45+

0

10

20

30P = 0.062



Low-grade High-gradeDysplasiaprogression

% C

XCR

4+ C

ells

CD45+

BE EAC0

100

200

300

400

Num

ber o

f cel

ls in

5 p

ower

of f

ield

s

CD45+

0

10

20

30

40

50





% o

f Via

ble

cells

A

B

pL2-IL1β chow 12 months pL2-IL1β HFD 9 months pL2-IL1β HFD 12 months

Dysplasia progression

CXCR4-eGFP 8 months CXCR4-eGFP/pL2-IL1β 8 months CXCR4-eGFP/pL2-IL1β 14 months

Low-grade High-grade Dysplasia progression

No tumor

C

Cxcr4 CD31DAPI

Low-grade Mid-grade High-grade

Figure 2.

Immune cells are the main contributors of CXCR4 expression in esophagus in pL2-IL1b mice. WNT3a is the critical niche factor. A, Representative picturesshow that a-SMAþ fibroblast (pink) did not express CXCR4 (brown). B, Representative pictures show that CD31þ endothelial cells (red) did not expressCXCR4 (green). C, Both LGD mice and HGD mice show inflammation infiltration (flow cytometric analysis, n ¼ 4–7). Patients with esophageal adenocarcinomaand Barrett's esophagus (BE) LGD patients have inflammation infiltration (IHC, n ¼ 5). D–G, Flow cytometric analysis of CXCR4 expression in immune cellpopulations in esophagus. More neutrophils express CXCR4 in HGD mice than LGD mice (n ¼ 5–7). Mice were sacrificed at 12-month time point. Markers forneutrophils: CD45þCD11bþLy6GþF480�. Markers for macrophages: CD45þCD11bþLy6G�F480þ. (Continued on the following page.)

Fang et al.





CXCR4+CD45+CD11b+

0

10

20

30

40

50*

LGD9 months

LGD12 months

pL2-IL1βchow mice

% C

XCR

4+ C

ells

CD45

0

20

40

60 *

LGD9 months

LGD12 months

pL2-IL1βchow mice

% C

XCR

4+ C

ells

WNT3a

0

1

2

3

4

5 *




F

WNT5a

0.0

0.5

1.0

1.5 *




DDC

t

DDC

t

I

Neutrophils

0

5

10

15

20 *




% C

XCR

4+ C

ells

CXCR4+Epcam+

0

2

4

6

8 **





% o

f Via

ble

cells

CD3+ T Cells

0

5

10

15 *




% C

XCR

4+ C

ells

G

H

J

CXCR4+CD45+CD11b+

0

10

20

30 *




% C

XCR

4+ C

ells

E

Only in immune cells

53%

Only in epithelial

cells3%

Both in immune cells and epithelial

cells42%

Not in immune cells nor epithelial

cells2%

CXCR4 Expression contribu�on

K

L

Figure 2.

(Continued. )H, Flow cytometric analysis of CXCR4þEpCAMþ cell populations in cardia and forestomach of different time point mice (pL2-IL1b 9 monthsand 12 months mice n ¼ 3, pl2-IL1b 14 months mice n ¼ 2). I, Flow cytometric analysis of CXCR4 expression in immune cells populations inesophagus in LGD mice at different time points (n ¼ 5). J, Real-time quantitative PCR shows that WNT5a is downregulated and WNT3a isupregulated in esophageal in high-fat diet (HFD) 12-month mice. K, Tumor microarray of 59 patients with esophageal adenocarcinoma. Many immunecells (opened arrows) express CXCR4 and some epithelial tumor cells (solid arrows) express CXCR4. L, Contribution of CXCR4 from immunecells or/and epithelial cells in tumor microarrays of 59 patients. Data are represented as mean � SEM.� , Two-tailed t test, P < 0.05.






numbers of a-SMAfibroblastswere also not themain contributorsto elevated CXCR4 expression during esophageal carcinogenesis,as previously suggested in other cancer models (6, 22). In addi-tion, CD31þ cells did not show any overlap with the GFP reportergene in a CXCR4-GFP reporter mouse crossed to the pL2-IL1bmouse model at different stages of carcinogenesis (Fig. 2B),indicating that endothelial cells are also not contributingsubstantially to the CXCR4þ cell pool.

In contrast, there were multiple CXCR4þ immune cells detect-able in esophageal adenocarcinoma patient biopsies (Fig. 1C,opened arrows). Mice with both LGD and HGD, respectively,showed a similar abundance of CD45þ immune cells in theinflamed esophagus, indicating a significant inflammatory infil-tration (Fig. 2C). Similar findings were also noted in patientswith Barrett's esophagus and esophageal adenocarcinoma(Fig. 2C). Interestingly, in the esophagus of 9- to 12-month-oldpL2-IL1b mice, we observed increasing numbers of CXCR4þ

leukocytes (CXCR4þCD45þ), particularly increased myeloid cell(CD45þCD11bþ), neutrophil (CD45þCD11bþF480�Ly6Gþ)andCD3þT-cell populations (Fig. 2C–G). Therewas nodifferencein B cells, ILCs, and NK-cell populations (Supplementary Fig.S1B–S1D). Moreover, we also observed significantly increasednumbers of CXCR4þmyeloid cells in LGD lesions from 9monthsto 12 months (pL2-IL1b mice on chow diet; Fig. 2I). Takentogether, these data suggest that immune cells might be the maincontributors to the elevated stromal CXCR4 expression, and aredetectable at early dysplastic stages, comprising a distinct pre-tumor microenvironment. In addition, increased CXCR4þ mye-loid cells were found in the spleen (Supplementary Fig. S1E),implying that the CXCR4þ myeloid cells in esophagus may berecruited in part from extramedullary sites. Together, these datasuggest that these elevated CXCR4þ immune cell populationsmight be the main contributors to a procarcinogenic inflamma-tory niche that is distinct from routine esophageal inflammation,and instead leads to dysplasia and cancer.

Barrett's esophagus is defined as replacement of the stratifiedsquamous epithelium in the distal esophagus with a metaplastic,intestinal-like columnar epithelium (23). In ourmurinemodel ofBarrett's esophagus and esophageal adenocarcinoma, the meta-plastic lesions likely originate from stem cells in the gastric cardia(19, 24), which over time appear to expand proximally into thesquamous esophagus, leading eventually to dysplasia. The triggerfor the activation and expansion of gastric cardia progenitors maybe cytokine/chemokine signals from infiltrating immune cells.We noted increasing numbers of CXCR4þ epithelial cells at theSCJ in the CXCR4-GFP reporter mouse crossed to the pL2-IL1bmice (CXCR4-GFP;pL2-IL1b) during the stepwise progressionfrom Barrett's esophagus to LGD and HGD (Fig. 1C) and mouseGFPþ epithelial cells (Fig. 2B). Many of these CXCR4�GFPþ cellswere proliferating (Ki67þ; Supplementary Fig. S2), and theirnumbers correlated with increasing grades of dysplasia. A similarincrease in CXCR4þ epithelial columnar cells was observedin patients with Barrett's esophagus, with strong IHC stainingparticularly evident in Barrett's tissue with HGD (Fig. 1C). Thus,we further investigated the correlation of epithelial progenitorcells and elevated CXCR4 expression in our mouse model usingFACS. Increasing numbers of CXCR4þ EpCAMþ cells weredetected in the cardia and forestomach ofmice withHGD lesions,compared with mice with LGD (Fig. 2H). In tumor microarraysfrom 59 patients with esophageal adenocarcinoma, CXCR4expression was restricted to infiltrating immune cells in 53% of

patients, in 3% of patients, CXCR4 was expressed only by epi-thelial tumor cells and in 42%of patients, both, immune cells andepithelial tumor cells contributed to the total CXCR4 expression(Fig. 2K and L). In summary, epithelial progenitor cells mightrepresent yet another contributor of elevatedCXCR4expression inlater stages of dysplasia, while immune cells are likely the maincontributor to elevated CXCR4 expression in early dysplasiadevelopment.

The cellular proliferation and differentiation of epithelial cellsdepends on a large array of signaling molecules such as canonicalWnt (Wingless-Type MMTV Integration Site Family) signaling(25). As it is not clear whether such activation of Wnt signalingis critical in Barrett's metaplasia, dysplastic, and esophageal ade-nocarcinoma development, but WNT3 is suggested to play a keyactivation role in breast, rectal, lung, and gastric cancers throughactivation of the WNT–b-catenin–TCF canonical signaling path-way (26),we further investigated theWnt signalingcomponents inour Barrett's esophagus mouse model. In contrast to our findingsin the gastric corpus,whereWnt5a-secreting innate lymphoid cells(ILC2) were critical to the development of diffuse gastric cancer(9), we found that in correlation to the increasing numbers ofCXCR4 immune cells, Wnt3a but not Wnt5a was unregulated inthe esophageal niche during accelerated tumorigenesis in ourBarrett's esophagus model (Fig. 2J), pointing to Wnt3a as apotentially important cancer niche factor in the esophagus.

Ex vivo fluorescence imaging and ex vivo autoradiography showincreased CXCR4 expression during dysplasia progression inpl2-IL1b mice

On the basis of the finding that local CXCR4 expressionincreased gradually during esophageal carcinogenesis, we hypoth-esized that CXCR4 expressionmighty be exploited as a biomarkerof disease progression. Thus, we conjugated a commerciallyavailable anti-mCXCR4 antibody with the NIR fluorescent dyeCy5.5 (AF680) and injected it intravenously into mice. Thespecificity of the fluorescent probe was validated in these studiesby coinjecting the CXCR4-Cy5.5 antibody with the CXCR4 inhib-itor AMD3100 (2mg/kg), which blocked binding by the antibody(data not shown). Ex vivo fluorescence imaging at 24 hours afterinjection indeed showed that the CXCR4-Cy5.5 antibody specif-ically accumulated in small dysplastic lesions in pL2-IL1b mice,and that uptake was significantly increased in mice with HGD(high-fat diet pL2-IL1b; Figs. 3A, i and ii and 3B). Some CXCR4-specific Cy5.5 fluorescence signal was also detected at the SCJ(Fig. 3A, iii and iv). While there was a moderate increase of signalthroughout the inflamed esophagus of pL2-IL1bmice, consistentwith this receptor marking chronic inflammation, there was aremarkable high level of accumulation in areas of tumorigenesis(yellow lesions, marked with arrows), indicating that high levelsof CXCR4 expression were very specific for the dysplastic lesionitself, reflecting more than simply an accumulation of chronicinflammatory cells.

[68Ga]pentixafor is currently being evaluated clinically for PETimaging of CXCR4 expression in patients with cancer (LIT;refs. 27–29), and displays pronounced selectivity for humanCXCR4. It does not bind to murine CXCR4, and thus is notsuitable for in vivo imaging of disease progression in the pL2-IL1btransgenic mouse model. Therefore, an alternative CXCR4 ligandwith high affinity for the murine CXCR4 receptor, [125I]TUM-4007, a peptidic probe with a molecular weight <2,000 Da, wasused to detect CXCR4 expression in murine Barrett's esophagus

Fang et al.





and esophageal adenocarcinoma via autoradiography. Mice wereinjected with approximately 1.85 MBq [125I]TUM-4007 with orwithout 2 mg/kg AMD3100, and animals were sacrificed 1 hourlater for biodistribution analysis. The 125I-labeled mCXCR4-targeted ligand showed significant accumulation in the liver,

along with moderate uptake in the spleen and stomach. Adecrease in tracer accumulation in liver, spleen, and stomach wasobserved in mice coinjected with 2 mg/kg of the antagonist,AMD3100 (Fig. 4C), demonstrating CXCR4 specificity of radi-oligand uptake in these tissues. For ex vivo autoradiography, mice

A

B C

pL2-IL1β HFDWT chow pL2-IL1β chow pL2-IL1β HFD

CXCR4-AF680 signal

0

10

20

30

40




WT HFD12 months

WT chow12 months

No tumor

Rat

io to

bac

kgro

und

lesions

No tumorMid-grade

dysplasia lesionsDysplasia progression

No tumorHigh-grade

dysplasia lesions

No tumorHigh-grade

lesionsHigh-grade

lesionsMid-grade

lesions

(A)pL2-IL1β chow WT HFD pL2-IL1β HFD WT chow

WT HFD

Esophagus

SCJ

SCJEsophagus

Lesions

(i)

(ii)

(iii)

Lesions

Esophagus

Lesions

Lesions

Esophagus

EsophagusEsophagus

SCJ

EsophagusBright field

EsophagusCy5.5

SCJBright field

(iv ) SCJ Cy5.5

No tumorHigh-grade

lesionsWT chow pL2-IL1β HFD

High-grade lesions

Mid-grade lesions

pL2-IL1β chow pL2-IL1β HFD

Figure 3.

Ex vivo—fluorescence imaging shows that CXCR4-targeted fluorescent antibody accumulates in small esophageal dysplasia lesions, and uptake graduallyincreases with dysplasia progression in pL2-IL1b mice. A, CXCR4-targeted fluorescence signal is high and specifically increased in esophageal tumor lesionsand is enhanced in high-grade tumor mice (i and ii, arrows). Specific accumulation of fluorescent anti-CXCR4 antibody is also found in squamo-columnarjunction (SCJ). This suggests that CXCR4 also accumulated in inflammation areas (iv), (dash lines). B, Quantification of Cy5.5 signal intensity. Representativequantification images of esophageal areas (dash lines) and background areas (white rectangle). Mice (12 months of age) were sacrificed at 24-hourpostinjection of Cy5.5-conjugated anti-CXCR4 antibody. Images shown here were all taken at 2-second exposure time. pL2-IL1b mice n ¼ 4. Wild-type (WT)mice n ¼ 1–2.






Autoradiography signal

0

50

100

150

200[125I]TUM4007[125I]TUM4007 + AMD3100



Mid-grade High-gradeDysplasiaprogression

Inte

nsity

[AU

]A

[125I]TUM4007

[125I]TUM4007 + AMD3100

pL2-IL1β HFDpL2-IL1β chowMouse 1 Mouse 2

Mouse 3 Mouse 4

Mid-grade dysplasia lesions High-grade dysplasia lesions

Mouse 5 Mouse 6

Mouse 6 Mouse 7

Blood Spleen Stomach Muscle0

1

2

3

4

5

6

High-grade tumor (pL2-IL1β HFD 12 months) [125I]TUM4007 only (n = 3)

High-grade tumor (pL2-IL1β HFD 12 months) coinjection of 2 mg/kg AMD3100 (n = 2)

Low-grade tumor (pL2-IL1β chow 12 months) [125I]TUM4007 only (n = 2)

Low-grade tumor (pL2-IL1β chow 12 months) coinjection of 2 mg/kg AMD3100 (n = 2)

Act

ivity

accu

mul

atio

n[%

iD/g

]

B C

(i)

(ii)

(iii)

(iv)

Figure 4.

PET tracer ex vivo autoradiography show that CXCR4 accumulated in small esophageal dysplasia lesions and it is gradually increased while dysplasiaprogresses in pL2-IL1b mice. A, (i and iii) Autoradiography signal is increased in HGD mice compared with LGD mice. CXCR4 specificity of tracer accumulationwas demonstrated by coinjection of the unlabeled CXCR4 antagonist AMD3100 (2 mg/kg). (ii and iv) hematoxylin and eosin staining of corresponding slicesdemonstrate inflammation in esophagi and SCJs of all mice. B, Quantification of autoradiography. C, Tracer biodistribution shows significant accumulationin spleen and stomach. CXCR4 specificity of tracer accumulation was demonstrated by coinjection of the unlabeled CXCR4 antagonist AMD3100 (2 mg/kg).Mice (12 months of age, n ¼ 2 in each group) were sacrificed 1 hour postinjection of app. 1.85 MBq [125I]TUM-4007. Organs of interest were dissected andcryosliced, and cryosections of stomach and esophagus imaged by autoradiography using a Phosphor Imager.

Fang et al.





were injected with [125I]TUM-4007 with or without AMD3100,sacrificed 1 hour postinjection, and CXCR4-mediated tracer accu-mulation in tissue cryosections from stomach and esophagus wasvisualized using autoradiography. Notably, high focal [125I]TUM-4007 uptake was seen in pL2-IL1b mice with dysplasia, with asignificant increase in tracer uptake in HGD (Fig. 4A). Moreimportantly, a significant decrease in tracer signal was found inmice coinjected with 2mg/kg of the inhibitor, AMD3100 (Fig. 4Aand B). These data strongly suggest that uptake of the CXCR4tracer is specific for tumor lesions and/or premalignant inflamedtissue, and correlates stronglywith adysplastic phenotype (Fig. 4Aand B).

CXCR4-targeted [68Ga]pentixafor PET in patients withmetastatic esophageal cancer

On the basis of our Barrett's esophagus mouse model data, wehypothesized that imaging of CXCR4 expression could beexploited as a marker for diagnosis and staging of esophagealadenocarcinoma in patients. As noted above, ex vivo fluorescenceimaging inpL2-IL1bmice showed thatCXCR4þ cells accumulatedearly on in the dysplastic lesion itself. The recent development ofthe CXCR4-targeted PET tracer [68Ga]pentixafor (28, 29) hasprovided for sensitive and high-contrast imaging of hCXCR4expression in patients suffering from lymphoma (30), multiplemyeloma (27, 31), SCLC (32), glioblastoma (33), and other solidtumors (34, 35). Therefore, we investigated the potential of[68Ga]pentixafor PET/CT for diagnosis and therapeutic monitor-ing of metastatic esophageal adenocarcinoma in a small patientcohort, and compared the findings with results using the meta-bolic tracer [18F]FDG, which is routinely used in the staging ofesophageal adenocarcinoma.

[68Ga]pentixafor-PET/CT was performed in five patients (onewoman, four men; mean age, 72 � 12.1 years) with esophagealcancer, and the studies confirmed that CXCR4 expression canbe detected in vivo in esophageal adenocarcinoma (Fig. 5A).Overall, nine PET/CT examinations were carried out.[68Ga]pentixafor was used in six and [18F]FDG in three scans. Inthree patients, both [68Ga]pentixafor- and [18F]FDG-PET/CTimaging were performed for intrapatient comparison. One pati-ent received [68Ga]pentixafor-PET/CT imaging before and afterchemotherapy. Another patient only received [68Ga]pentixafor-PET/CT. Three of these patients showed metastasis. The evalua-tion of the SUV values is shown in Table 1. Representativepatient data demonstrated strong signal intensity for both[18F]FDG-PET (n ¼ 3) and [68Ga]pentixafor-PET (n ¼ 5),but the latter appeared to show greater specificity; thus, whileFDG-PET had a strong signal in the tumor region (Fig. 5,solid arrows), it also showed intense signals in the liver. Inaddition, CXCR4-mediated tracer uptake in the spleen wasobserved in patients with tumors (Fig. 5B, open arrow), con-firming the observations in our mouse studies. Moreover,[68Ga]pentixafor-PET/CT images of a patient with lymph nodemetastases indicated that CXCR4-targeted PET imaging mightpotentially be used to monitor metastasis in vivo (Fig. 5B).Furthermore, after receiving chemotherapy, the CXCR4 signalin both primary tumor and lymph node metastasis decreased(Fig. 5B). Finally, CXCR4 IHC of an esophageal adenocarcino-ma from a patient imaged by [68Ga]CXCR4-PET/CT showedinfiltration with CXCR4-expressing immune and epithelial cells(Supplementary Fig. S3), confirming that we indeed couldimage CXCR4 in patients with esophageal adenocarcinoma.

DiscussionIn this study, we show that local CXCR4 expression increases

during the progression of dysplasia in a mouse model of Barrett'sesophagus and esophageal adenocarcinoma. Mice harboringHGDdemonstrated increased CXCR4 expression in preneoplastictissue, compared with LGD. Interestingly, the increase in CXCR4expression was observed largely in immune cells (neutrophilsand T cells), but not fibroblast or endothelial cells, consistentwith earlier observations that neutrophils are a key contributorto esophageal carcinogenesis (19). Increased numbers of neu-trophils have been observed in patients with myxofibrosar-coma, gastric carcinoma, and melanoma (36–38). Neutrophilshave been suggested to contribute to tumor cell growth andmetastasis (39, 40), and increased neutrophil abundance pre-dicts worse metastasis-specific survival in patients with breastcancer (41, 42). In esophageal cancer, a high neutrophil–lymphocyte ratio predicts a worse disease-free overall survivalin surgically treated patients with esophageal squamous cellcarcinoma (43), and in REAL-2–treated patients with advancedesophagogastric cancer (44).

It is likely that the CXCR4þ immune cells are recruited in partfrom the spleen, where CXCR4-mediated uptake of CXCR4-targeted imaging probes was observed both in humans and inour mouse model. Extramedullary contributions to the tumormicroenvironment have been previously demonstrated, withstudies showing that neutrophil precursors can physically relo-cate from the spleen to the tumor stroma (45). The stromalmicroenvironment of tumors includes a mixture of hemato-poietic and mesenchymal cells, and clinical evidence supportsthe notion that these cells contribute to the development of acancer (46). Specific targeting of such an environment usingdedicated imaging probes may be used in the assessment ofearly tumor development, given that the recruitment ofCXCR4þ neutrophils appears strongly associated with progres-sion to HGD.

Furthermore, more CXCR4-expressing proliferating epithelialcells were observed in HGD mice, suggesting that epithelialprogenitor cells also contribute to elevated CXCR4 expression inthe later stages of dysplasia. In the mouse, CXCR4 is expressed inthe proliferative zone of the cardia glands, such that the expansionin Barrett's esophagus again points to a cardia origin for thismetaplastic lesion. As the numbers of infiltrating CXCR4þ

immune cells were elevated in the early stages of dysplasia,the epithelial progenitors in the gastric cardia may possibly beactivated by signals from the CXCR4þ immune cells or otherstromal cells in the pretumorigenic microenvironment. Furtherstudies, however, will be needed to define the source ofthe chemokine ligand, CXCL12 or SDF1, which presumablyregulates themigration and expansionofCXCR4þ cells inBarrett'sesophagus lesions.

The contribution of the recruited immune cells to the micro-environment, their interaction with progenitor epithelial cells,and their overall importance for tumor development or progres-sion remain unresolved. Hayakawa and colleagues recentlyreported that a CXCL12/CXCR4 perivascular niche in diffuse-typegastric carcinogenesis supports normal and neoplastic stem cellsthrough Wnt5a production (9). Given that Barrett's esophagus/esophageal adenocarcinoma derives from expansion of gastriccardia progenitors (19, 47), the role of the stromal niche seemshighly relevant. Hayakawa and colleagues also demonstrated that






a population of innate lymphoid cells (ILCs) expresses CXCR4and that these are specifically recruited to the site of diffuse gastriccancer (DGC) arising in the corpus, and that depletion of suchcells can inhibit tumorigenesis (9). Given the close relationshipbetween esophageal and gastric cancer (48), we investigated thepossible similarity between CXCR4þ immune cells in Barrett's

esophagus and esophageal adenocarcinoma. However, in con-trast to diffuse gastric cancer, the majority of CXCR4þ immunecells were not ILC2s but CD11bþmyeloid cells, and instead of anupregulation of Wnt5a as was observed in DGC, we found thatWnt3a was increased in the esophageal in our Barrett's esophagusmouse model. In addition, we recently showed that Wnt3a

Staging

Follow-up chemotherapy

tumorPrimary Lymph node metastasisB

Pa�ent 1 Pa�ent 2A[68Ga]pen�xafor

[18F]FDG

[68Ga]pen�xafor

[18F]FDG

Staging

Follow-up chemotherapy

Figure 5.

CXCR4 focal tracer accumulation in esophageal cancer and in metastasis sites in human patients. A, Transversal images of [68Ga]CPCR4-PET/CT and[18F]FDG-PET/CT showing high tracer uptake in patientswith esophageal cancer (solid arrows), indicating that CXCR4 can be detected in vivo.B, [68Ga]CPCR4-PET/CT of a patient with esophageal cancer and local regional lymph node metastasis. Primary tumor and lymph node metastasis exhibit high tracer uptake.Open arrow, spleen.

Fang et al.





improved Barrett's organoid growth in 3Dorganoid cultures (49),underlining the likely contribution of Wnt3a to columnar epi-thelial expansion in the Barrett's esophagusmousemodel. Wnt3amight well represent an important niche factor in Barrett's esoph-agus/esophageal adenocarcinoma, which would fit with its intes-tinal phenotype (50). Recent findings from our group haveindicated that myeloid cells likely represent an importantsource of Wnt regulating proliferation in the gastrointestinaltract (51). In the canonical Wnt signaling pathway, Wnt proteinsbind to the Frizzled/LRP receptor complex at the cell surface andfurther inhibit the degradation and subsequently accumulation ofb-catenin in the cytoplasmandnucleus (52). It has been suggestedthat noncanonical Wnt signaling might inhibit Wnt/b-cateninsignaling in developmental contexts. For example, Wnt5a expres-sion can inhibit the constitutively high Wnt/b-catenin signalingactivity of SW48 colon cancer cells (53). WNT5a may also act astumor suppressor (54). Taken together, in ourmodel, recruitmentof stem cells and potential differentiation into intestinal meta-plasia may rely more on myeloid cells and Wnt3a expression,similar to our current understanding for intestinal stem cells.

CXCR4-mediated probe accumulation was detected in bothmurine Barrett's esophagus and esophageal tumors by ex vivofluorescence and tissue autoradiography. Moreover, in PET/CTimaging in human patients, the CXCR4-targeted PET probe[68Ga]pentixafor accumulated in esophageal cancer tissue and inlymph node metastatic sites in esophageal adenocarcinomapatients. These data suggest that a distinct CXCR4þ immune orepithelial cell population might accumulate in tumors, althoughwe have not been able yet to demonstrate a major fraction oftumor cells to be CXCR4þ. Uptake of the CXCR4-targeted PETprobe in malignant lesions was markedly reduced followingchemotherapy, allowing us to hypothesize that advanced diseasemight be potentially monitored in this manner during treatment,similar to [18F]FDG-PET monitoring of treatment response (55),although confirmation of these preliminary data by moredetailed studies is needed. Taken together, the initial resultsobtained from esophageal adenocarcinoma patients using[68Ga]pentixafor-PET imaging are promising, and while compa-rable with standard [18F]FDG-PET with respect to imagingcontrast and sensitivity of tumor detection, [68Ga]pentixafor-PETprovides complementary biochemical information. Moreover, incontrast to [18F]FDG, which accumulates in proliferating tumortissue, [68Ga]pentixafor-PET provides information on the tumorniche, and therefore might offer novel opportunities for cancerprevention and detection of early metastasis, although confirma-tion of these preliminary observations is needed. A much largerseries of case studies will be required to confirm the ability todetect metastatic sites, and to determine whether PET-CT imaging

can in fact detect early cancers or even dysplastic lesions. Giventhat in the mouse model, CXCR4þ immune cells are recruitedduring the process of carcinogenesis, we can speculate that such acell typemayhelp define the pretumor and/or premetastatic nichethat precedes the local homing and proliferation of tumor cells.Given that Barrett's dysplasia likely involves an expansion ofgastric cardia progenitors into the esophagus, the CXCR4þ

immune cells may be crucial for the survival and expansion ofmigrating epithelial progenitors. In conclusion, CXCR4 (over)expression may characterize both tumor cells in esophagealtumors as well as cells in the local tumor microenvironment,making this receptor a promising and relatively specificmoleculartarget for the detection, staging, and monitoring of esophagealadenocarcinoma in all stages of the disease.

Disclosure of Potential Conflicts of InterestH. Wester is a consultant/advisory board member for and holds ownership

interest (including patents) in Scintomics GmbH. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: H.-Y. Fang, N.S. M€unch, T.C. Wang, M. QuanteDevelopment of methodology: H.-Y. Fang, N.S. M€unch, M. Schottelius,J. Ingermann, H. Liu, S. Stangl, G. Multhoff, A.R. Sepulveda, H.-J. WesterAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H.-Y. Fang, N.S. M€unch, M. Schottelius, J. Ingermann,H. Liu, M. Schauer, S. Stangl, G. Multhoff, K. Steiger, A.A. K€uhl, M. QuanteAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.-Y. Fang, N.S. M€unch, M. Schottelius, M. Schauer,S. Stangl, K. Steiger, C. Gerngroß, M. Jesinghaus, M. QuanteWriting, review, and/or revision of the manuscript: H.-Y. Fang, N.S. M€unch,M. Schottelius, S. Stangl, G. Multhoff, C. Gerngroß, W. Weichert, A.A. K€uhl,A.R. Sepulveda, H.-J. Wester, T.C. Wang, M. QuanteAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N.S. M€unch, K. Steiger, C. Gerngroß,H.-J. WesterStudy supervision: H.-Y. Fang, N.S. M€unch, H.-J. Wester, M. Quante

AcknowledgmentsThe research leading to these results has received funding from the

Deutsche Forschungsgemeinschaft (DFG) under grant agreement no. SFB824, has been funded by the Deutsche Krebshilfe with in the Max EderProgram (to M. Quante), and 2 U54 CA163004-06 (principal investigator;The role of the microenvironment in Barrett's esophagus, within the BETR-Net program).

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 June 24, 2017; revised October 17, 2017; accepted November 30,2017; published OnlineFirst December 5, 2017.

Table 1. Patient characteristics and [68Ga]pentixafor-PET/CT imaging results

Patient Sex Age Tracer ModalityInjected dose,

MBqWaiting time,

min SUVmax primarius SUVmean primarius SUVmax metastasisSUV

metastasis

1 M 88 FDG PET/CT 300 72 17.6 11.3 — —

CPCR4 PET/CT 244 62 4.4 2.7 — —

2 M 54 CPCR4 PET/CT 110 45 4.9 3.1 5 3.23 W 74 CPCR4 PET/CT 191 50 5.7 3.9 11.2 7.4

CPCR4 PET/CT 184 50 3.4 2.1 6.3 4.24 M 72 FDG PET/CT 422 64 7 4.5 — —

CPCR4 PET/CT 112 61 5.2 3.3 — —

5 M 74 FDG PET/CT 362 60 13.4 9.1 17.1 10.4CPCR4 PET/CT 139 67 6.2 3.7 6.5 3.8






References1. Scotton CJ, Wilson JL, Scott K, Stamp G, Wilbanks GD, Fricker S, et al.

Multiple actions of the chemokine CXCL12 on epithelial tumor cells inhuman ovarian cancer. Cancer Res 2002;62:5930–8.

2. Neve Polimeno M, Ierano C, D'Alterio C, Simona Losito N, NapolitanoM, Portella L, et al. CXCR4 expression affects overall survival of HCCpatients whereas CXCR7 expression does not. Cell Mol Immunol 2015;12:474–82.

3. Luker KE, Lewin SA,Mihalko LA, Schmidt BT,Winkler JS, Coggins NL, et al.Scavenging of CXCL12 by CXCR7 promotes tumor growth and metastasisof CXCR4-positive breast cancer cells. Oncogene 2012;31:4750–8.

4. Wang DF, Lou N, QiuMZ, Lin YB, Liang Y. Effects of CXCR4 gene silencingby lentivirus shRNA on proliferation of the EC9706 human esophagealcarcinoma cell line. Tumour Biol 2013;34:2951–9.

5. Lu CL, Ji Y, Ge D, Guo J, Ding JY. The expression of CXCR4 and itsrelationship with matrix metalloproteinase-9/vascular endothelial growthfactor in esophageal squamous cell cancer. Dis Esophagus 2011;24:283–90.

6. Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, NaeemR, et al. Stromal fibroblasts present in invasive human breast carcinomaspromote tumor growth and angiogenesis through elevated SDF-1/CXCL12secretion. Cell 2005;121:335–48.

7. Fang HY, Hughes R, Murdoch C, Coffelt SB, Biswas SK, Harris AL, et al.Hypoxia-inducible factors 1 and 2 are important transcriptional effectors inprimary macrophages experiencing hypoxia. Blood 2009;114:844–59.

8. Nieto JC, Canto E, Zamora C, Ortiz MA, Juarez C, Vidal S. Selective loss ofchemokine receptor expression on leukocytes after cell isolation. PLoSOne2012;7:e31297.

9. Hayakawa Y, AriyamaH, Stancikova J, Sakitani K, Asfaha S, Renz BW, et al.Mist1 expressing gastric stem cells maintain the normal and neoplasticgastric epithelium and are supported by a perivascular stem cell niche.Cancer Cell 2015;28:800–14.

10. Bendall LJ, Baraz R, Juarez J, ShenW, Bradstock KF. Defective p38mitogen-activated protein kinase signaling impairs chemotaxic but not proliferativeresponses to stromal-derived factor-1alpha in acute lymphoblastic leuke-mia. Cancer Res 2005;65:3290–8.

11. Shibata W, Ariyama H, Westphalen CB, Worthley DL, Muthupalani S,Asfaha S, et al. Stromal cell-derived factor-1 overexpression induces gastricdysplasia through expansion of stromal myofibroblasts and epithelialprogenitors. Gut 2013;62:192–200.

12. Everhart JE, RuhlCE. Burdenof digestive diseases in theUnited States part I:overall and upper gastrointestinal diseases. Gastroenterology 2009;136:376–86.

13. Kaifi JT, Yekebas EF, Schurr P, Obonyo D, Wachowiak R, Busch P, et al.Tumor-cell homing to lymph nodes and bone marrow and CXCR4expression in esophageal cancer. J Natl Cancer Inst 2005;97:1840–7.

14. Wu J, Wu X, Liang W, Chen C, Zheng L, An H. Clinicopathological andprognostic significance of chemokine receptor CXCR4 overexpression inpatients with esophageal cancer: a meta-analysis. Tumour Biol 2014;35:3709–15.

15. Lu CL, Guo J, Gu J, Ge D, Hou YY, Lin ZW, et al. CXCR4 heterogeneousexpression in esophageal squamous cell cancer and stronger metastaticpotential with CXCR4-positive cancer cells. Dis Esophagus 2014;27:294–302.

16. Drenckhan A, Kurschat N, Dohrmann T, Raabe N, Koenig AM, Reichelt U,et al. Effective inhibition of metastases and primary tumor growth withCTCE-9908 in esophageal cancer. J Surg Res 2013;182:250–6.

17. Gros SJ, Kurschat N, Drenckhan A, Dohrmann T, Forberich E, EffenbergerK, et al. Involvement of CXCR4 chemokine receptor in metastastic HER2-positive esophageal cancer. PLoS One 2012;7:e47287.

18. Falk GW. Barrett's esophagus. Gastroenterology 2002;122:1569–91.19. QuanteM, BhagatG, Abrams JA,Marache F,GoodP, LeeMD, et al. Bile acid

and inflammation activate gastric cardia stem cells in a mouse model ofBarrett-like metaplasia. Cancer Cell 2012;21:36–51.

20. Fitzgerald RC, Abdalla S,Onwuegbusi BA, Sirieix P, Saeed IT, BurnhamWR,et al. Inflammatory gradient in Barrett's oesophagus: implications fordisease complications. Gut 2002;51:316–22.

21. Stangl S, Varga J, Freysoldt B, Trajkovic-Arsic M, Siveke JT, Greten FR, et al.Selective in vivo imaging of syngeneic, spontaneous, and xenograft tumorsusing a novel tumor cell-specific hsp70 peptide-based probe. Cancer Res2014;74:6903–12.

22. Quante M, Tu SP, Tomita H, Gonda T, Wang SS, Takashi S, et al.Bone marrow-derived myofibroblasts contribute to the mesenchymalstem cell niche and promote tumor growth. Cancer Cell 2011;19:257–72.

23. Spechler SJ, Fitzgerald RC, Prasad GA, Wang KK. History, molecularmechanisms, and endoscopic treatment of Barrett's esophagus. Gastroen-terology 2010;138:854–69.

24. Paull A, Trier JS, Dalton MD, Camp RC, Loeb P, Goyal RK. The histologicspectrum of Barrett's esophagus. N Engl J Med 1976;295:476–80.

25. Gregorieff A, Pinto D, Begthel H, Destree O, Kielman M, Clevers H.Expression pattern of Wnt signaling components in the adult intestine.Gastroenterology 2005;129:626–38.

26. Katoh M. Molecular cloning and characterization of human WNT3. Int JOncol 2001;19:977–82.

27. Philipp-Abbrederis K, Herrmann K, Knop S, Schottelius M, Eiber M,L€uckerath K, et al. In vivo molecular imaging of chemokine receptorCXCR4 expression in patients with advanced multiple myeloma. EMBOMol Med 2015;7:477–87.

28. Gourni E,DemmerO, SchotteliusM,D'Alessandria C, Schulz S,Dijkgraaf I,et al. PET of CXCR4 expression by a (68)Ga-labeled highly specific targetedcontrast agent. J Nucl Med 2011;52:1803–10.

29. DemmerO,Gourni E, Schumacher U, Kessler H,WesterHJ. PET imaging ofCXCR4 receptors in cancer by a new optimized ligand. ChemMedChem2011;6:1789–91.

30. Wester HJ, Keller U, Schottelius M, Beer A, Philipp-Abbrederis K,Hoffmann F, et al. Disclosing the CXCR4 expression in lymphopro-liferative diseases by targeted molecular imaging. Theranostics 2015;5:618–30.

31. Herrmann K, Lapa C, Wester HJ, Schottelius M, Schiepers C, Eberlein U,et al. Biodistribution and radiation dosimetry for the chemokine recep-tor CXCR4-targeting probe 68Ga-pentixafor. J Nucl Med 2015;56:410–6.

32. Lapa C, L€uckerath K, Rudelius M, Schmid JS, Schoene A, Schirbel A, et al.[68Ga]Pentixafor-PET/CT for imaging of chemokine receptor 4 expres-sion in small cell lung cancer - initial experience. Oncotarget 2016;7:9288–95.

33. Lapa C, L€uckerath K, Kleinlein I, Monoranu CM, Linsenmann T, Kessler AF,et al. (68)Ga-Pentixafor-PET/CT for imaging of chemokine receptor 4expression in glioblastoma. Theranostics 2016;6:428–34.

34. Vag T, Gerngross C, Herhaus P, Eiber M, Philipp-Abbrederis K, Graner FP,et al. First experience with chemokine receptor CXCR4-targeted PET imag-ing of patients with solid cancers. J Nucl Med 2016;57:741–6.

35. Bluemel C, Hahner S, Heinze B, Fassnacht M, Kroiss M, Bley TA, et al.Investigating the chemokine receptor 4 as potential theranostic target inadrenocortical cancer patients. Clin Nucl Med 2017;42:e29–34.

36. Mentzel T, Brown LF, Dvorak HF, Kuhnen C, Stiller KJ, KatenkampD, et al.The association between tumour progression and vascularity in myxofi-brosarcoma and myxoid/round cell liposarcoma. Virchows Arch 2001;438:13–22.

37. Eck M, Schmausser B, Scheller K, Brandlein S, Muller-Hermelink HK.Pleiotropic effects of CXC chemokines in gastric carcinoma: differencesin CXCL8 and CXCL1 expression between diffuse and intestinal types ofgastric carcinoma. Clin Exp Immunol 2003;134:508–15.

38. Mhawech-Fauceglia P, Kaya G, Sauter G, McKee T, Donze O, Schwaller J,et al. The source of APRIL up-regulation in human solid tumor lesions.J Leukoc Biol 2006;80:697–704.

39. Coffelt SB, KerstenK,DoornebalCW,Weiden J, VrijlandK,HauCS, et al. IL-17-producing gammadelta T cells and neutrophils conspire to promotebreast cancer metastasis. Nature 2015;522:345–8.

40. Tazzyman S, Barry ST, Ashton S, Wood P, Blakey D, Lewis CE, et al.Inhibition of neutrophil infiltration into A549 lung tumors in vitro andin vivo using a CXCR2-specific antagonist is associatedwith reduced tumorgrowth. Int J Cancer 2011;129:847–58.

41. Noh H, Eomm M, Han A. Usefulness of pretreatment neutrophil tolymphocyte ratio in predicting disease-specific survival in breast cancerpatients. J Breast Cancer 2013;16:55–9.

42. Azab B, Bhatt VR, Phookan J, Murukutla S, Kohn N, Terjanian T, et al.Usefulness of the neutrophil-to-lymphocyte ratio in predicting short- andlong-term mortality in breast cancer patients. Ann Surg Oncol 2012;19:217–24.


Fang et al.




43. Jung J, Park SY, Park SJ, Park J. Prognostic value of the neutrophil-to-lymphocyte ratio for overall and disease-free survival in patients withsurgically treated esophageal squamous cell carcinoma. Tumour Biol2015;37:7149–54.

44. Grenader T, Waddell T, Peckitt C, Oates J, Starling N, CunninghamD, et al.Prognostic value of neutrophil to lymphocyte ratio in advanced oeso-phago-gastric cancer: exploratory analysis of the REAL-2 trial. Ann Oncol2016;27:687–92.

45. Cortez-Retamozo V, Etzrodt M, Newton A, Rauch PJ, Chudnovskiy A,Berger C, et al. Origins of tumor-associated macrophages and neutrophils.Proc Natl Acad Sci U S A 2012;109:2491–6.

46. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7.

47. Wang X, Ouyang H, Yamamoto Y, Kumar PA, Wei TS, Dagher R, et al.Residual embryonic cells as precursors of a Barrett's-like metaplasia. Cell2011;145:1023–35.

48. Hayakawa Y, Sethi N, Sepulveda AR, Bass AJ, Wang TC. Eosophagealadenocarcinoma and gastric cancer: should we mind the gap? Nat RevCancer 2016;16:305–18.

49. PastulaA,MiddelhoffM,Brandtner A, TobiaschM,H€ohl B,NuberAH, et al.Three-dimensional gastrointestinal organoid culture in combination

with nerves or fibroblasts: a method to characterize the gastrointestinalstem cell niche. Stem Cells Int 2016;2016:3710836.

50. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, et al.Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.Nature 2011;469:415–8.

51. Saha S, Aranda E, Hayakawa Y, Bhanja P, Atay S, Brodin NP, et al.Macrophage-derived extracellular vesicle-packagedWNTs rescue intestinalstem cells and enhance survival after radiation injury. Nat Commun2016;7:13093.

52. Logan CY, Nusse R. The Wnt signaling pathway in development anddisease. Annu Rev Cell Dev Biol 2004;20:781–810.

53. Topol L, JiangX,ChoiH,Garrett-Beal L,CarolanPJ, YangY.Wnt-5a inhibitsthe canonical Wnt pathway by promoting GSK-3-independent beta-cate-nin degradation. J Cell Biol 2003;162:899–908.

54. Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, BittnerM, et al.Wnt5a signaling directly affects cell motility and invasion of metastaticmelanoma. Cancer Cell 2002;1:279–88.

55. Lordick F,Ott K, Krause BJ,WeberWA, Becker K, SteinHJ, et al. PET to assessearly metabolic response and to guide treatment of adenocarcinoma of theoesophagogastric junction: the MUNICON phase II trial. Lancet Oncol2007;8:797–805.






2018;24:1048-1061. Published OnlineFirst December 5, 2017.Clin Cancer Res Hsin-Yu Fang, Natasha Stephens Münch, Margret Schottelius, et al. Barrett's Dysplasia and Esophageal Adenocarcinoma

Is a Potential Target for Diagnostic PET/CT Imaging inCXCR4

Updated version

10.1158/1078-0432.CCR-17-1756doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/12/05/1078-0432.CCR-17-1756.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/24/5/1048.full#ref-list-1

This article cites 55 articles, 12 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/24/5/1048.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/24/5/1048To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-1756

http://clincancerres.aacrjournals.org/content/suppl/2017/12/05/1078-0432.CCR-17-1756.DC1

http://clincancerres.aacrjournals.org/content/24/5/1048.full#ref-list-1

http://clincancerres.aacrjournals.org/content/24/5/1048.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/24/5/1048


cxcr4 isapotentialtargetfordiagnosticpet/ct …...ageal dysplasia and cancer highlight the potential...

Documents