Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis · Clever-1/Stabilin-1, also known as...

Cancer Therapy: Preclinical

Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis

Marika Karikoski1, Fumiko Marttila-Ichihara1, Kati Elima1,2, Pia Rantakari1,3, Maija Hollm�en1, Tiina Kelkka1,Heidi Gerke1, Ville Huovinen1, Heikki Irjala1,4, Rikard Holmdahl1, Marko Salmi1,5,6, and Sirpa Jalkanen1,5,6

AbstractPurpose: Immunosuppressive leukocytes and vasculature are important host cell components regulating

tumor progression. Clever-1/Stabilin-1, amultifunctional scavenger and adhesion receptor, is constitutively

present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is

unknown.

Experimental Design: Here, we generated full Clever-1 knockout mice and cell-specific ones lacking

Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16

melanoma and EL-4 lymphoma.

Results: Clever-1–deficient mice had smaller primary and metastatic tumors than wild-type (WT)

controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-

1 selectively inmacrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor

progression inWTmice. Both genetically and therapeutically induced absence of functional Clever-1 led to

diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated

binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1,

and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity.

Conclusion: This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new

immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating

lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The

ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeu-

tically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and

lymphatic spread in cancer. Clin Cancer Res; 20(24); 6452–64. �2014 AACR.

IntroductionThe ability to invade, induce angiogenesis, and avoid

immune destruction are important hallmarks of cancer cells(1). Tumor cells typically invade locally through the extra-cellular matrix. The cells, which can intravasate into thepreexisting or tumor-induced neoangiogenic blood or lym-phatic vessels, can formmetastases in draining lymphnodes(LN) and at distant sites. Because of differences in theendothelial and vessel wall structure, lymphatic vesselslikely offer pathways of lower resistance for migrating cells,

which is associated with the fact that approximately 80% ofmetastasizing tumors preferentially spread via the lymphat-ic system (2).

In addition to the intrinsic properties of tumor cells andthe vasculature, the progression of cancer is also dependenton thequality andquantity of antitumor immune responses(1). Lymphocytes continuously patrol through the blood,lymphoid tissues, and lymphatic vasculature during cancerimmune surveillance. Proinflammatory and cytotoxicimmune responses are useful in limiting tumor progression,whereas anti-inflammatory immune cell types often para-doxically promote tumor growth. For instance, regulatory Tcells (Treg) and type II macrophages can cause immuno-suppression, which is one of the main obstacles in success-ful cancer treatment (3–5). The same cell types also producemultiple proangiogenic molecules and thereby contributeto the angiogenic switch in cancer (6).

Clever-1/Stabilin-1, also known as Feel-1, is a multi-functional molecule conferring scavenging ability on asubset of type II macrophages (5, 7–10). In these cells, it isinvolved in receptor-mediated endocytosis and recycling,intracellular sorting, and transcytosis of altered andnormal self-components (11). Although other leukocytetypes (granulocytes and lymphocytes) are Clever-1–neg-ative, it is also constitutively expressed on afferent and

1MediCity Research Laboratory, Institute of Biomedicine, University ofTurku, Turku, Finland. 2Department of Medical Biochemistry and Genetics,Institute of Biomedicine, University of Turku, Turku, Finland. 3Departmentof Physiology and Turku Center for Disease Modeling, Institute of Biomed-icine, University of Turku, Turku, Finland. 4Department of Otorhinolaryn-gology, Turku University Hospital, Turku, Finland. 5National Institute forHealth and Welfare, Turku, Finland. 6Department of Medical Microbiologyand Immunology, University of Turku, Turku, Finland.

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

Corresponding Author: Sirpa Jalkanen, MediCity Research Laboratory,University of Turku, Tykist€okatu 6A, FIN-20520Turku, Finland. Phone: 358-2-333-7007; Fax: 358-2-333-7000; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1236

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(24) December 15, 20146452

on October 19, 2020. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 15, 2014; DOI: 10.1158/1078-0432.CCR-14-1236

http://clincancerres.aacrjournals.org/

efferent lymphatic endothelial cells, on sinusoidal endo-thelial cells in the liver and spleen, and on high endo-thelial venules (8, 12–14). Moreover, upon inflamma-tion, it can be induced on blood vessel endothelium,where it mediates the trafficking of lymphocytes, granu-locytes, and monocytes from the blood into the inflamedtissue (13, 15–17).Clever-1–positive lymphatics and macrophages are

found in human cancers and high number of Clever-1–positive macrophages is associated to shorter disease-specific survival in colorectal cancers of advanced stage(15, 18). This work was designed to elucidate the role ofClever-1 in tumor immunity and in tumor cell trafficking,and to test whether it can be used as a therapeutic targetin preclinical settings. We generated universal and cell-type selective Clever-1–deficient mice to analyze tumorgrowth, lymphatic spread, and antitumor immuneresponses. We also therapeutically targeted Clever-1 byantibodies during tumor growth in wild-type (WT) mice.The results showed that Clever-1 controls tumor progres-sion by mediating leukocyte-subtype selective entrance oftumor-infiltrating leukocytes (TIL) from the blood intothe tumor.

Materials and MethodsAnimalsSix- to 12-week-old C57BL/6 WT mice were used in

antibody experiments. Clever-1 full knockout (Clever-1�/�)mice,micelackingClever-1inmonocytes/macrophages(Clev-er-1fl/flLyz2Cre(Cre)/(Cre); called hereafter MACROclever�/�),and mice without Clever-1 in vascular endothelium (Clever-1fl/flTie2Cretm(Cre)/þ; called hereafter ENDOclever�/�) andtheir WT littermates as controls (as recommended to be arequirement in mouse studies; ref. 19) were used in C57BL/6N;129SvJ mixed background. In certain experiments,

NADPH oxidase–deficient mice (Ncfmut) and New Zealandwhite (NZW) rabbits were used.

The Clever-1/Stab-1 conditional targeted allele, withrecombination sites (loxP) before and after exon 1, wasconstructed by recombination technology. ConditionalClever-1fl/fl mice were generated by homologous recombi-nation inmice. Clever-1fl/flmicewere bred toCAG-Cre (20),Tie2-Cre (21), and Lyz2-Cre (22) mice, to delete exon 1 oftheClever-1/Stab-1 locus in all cells, vascular endothelial, ormyeloid compartments (neutrophils are inherently Clever-1–negative), respectively (Fig. 2A and SupplementaryMate-rials and Methods).

All animal studies were done in adherence with the rulesand regulations of The Finnish Act on Animal Experimen-tation (62/2006), performed in compliance with the 3Rsprinciple and accepted by the local Committee for AnimalExperimentation (Animal licence number 3791/04.10.03/2011).

Tumor cell linesKCA, a human lymphoblastoid cell line, was a kind gift

from E. Engleman (Stanford University, Stanford, CA) in1984 and its human origin was authenticated in 2011 byusing antibodies against human antigens. Mouse B16-F10-luc-G5 melanoma cell line containing a luciferase constructandmouse EL-4 T lymphomawere purchased fromXenogenin 2008 and their authentication is based on color (mela-noma is black) and their luciferase activity in each experi-ment. Tumor cells were cultured in RPMI-1640 (KCA andEL-4) and MEM/HBSS (B16 melanoma; HyClone) supple-mented with 10% FBS (Invitrogen, Gibco), nonessentialamino acids (Biologial Industries), 200mmol/L L-glutamine(B10 Whittaker), 1 mmol/L sodium pyruvate (Invitrogen,Gibco), and MEM vitamin solution (Invitrogen, Gibco).

Cancer modelsB16-F10-luc-G5 melanoma cells (4 � 105 cells) or EL-4

lymphoma cells (5 � 106 cells) in 30 mL of RPMI (GIBCO)were injected subcutaneously into the left ear or in certainexperiments (1 � 106 cells in 20 mL) into the footpads ofmice. Ear tumors metastasized to the cervical LNs, whereasthe footpad ones spread to popliteal LNs. Tumor growthwas assessed by luciferase bioluminescence measurementstwice a week, using a previously described methodology(23) and also by an electronic caliper (Mitutoyo). Thevolume of the tumor was calculated according to the for-mula V ¼ p/6 � (shortest diameter)2 � (longest diameter)as described previously (24).

The popliteal LN metastases were visually scored at theend of the experiment as follows: 0, no visible metastasis; 1,metastases (black spots) encompassing less than half of theLN surface; 2, metastases covering more than 50% but lessthan100%of LN; 3,whole LNblack, but still of normal size;and 4, enlarged, fully black LN.

The antibody treatments in melanoma-bearing WT micewere done with anti-Clever-1 (originally a kind gift fromS. Goerdt [Department of Dermatology, Venereology andAllergology, University Medical Center andMedical Faculty

Translational RelevanceHuman cancers with high number of Clever-1–posi-

tive macrophages are associated with poor prognosis inadvanced cancers. However, its mode of action in con-trolling cancer behavior and potential to use it as atherapeutic target has remained unknown. In this work,we have for the first time demonstrated the immuno-suppressive role of Clever-1 in cancer behavior usinggene-targeted mice. Most importantly, we have shownthat cancer growth and metastases can be efficientlyprohibited with an antibody therapy against Clever-1without any obvious side effects. The inhibitory effects ofanti-Clever-1 antibody therapy on progression of pri-mary andmetastatic tumors suggest that this molecule isa new immunomodulatory target for cancer immuno-therapy. Because it has a unique mode of action, it mayhave potential benefits in comparison with currentlyavailable immunomodulating drugs.

Clever-1 Regulates Cancer Progression

www.aacrjournals.org Clin Cancer Res; 20(24) December 15, 2014 6453




Mannheim, Heidelberg University, Mannheim, Germany]and then purchased from InVivo Biotech; ref. 25) or NS-1control antibody (produced by InVivo Biotech) using pro-phylactic and therapeutic protocols. In the prophylacticsettings, the mice were treated with by subcutaneous injec-tions of the mAbs (50 mg) into the ear 1 day before tumorinjection followed by intraperitoneal dosing (100 mg/injec-tion) starting 1 day after tumor injection and then repeatedevery third day. In the therapeutic experiments, no localantibodies were given and the intraperitoneal antibodytherapy was first started 3 days after B16 melanoma cellinjections (1 � 106 cells in 20 mL).

Ex vivo adhesion assaysTILs were isolated frommelanomas grown in nontreated

WT mice using previously described methodology (26).Briefly, the isolated tumors were minced to small piecesand digested with collagenase D (1 mg/mL, þ37�C, 40minutes; in the presence of DNase I). The released cellswere purified using anti-CD45-PE antibody (BD Pharmin-gen) and magnetic cell sorting (Mouse PE selection kit,EasySep; STEMCELL Technologies). CD4þ cells were sepa-rated from the blood of WT mice using the EasySep mouseCD4þ T-cell preenrichment kit according to the instructionsof themanufacturer.Whole blood after erythrocyte lysiswasused as a source of monocytes.

Melanomaswere collected from anti-Clever-1 and controlantibody-treated mice on day 14, snap-frozen, and sec-tioned. The ex vivo adhesion assays were performed aspreviously described (27). Briefly, isolated TIL and CD4þ

cells from blood were allowed to bind to vessels in themelanoma sections for 30 minutes at þ7�C under rotatoryconditions. In the second set of assays, blood leukocytesfrom tumor-bearingmice were preincubated with anti-Clev-er-1 or control antibody for 30 minutes and washed twicebefore application onto the tissue sections. The nonboundcellswere then gently decantedoff from the sections, and theadherent cells were fixed in 1% glutaraldehyde. The numberof leukocytes bound to tumor vessels was counted underdark-field microscopy. These conditions allowed us to dis-criminate the TIL (small and phase-bright), tumor-infiltrat-ingmyeloid cells (mainlymacrophages, which are large andhave a ruffledappearance), andbloodmonocytes (largewitha ruffled appearance; ref. 28). At least 100 vessels from threeindependent tumors in both treatment groupswere countedand the average number of leukocytes bound per vessel incontrol treated mice was used to define 100% adherence.

ImmunohistochemistryAcetone-fixed frozen sections of the primary tumors and

metastases of themice inoculatedwithmelanoma cellswerestained with rat mAbs against macrophage mannose recep-tor (MRC), MR5D3, a marker for type II macrophages (,akind gift from L. Martinez-Pomares, University of Notting-ham, Nottingham, United Kingdom; ref. 29), PV-1 antigen(blood vessel antigen, Meca-32, a kind gift from E. Butcher,Stanford University), CD31 (a marker of both blood andlymphatic vessels; BD Pharmingen), CD3 (BD Pharmin-

gen), CD8 (Caltag), SPARC (R&D Systems), and rabbitpolyclonal antibody against LYVE-1 (a lymphatic endothe-lium-specific marker; Reliatech), or with a negative controlmAb (Hermes-1 against human CD44) or normal rabbitserum. FITC-conjugated anti-rat Ig (Sigma) or FITC-conju-gated anti-rabbit Ig (Sigma) diluted in PBS containing 5%normalmouse serumwas used as the second-stage antibody.Tumor tissues, skin, metastases, and LN sections were alsostained using biotinylated anti-Clever-1 (1.26) followed byStreptavidin–Alexa Fluor 546. Alternatively, 9–11 (rat anti-human Clever-1, which cross-reacts with the mouse homo-log; ref. 30) or 3–372 (mouse anti-human Clever-1, whichcross-reacts with the rabbit homolog) followed by FITC-conjugated anti-rat Ig or mouse Ig was used (13). Fordouble stainings, anti-LYVE-1 or anti-PV-1 followed byAlexa Fluor 546–conjugated anti-rabbit IgG (Invitrogen)or Alexa Fluor 546–conjugated anti-rat IgG (Invitrogen),respectively, was used together with Alexa Fluor 488–conjugated anti-Clever-1 (9–11). Anti-FoxP3 (a surrogatemarker for Tregs from eBioscience) stainings were donefrom frozen sections fixed with 2% paraformaldehyde,using peroxidase-conjugated rabbit anti-rat Ig (Dako) and3,30-diaminobenzidine hydrochloride for visualization.

TACS 2TdT-Blue Label In Situ Apoptosis Detection Kit(Trevigen) was used according to the manufacturer’sinstructions to detect apoptosis on tumor sections. Thenumbers of apoptotic cells/HPF (the whole section, 9–24fields/slide depending on the size of the tumor) werecounted.

The stainings were analyzed using Olympus BX60micro-scope and cell

^D version 2.6 software (Soft Imaging Solu-

tions GmbH). Intensity of SPARC staining was analyzedusing ImageJ software.

Coculture experimentsBlood monocytes and peritoneal macrophages from

WT mice were added to the upper compartments ofTranswell (transparent polyester membranes, pore size0.4 mm; Corning) in medium containing 20 mg/mL anti-Clever-1 (1.26) antibody or negative control antibody,and B16 melanoma (50,000 cells) were simultaneouslyplated to the lower compartments. After 3 and 7 days ofcoculturing, the methanol-fixed membranes with adher-ent monocytes/macrophages were stained with 5D3against MR, 9–11 against Clever-1, and negative controlantibody followed by FITC-anti-rat Ig second stage. Per-centages of positive cells were counted under a fluores-cence microscope (Olympus BX60).

Immune array qPCRIsolated CD45þ TIL and nonhematopoetic CD45� cells

from B16 melanomas from anti-Clever-1 or control anti-body treated mice (n ¼ 6) were pooled in groups of two.Total RNA was isolated [Nucleo-Spin RNAII Total RNAIsolation Kit (Macherey-Nagel)] and reverse-transcribedusing iScript cDNA Synthesis Kit (BioRad). Equalamounts of samples were loaded into TaqMan MouseImmune Array Microfluidic Cards (Applied Biosystems)

Karikoski et al.

Clin Cancer Res; 20(24) December 15, 2014 Clinical Cancer Research6454




and run using a 7900HT Fast Real-Time PCR System(Applied Biosystems) in the Finnish Microarray andSequencing Center, Center for Biotechnology (Turku,Finland). The results were normalized using 18S RNA asan endogenous control. The results were analyzed withSDS 2.3 and DataAssist v3.0 software using relative quan-tification (RQ).

Tumor cell migration via lymphaticsRabbits were treated with 2 mg/kg of 3–372 or control

antibody intravenously ondays�1 and0.CFSE-labeledKCAlymphoma cells (40 � 106 cells labeled with 0.5 mmol/LCSFE for 20 minutes at 37�C followed by three washings)together with additional 0.5-mg dose of antibodies wereinjected subcutaneously into the footpads at day 0. After24 hours, the popliteal LNs were collected and cell suspen-sions were analyzed by flow cytometry (FACSCalibur; BDBiosciences) to quantify the numbers of immigrated CFSE-positive cells.

Statistical analysesThe two-tailed Student test and Mann–Whitney U test

were used. P values < 0.05 were considered significant.

ResultsInduction of Clever-1 in tumor vasculature andmacrophages

In normal WT mice, Clever-1 is expressed in lymphaticsand faintly in high endothelial venules but is practicallyabsent on flat-walled vessels and macrophages inLNs (Fig. 1A). The expression pattern of Clever-1 wasaltered in B16 melanoma-bearing mice. The majorityof the intratumoral vessels in primary tumors and LNmetastases were enlarged with a widely open lumen and,unlike the normal flat-walled vessels, they expressedClever-1 (Fig. 1B). Moreover, a subset of Clever-1–posi-tive macrophages not seen in normal LNs was detectedin the primary tumors and LN metastases (Fig. 1C).All Clever-1–positive macrophages were MRC–positive

Figure 1. Clever-1 is induced ontumor vasculature and a subset oftumor macrophages. A, two-colorimmunofluorescence staining ofClever-1 (green) on LNs with anti-LYVE-1 (red) or anti-PV-1 (red) ofWT mice. Thick arrows point tolymphatic vessels and a thin arrowpoints to a high endothelial venuleand an arrowhead to a PV-1–positive flat-walled venule. B,two-color staining of Clever-1 withbiotinylated 1.26 antibody (red) andPV-1 with Meca-32 antibody(green) identifies the tumor vessels.Vascular positivity was confirmedwith another monoclonal antibody(9–11) against the N-terminal 3 Kbfragment of Clever-1. The vesselsare indicated by thin arrows andsome macrophages (positive forClever-1) by open arrows. C,immunofluorescence staining ofmelanoma metastases. Doublestaining with anti-MRC (green) andanti-Clever-1 (red). Bars, 100 mm in(A and B) and 20 mm in (C).






(a prototype marker for type II macrophages), but onlyabout 50% of MRC-positive macrophages coexpressedClever-1. Tumor cells induced Clever-1 expression inmacrophages, because Clever-1–positive macrophageswere not found in enlarged draining LNs after a 17-daysubcutaneous OVA immunization (Supplementary Fig.S1). In kinetic analyses of tumors on days 3, 6, and 10, thefirst MRC-positive macrophages were seen in day 6 sam-ples, whereas Clever-1 was first detected on macrophagesin day 10 samples. Clever-1–positive monocytes were alsopresent in the blood of tumor-bearing mice. These resultsindicate that at least two different subpopulationsof MRC-positive macrophages (MRCþ/Clever-1þ andMRCþ/Clever-1�) exist in cancer but the former is notinduced during non-cancer-related immune response.

To study, whether the lack of Clever-1 has an impacton tumor development and metastasis, we generatedClever-1 full knockout mice (Clever-1�/�), mice lackingClever-1 in macrophages (MACROclever�/� mice), andmice lacking this molecule in blood vessel endothelium(ENDOclever�/� mice; Figs. 2A and SupplementaryFig. S2). All cell types in tumors of Clever-1�/� micecompletely lacked Clever-1 expression, as expected (Fig.2B). In tumors of MACROclever�/� mice, Clever-1expression in the lymphatics and blood vessels wascomparable with tumor-bearing WT mice, whereasalmost all Clever-1–positive macrophages had disap-peared (Fig. 2C). Tumors of ENDOclever-1�/� mice, onthe other hand, did not have visible Clever-1 expressionon PV-1–positive blood vessels but macrophages and the

Figure 2. Generation andendothelial phenotype of differentClever-1 knockout mice. A,schematic presentation ofthe generation of differentClever-1–deficient mice. Forverification of Clever-1/Stab-1targeting (50-end screening), DNAwas digested with BamHI andhybridizedwith probeA (solid blackline). Southern blot analysis ofgenomic DNA isolated fromWT (Clever-1þ/þ) and fromheterozygous (Clever-1þ/�

NEOþ/�) embryonic stem cellclones. B, immunofluorescencestainings of tumors of Clever�/�

mice showing the lack of Clever-1on PV-1–positive (red) vessels. C,immunofluorescence staining oftumors of MACROclever-1�/�

showing Clever-1 (green) positivityon PV-1–positive (red) bloodvessels (thin arrows). D,immunofluorescence stainingof ENDOclever�/� mice showingClever-1 (green) positivity only onmacrophages (open arrow) andLYVE-1 (red)–positive lymphatics(arrow) in tumors. Staining with anegative control antibody is shownin the insets. Bars, 100 mm.

Karikoski et al.





majority of lymphatic vessels brightly expressed Clever-1(Fig. 2D).

Retarded tumor growth and metastatic disseminationin absence of Clever-1The primary B16 tumors remained significantly smaller

in Clever-1�/� mice than in WT mice and the same trendwas seen in MACROclever�/� mice when compared withWTmice on day 14 (P¼ 0.057; Fig. 3A). However, the sizesbetween MACROclever�/� tumors were significantly smal-ler on day 9/10 than theWT tumors (P¼ 0.009). In the nextexperiment, primary tumors of also ENDOclever�/� micetended to remain smaller than those of WT mice (Fig. 3B)and were comparable with the size of tumors in Clever�/�

mice (see Fig. 3A for comparison). If the size of tumors inENDOclever�/� mice was compared with size of all WTtumors in this set of experiments, the difference was statis-tically significant (P ¼ 0.046) at the end of the experiments(day 14). In contrast, metastases remained small only in thefull Clever�/� mice while the size of the metastases in thecell type–specific knockouts did not differ from those ofthe WT mice (Fig. 3C). Thus, Clever-1 is needed fornormal progression of primary melanoma tumors in vivoboth on macrophages and vascular endothelium.Moreover, because lymphatics in ENDOclever�/� andMACROclever�/� mice are Clever-1–positive and full Clev-er-1�/� lacking Clever-1 also on lymphatics have only smallmetastases, Clever-1 in lymphatics seems to be important inmediating the spread of tumor cells into the draining LNs.As the future aim is to test the potential of Clever-1 as a

target in human malignancies, we used another cancermodel to test whether anti-Clever-1 mAb interferes withcancer cell migration via the lymphatics. For these experi-ments, we selected rabbit as a model, as we then could useanti-human Clever-1 mAb 372, which cross-reacts withrabbit Clever-1 (Supplementary Fig. S3A), and Clever-1–negative human lymphoma cells (Supplementary Fig. S3B).The blockade of lymphatic Clever-1 with this cross-reactingmAb in this model efficiently blocked the trafficking oflymphoma cells from the footpad to the draining poplitealLNs (Fig. 3D). This supports the notion that lymphaticClever-1 may promote metastasis formation.

Reduced tumor growth during antibody therapyTo test whether Clever-1 can be used as a therapeutic target

in cancer, we next used function-blocking antibody againstmouse Clever-1. We injected B16 melanoma and EL-4 lym-phoma cells, both of which are Clever-1–negative (Supple-mentaryFig. S4AandS4B), subcutaneously intopinnaof earsto allow in vivo imaging of metastasis in the neck. In the B16model, both the primary tumors and the metastases wereabout 70% smaller in mice treated with anti-Clever-1 anti-body in comparison with the control-treated animals, whenthe treatment was started before the tumor cell injections(Fig. 4A and B and Supplementary Fig. S4C).In the EL-4 lymphoma model, tumor growth was

observed in 9/11 control-treated and 9/10 anti-Clever-1–treated mice. Among the mice with detectable tumor

growth, the primary tumors remained significantly smallerin the anti-Clever-1–treated group (Fig. 4C). The EL-4metastases in draining LNs were on average 49% smalleron day 11 (P < 0.01) and 26% smaller on day 14 in the anti-Clever-1–treated group than in the control antibody-treatedgroup, but this difference did not reach statistical signifi-cance (Fig. 4C).

To evaluate the efficacyofClever-1blockade in a clinicallyrelevant therapeutic setting, we then let the B16 tumorsgrow for 3 days before starting the antibody therapy. In thismodel, the anti-Clever-1 antibody treatment also led to astatistically significant reduction in primary tumors andmetastases on day 20 (Fig. 4D). Thus, prophylactic andtherapeutic neutralization of Clever-1 by mAbs attenuatestumor growth in vivo.

Clever-1 is needed for intratumoral accumulation oftype II macrophages and FoxP3-positive T cells but notfor neoangiogenesis

Next, we analyzed whether decreased tumor growth inthe absence of Clever-1 would be associated with alteredtumor neo(lymph)angiogenesis or leukocyte infiltration.In the tumors of Clever-1�/� mice, the density of F4/80and MRC-positive macrophages and FoxP3-positive Tcells was diminished and no difference was found in thenumbers of intratumoral CD3þ and CD8þ T cells or inCD31þ vessels (Fig. 5A). Number of MRC-positive macro-phages and FoxP3-positive cells were also reduced both inthe primary tumors and metastases of anti-Clever-1 mAb-treated WT mice in comparison with control mAb-treatedmice (Fig. 5B). Moreover, by comparing tumors of over-lapping sizes in anti-Clever-1 and control-treated groups,we found that the diminished numbers of intratumoraltype II macrophages subsequent to the antibody therapywas not dependent on the size of the tumors (Supple-mentary Fig. S5A). The reduction of MRC- and FoxP3-positive cells was selective because the numbers of intra-tumoral CD3þ and CD8þ cells were comparable in bothtreatment groups (Fig. 5B).

Although Clever-1 has been reported to play a role inangiogenesis in vitro (7) the numbers of intratumoral bloodvessels (CD31þ and PV-1–positive) and lymphatics(CD31þ and PV-1–negative) were comparable after anti-Clever-1- and control antibody treatments inWTmice (Fig.5B). Together, these data thus show that the number ofregulatory immune cell types is diminished subsequent totargeting Clever-1, but both the blood and lymphatic vas-culature remain unchanged (Fig. 5).

Anti-Clever-1 mAb treatment does not alter scavengingof SPARC or macrophage polarization

As Clever-1 on macrophages mediates the uptake ofSPARC (9), an extracellular matrix molecule regulatingtumor growth, the amount of SPARC could contribute tothe reduced tumor growth in anti-Clever-1–treated mice.However, the expression of SPARC was found to be similarin tumors of anti-Clever-1- and control antibody-treatedmice (Supplementary Table S1).






Because anti-Clever-1 treatment significantly decreasedthe number of MRC-positive macrophages (Fig. 5B) theantibody might lead to the depletion of MRCþClever-1þ

macrophages by antibody-dependent cell-mediated cyto-toxicity or complement activation. However, this was notthe case because after anti-Clever-1 and control mAbadministration, similar proportion of intratumoral type IImacrophages in primary tumors coexpressed Clever-1(51.7% � 5.9%, n ¼ 5 and 52.2% � 6.4%, n ¼ 4 of type

II MRC-positive macrophages were Clever-1–positive inanti-Clever-1 mAb and in control mAb-treated mice,respectively).

To analyze the potential effect of anti-Clever-1 mAb onmacrophage polarization, we used in vitro polarization ofblood monocytes and peritoneal macrophages from WTmice. Presence of B16melanoma cells polarized the mono-cytes/macrophages toward type II cells significantly betterthan the culture medium alone (Supplementary Fig. S5B

0

0.2

0.4

0.6

0.8

1

1.2

1.4 P = 0.03

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

WT Clever-1–/–

MACROclever–/– ENDOclever–/–

0

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

14,000,000

16,000,000

2 7 10 14

Time (days)

Pho

tons

/s

A B

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

14,000,000

16,000,000

Pho

tons

/s

2 7 10 14

Time (days)

C P = 0.03

P = 0.015

0

0.5

1

1.5

2

2.5

3

Met

asta

sis

scor

e

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

Day 14Day 14

Day 14

D

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control α-Clever

P < 0.005

Rel

ativ

em

igra

tion

Figure 3. Clever-1 deficiency leads to retarded tumor growth. A, combined results of three independent experiments demonstrating the size of primary tumorsas relative luciferase counts�SEMofB16melanoma inWT (n¼18), full Clever-1�/� (n¼11), andMACROclever�/� (n¼21)mice at the end of the experiments(day 14). Inset, the raw values of one experiment are shown as an example. B, relative size of B16 melanoma in WT (n ¼ 6) and ENDOclever�/� (n ¼ 9) miceat the end of the experiment (day 14). Kinetics of the tumor development is shown in the inset in raw values. C, combined results of all experimentsdemonstrating the size ofmetastases of B16melanomas in different genotypes. Themetastases have been scored at the end of the experiments as explainedin the Materials and Methods. D, lymphoma cell migration in the rabbit. Fluorescently labeled KCA lymphoma cells were subcutaneously injected into thefootpads of rabbits treated with either anti-Clever-1 (n ¼ 8) or control antibody (n ¼ 9), and their appearance in the draining LNs was detected by flowcytometry. The results are presented as relative migration (mean � SEM). Migration after control antibody treatment is 1.0 by definition.

Karikoski et al.





Rel

ativ

elu

cife

rase

biol

umin

esce

nce

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

A

C

B

0

0.5

1

Day 140

0.5

1

Day 10

Primary tumor metastasis

Day 14

Control -Cleverα

P = 0.01 P < 0.01 P < 0.01

0

0.2

0.4

0.6

0.8

1

Day 7 Day 11 Day 14

Rel

ativ

evo

lum

eof

the

tum

or

P < 0.01 P < 0.011.2

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

0

0.2

0.4

0.6

0.8

1

1.2

Day 14

Primary tumor

Day 20

D

Day 14

Metastasis

Day 200

0.2

0.4

0.6

0.8

1

1.2

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

P < 0.0001P < 0.0001

α-CleverControl

02468

10

Day 0 Day 1 Day 4 Day 7 Day 10 Day 14

01,0002,0003,0004,0005,0006,000

Day 4 Day 7 Day 10 Day 14

mm

3

x10

phot

ons/

s5

0

0.2

0.4

0.6

0.8

1

1.2

Metastasis

Day 7 Day 11 Day 14

Rel

ativ

elu

cife

rase

biol

umin

esce

nce

P < 0.01P < 0.05

Primary tumor

Figure 4. Anti-Clever-1 antibody treatment diminishes primary tumor growth and metastases. A, B16-luc melanoma cells were injected subcutaneously intothe ear. Growth of the primary tumor and the development of metastases were assessed by bioluminescence. Relative size (mean � SEM) of the primarytumor after 10 and 14 days in the two treatment groups (n ¼ 12 in both groups, combined results of two experiments) and relative size (mean � SEM) ofmetastases at the end of the experiment (day 14). The size (assessed by luciferase bioluminescence counts) of the primary tumor and metastasesin the control-treated group is 1.0 by definition. Results of one experiment with raw values are shown in the inset. B, examples of tumors in animals treatedwithanti-Clever-1 or control antibody.White arrowspoint to the injection site (primary tumor) andyellowarrows to theneckmetastases.Note that oneanti-Clever-1antibody–treated mouse does not have a detectable tumor at the site of injection and the other does not have neck metastases. C, kinetics of EL-4T-cell lymphoma growth in anti-Clever-1- and control antibody-treated animals. Only animals with tumor growth were included (9/11 in control antibody-treated and 9/10 in anti-Clever-1–treated groups). The combined data of two experiments are presented as the relative volume of the tumor and relativebioluminescence counts of the metastases (mean � SEM). The control-treated group is 1.0 by definition. Results of one experiment with raw valuesare shown in the inset. D, development of primary tumor andmetastases when the antibody treatment was started 3 days after the injection of B16melanomacells (day 14, n ¼ 12 in both groups, and day 20, n ¼ 6 in both groups). The data are presented as in A.






Karikoski et al.





and S5C). Notably, the presence of saturating anti-Clever-1mAb concentrations had no effect on this process. Together,these data strongly suggest that the antibody treatment doesnot deplete macrophages and has no direct effect on theirM1/M2 polarization.

Clever-1 mediates binding of TIL to tumor vasculatureBecause Clever-1 is involved in the extravasation of

leukocytes to lymphoid organs and into sites of inflam-mation (17), we tested whether entrance of TIL (or that oftheir precursors) was inhibited during the antibody ther-apy. We collected tumors from both anti-Clever-1- andcontrol antibody-treated animals and tested the bindingof TIL and peripheral blood CD4þ T cells isolated fromnontreated WT mice to vessels in those tumors ex vivo.Both tumor-infiltrating myeloid cells and lymphocytesbound less efficiently to tumor vessels of anti-Clever-1–treated animals. Also, the adhesion of CD4þ blood lym-phocytes to the tumor vasculature of Clever-1–treatedmice was reduced in comparison with those of controlantibody-treated mice (Fig. 6A). Moreover, to test wheth-er monocyte Clever-1 contributes to adherence to theendothelium, we treated blood monocytes and lympho-cytes (lymphocytes served as controls as they are Clever-1–negative) collected from tumor-bearing mice withanti-Clever-1 mAb in vitro before the adhesion assay.Anti-Clever-1 mAb inhibited monocyte binding to tumorvessels by 70%, whereas no inhibition was seen in lym-phocyte binding (Fig. 6B). Thus, anti-Clever-1 therapymay prevent leukocyte entrance into the tumors by tar-geting Clever-1 both on the vascular endothelium andmonocytes and is well in line with the results obtainedwith MACROclever�/� and ENDOclever�/� mice.

Anti-Clever-1 treatment is responsible for increasedimmune activation and apoptosis in tumorsTo analyze whether decreased numbers of immunosup-

pressing leukocyte types in tumors after anti-Clever-1 treat-ment associates with increased antitumor immuneresponse, we performed qPCR immune arrays. Within theTIL (CD45þ) population (Fig. 6C), anti-Clever-1 treatmentincreased the expression of many activation markers (e.g.,proinflammatory CCL3, IL1 and IL6). In the non-hemato-poetic cell population of the tumors (CD45� cells, mainlycontaining tumor cells together with some non-hemato-poietic normal cells such as endothelial cells), inflamma-tion-induced endothelial markers such as E- and P-selectinwere decreased (Fig. 6D). In the CD45� population, the

expression of proapoptotic Smad7 was increased and theexpression of the antiapoptotic marker Bcl2-like protein 1(Bcl2l1) was decreased. The enhanced apoptosis of thetumors subsequent to anti-Clever-1 therapy was also con-firmed by in situ apoptosis detection. The mean number ofapoptotic cells in tumors of anti-Clever-1 antibody–treatedmice (n ¼ 4) was 46.4 � 4.8/high-power field (HPF) and15.7� 5.7/HPF in tumors of control antibody-treated mice(n ¼ 5; P ¼ 0.005).

Anti-Clever-1 therapy does not aggravate autoimmuneinflammations

As anti-Clever-1 treatment leads to immune activation incancer, it might aggravate inflammation in other settings.However, in neutrophil- and lymphocyte-dominated mod-els of arthritis, induced by anti-collagen II antibodies(CAIA) and collagen (CIA), respectively, the anti-Clever-1mAb therapy did not modify the disease course (severity orincidence). Similar results were also obtained in arthritisexperiments performed with Ncf1-mutated mice, whichnormally display a severe form of CIA (Supplementary Fig.S6). Thus, anti-Clever-1 antibody treatment appears to havedifferent immunomodulatory effects in inflammation relat-ed to cancer and autoimmune diseases.

DiscussionIn thiswork,we generated conditional Clever-1–deficient

mice to study the role of this multifunctional molecule intumor progression in vivo. We found that the progression ofmelanoma tumors was attenuated in the absence of Clever-1 from all cells, from macrophages, or from vascular endo-thelial cells. Prophylactic, and most importantly, therapeu-tic treatment ofWTmice with anti-Clever-1mAbs inhibitedtumor progression. Clever-1 was induced in tumor vascu-lature and in monocytes of tumor-bearing mice. Duringtumorigenesis, Clever-1 likely contributes to the extravasa-tion of TILs, because blocking of endothelial andmonocyteClever-1 impaired adhesion of blood-borne leukocytes tovascular endothelium.

Tumors in Clever-1–deficient mice and in WT mice trea-ted with anti-Clever-1 mAbs had lower numbers of MRC-positive macrophages than tumor-bearing control mice.MRC is regarded as one of the best phenotypic markers oftype II macrophages, which are known to play profoundimmunosuppressive functions in tumors (31). These cellsmost likely enter the tumors as monocytes from blood, andthen polarize to type II macrophages under the local influ-ence of tumor microenvironment, although differentiation

Figure 5. Clever-1 deficiency and antibody therapy causes reduction in type II macrophages and FoxP3-positive lymphocytes in B16 melanoma. The resultsare presented as themean number of positive cells�SEM/HPF. A, number of different cell populations in primary tumors andmetastases ofWTandClever�/�

(KO) mice. Numbers of F4/80-positive macrophages, MRC-positive macrophages, Foxp3-positive lymphocytes, CD3þ T cells, CD8þ T cells and CD31þ

vessels are shown as indicated. B, number of different cell populations in primary tumors and metastases in anti-Clever-1–treated and control antibody-treated mice. Numbers of FoxP3-positive lymphocytes, MRC-positive macrophages, CD3þ T cells, CD8þ T cells, CD31þ vessels with examples ofimmunofluorescence staining of primary tumors and metastases using anti-CD31 antibody in anti-Clever-1–treated and control antibody-treated mice andnumber of PV-1–positive vesselswith examples of immunofluorescence staining of primary tumors andmetastases using anti-Meca-32 antibody (against PV-1) in anti-Clever-1–treated and control antibody-treatedmice are shown as indicated (3–5 tumors/genotype/treatment analyzed; 5 fields/tumor counted for allothers except >30 fields/tumor for FoxP3-positive cells). Bar, 100 mm.






of local tissue-resident macrophages may also contribute(32, 33). The key role of tumor-dependent differentiation isalso supported by our findings showing thatmonocytes andmacrophages cocultured with B16melanoma cells polarizetoward type II macrophages in vitro. Importantly, anti-Clever-1 antibody did not interfere with the polarizationprocess. Also, the kinetics of appearance of MRC- andClever-1–positivemacrophages inmelanoma is compatiblewith the idea of differentiation-induced induction of Clev-er-1 on type II macrophages. Our in vivo results clearlyshowed that the intratumoral monocytes/macrophagesneed more than 3 days to differentiate to MRC-positivetype II macrophages, differentiation to Clever-1–positiveones takes even longer (more than 6 days), and the anti-Clever-1 treatment does not interfere with these kinetics(data not shown). The relatively late induction of Clever-1in tumor macrophages has been found in other models aswell (33). The anti-Clever-1 mAb did not deplete targetantigen-positive macrophages in vitro. This is consistentwith our earlier experiments demonstrating that in vivoantibody treatment with anti-Clever-1 does not cause vas-

cular damage or affect leukocyte counts (17). This is ofcourse notwithstanding with the possibility that Clever-1ligation might alter the functional responsiveness (e.g.,immunosuppressive functions) of targeted macrophages.

Type II macrophages also induce Tregs both via directcontacts (34) and via soluble mediators (32). For example,typical high IL10productionof type IImacrophages leads tothe expansion of Tregs, which, in turn, produce additionalsuppressive factors (4) and dampen the immune attackagainst the tumors, including B16 melanomas (35). Thus,anti-Clever-1 mAb therapymay initially lead to diminishednumbers of intratumoral type II macrophages. They, inturn, deliver suboptimal signals for maintaining normalnumbers of Tregs, which was seen as reduced numbers ofFoxP3-positive cells. Slower tumor growth and increasedapoptosis after Clever-1 mAb treatment was also supportedby qPCR analyses showing an increase in expression ofSmad7 and a decrease of BcL2l1 in the tumor cell–contain-ing population.

We postulate that the primary effect of the anti-Clever-1treatment is on blocking of Clever-1 function, that is,

A B

0

1

2

3

4

5

6

RQ

CD45+ cells

Ccl

3

Ccr

7

CD

3eN

fkb1

lkbk

b

Nfk

b2

Sta

t3C

D38

Veg

faS

ocs1

Hm

ox1

Csf

1 Il6Il1b

Il1a

CD

68C

D80

Tgfb

1

Tnf

Sel

e

C

α-Clever in vivoControl mAb in vivo

D

0

0.5

1S

mad

7S

kiE

dn1

Bcl

2l1

Ccl

2N

fkb2

CD

38P

tprc Il6

Tgfb

1Tn

fC

sf1

Il1b

Ccl

5C

D86 Fn1

Sel

eC

D68

Ccr

2E

ce1

H2-

Eb1

Soc

s1C

D34

Ptg

s2N

os2

Sel

pC

cr7

Il1a

RQ 1.5

2

2.5

3

Nonhematopoietic cells

120

100

80

60

40

20

0

TILMyeloid Lymphocytes

BloodCD4+

P < 0.0001 P < 0.0001 P < 0.0001

%of

Con

trol

bind

ing

120

100

80

60

40

20

0

%of

Con

trol

bind

ing

P < 0.002

LymphocytesMonocytes

α-Clever on leukocytesControl mAb on leukocytes

Figure6. InducibleClever-1on tumor vasculature andbloodmonocytes of tumor-bearingmicemediates leukocyte-endothelial cell interaction, and changes inTIL populations are reflected in their inflammatory status. A, binding of two major populations of TIL, myeloid cells and lymphocytes, and CD4þ cellsfrom the blood (both from untreated melanoma-bearing WT mice) to vessels in melanomas obtained from mice treated in vivo with anti-Clever-1 (n ¼ 3) orcontrol antibody (n ¼ 3) analyzed using ex vivo–frozen section assays. The number of leukocytes bound to control antibody-treated melanomas was set as100% by definition and the data are presented as the mean � SEM of control binding. B, binding of blood monocytes and lymphocytes from tumor-bearingmice to tumor vasculature using ex vivo–frozen section assays. Blood leukocytes were preincubated with anti-Clever-1 or control antibody and washedbefore the assay. Analyses were done as in A. Note that in A the Clever-1 blockage is on endothelium and in B on leukocytes (monocytes). Binding ofmonocytes (large ruffled, two pointed out by arrows) and lymphocytes (small, two indicated by open arrows) to tumor vasculature (surrounded by a dashedline) in an ex vivo–frozen section assay is shown as an example on the right. C and D, mRNA expression of the indicated target genes in CD45þ TILs (C)and non-hematopoietic CD45� cells (D) of anti-Clever-1 antibody-treated mice. The corresponding expression levels in the control antibody-treatedmice are set to 1.0 (dotted line). Only the genes with RQ > 2 or < 0.6 are depicted. (n ¼ 6 mice analyzed in both groups).


Karikoski et al.




leukocyte adhesion to Clever-1 molecule on tumor endo-thelium in agreement with the reported adhesive functionof Clever-1 in leukocyte transmigration (15, 16). We foundaberrant Clever-1 expression in intratumoral vessels in themelanoma model. The fact that anti-Clever-1 antibodytreatment inhibited TIL and blood leukocyte binding totumor endothelium in in vitro assays supports this inter-pretation. It was technically not possible to formally verifythis hypothesis in vivo, because leukocyte accumulation inthe tumor is slow when compared with LNs in an appro-priate time window to allow reliable discrimination ofhoming from local proliferation. Also in humans, Clever-1 is induced both in intratumoral and peritumoral bloodvessels and it supports cancer cell binding to tumor vascu-lature in in vitro assays (15). The numbers of Tregs and typeII macrophages, but not other leukocyte subtypes, werereduced in the tumors after Clever-1 mAb treatment. Thisimplies that counter-receptors forClever-1,which remain tobe identified, may be selectively expressed on differentleukocyte subtypes.The resultswith cell type–specificClever-1–deficientmice

indicate that Clever-1 in tumor endothelium and mono-cytes/macrophages is decisive in the growth of the primarytumors, whereas Clever-1 in lymphatics contributes tomigration of the cancer cells into the draining LNs. Therole of Clever-1 on lymphatics is in line with our earlierstudies, which have demonstrated the importance of Clev-er-1 in the trafficking of normal lymphocytes via afferentlymphatics into the draining LNs (17). Here, we used anindependent model for analyzing migration of subcutane-ously injected cancer cells via the afferent lymphatics todraining LNs and found nearly 70% decrease in the pres-ence of function-blocking anti-Clever-1 mAb. This furtherstrengthens the notion that metastasizing tumor cells mayuse Clever-1 in lymphatics for trafficking.In conclusion, we report here aberrant expression of

Clever-1 in tumor blood vessels and its central role inbinding of TILs. The inhibitory effects of anti-Clever-1antibody therapy on progression of primary and metastatictumors suggest that this molecule may be a new immuno-

modulatory target for cancer immunotherapy. Because anti-Clever-1 treatment, unlike, for example, CTLA-4 inhibitors(36), did not aggravate autoimmune reactions, and becauseit has a unique mode of action, it may have potentialbenefits in comparison with currently available immuno-modulating drugs.

Disclosure of Potential Conflicts of InterestM. Salmi and S. Jalkanen have ownership interest (including patents) in

Faron Pharmaceuticals. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: K. Elima, P. Rantakari, V. Huovinen, H. Irjala,R. Holmdahl, M. Salmi, S. JalkanenDevelopment of methodology: M. Karikoski, F. Marttila-Ichihara,V. Huovinen, H. IrjalaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Karikoski, F. Marttila-Ichihara, P. Rantakari,T. Kelkka, V. Huovinen, R. Holmdahl, M. Salmi, S. JalkanenAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Karikoski, F. Marttila-Ichihara,K. Elima, P. Rantakari, M. Hollm�en, T. Kelkka, V. Huovinen, H. Irjala,R. Holmdahl, M. Salmi, S. JalkanenWriting, review, and/or revision of the manuscript: K. Elima,M. Hollm�en, T. Kelkka, V. Huovinen, H. Irjala, R. Holmdahl, M. Salmi,S. JalkanenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F. Marttila-Ichihara, H. Gerke,V. HuovinenStudy supervision: H. Irjala, M. Salmi, S. Jalkanen

AcknowledgmentsThe authors thank Sari M€aki, Riikka Sj€oroos, and Maritta Pohjansalo for

technical and Anne Sovikoski-Georgieva for secretarial help.

Grant SupportThis work was supported by the Finnish Academy, the Finnish Cancer

Union, the Sigrid Juselius Foundation, Arvo and Inkeri Suominen Founda-tion, and the Heart and Cancer Foundation of Kirsti and Tor Johansson.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 20, 2014; revised August 25, 2014; accepted August 25,2014; published OnlineFirst October 15, 2014.

References1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.

Cell 2011;144:646–74.2. Wilting J, Hawighorst T, Hecht M, Christ B, Papoutsi M. Development

of lymphatic vessels: tumour lymphangiogenesis and lymphatic inva-sion. Curr Med Chem 2005;12:3043–53.

3. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fostersimmune privilege and predicts reduced survival. Nat Med 2004;10:942–9.

4. Zou W. Regulatory T cells, tumour immunity and immunotherapy. NatRev Immunol 2006;6:295–307.

5. Kzhyshkowska J, Gratchev A, Goerdt S. Stabilin-1, a homeostaticscavenger receptor with multiple functions. J Cell Mol Med 2006;10:635–49.

6. Lin EY, Pollard JW. Tumor-associated macrophages pressthe angiogenic switch in breast cancer. Cancer Res 2007;67:5064–6.

7. Adachi H, Tsujimoto M. FEEL-1, a novel scavenger receptor with invitro bacteria-binding and angiogenesis-modulating activities. J BiolChem 2002;277:34264–70.

8. Prevo R, Banerji S, Ni J, Jackson DG. Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelialvenule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEV-ER-1). J Biol Chem 2004;279:52580–92.

9. Kzhyshkowska J, Workman G, Cardo-Vila M, Arap W, Pasqualini R,Gratchev A, et al. Novel function of alternatively activated macro-phages: stabilin-1-mediated clearance of SPARC. J Immunol 2006;176:5825–32.

10. Kzhyshkowska J, Gratchev A, Schmuttermaier C, Brundiers H,Krusell L, Mamidi S, et al. Alternatively activated macrophagesregulate extracellular levels of the hormone placental lactogen viareceptor-mediated uptake and transcytosis. J Immunol 2008;180:3028–37.






11. Kzhyshkowska J, Gratchev A, Martens JH, Pervushina O, Mamidi S,Johansson S, et al. Stabilin-1 localizes to endosomes and the trans-Golgi network in human macrophages and interacts with GGA adap-tors. J Leukoc Biol 2004;76:1151–61.

12. Goerdt S, Walsh LJ, Murphy GF, Pober JS. Identification of a novelhigh molecular weight protein preferentially expressed by sinusoidalendothelial cells in normal human tissues. J Cell Biol 1991;113:1425–37.

13. Irjala H, Elima K, Johansson EL, Merinen M, Kontula K, Alanen K, et al.The same endothelial receptor controls lymphocyte traffic both invascular and lymphatic vessels. Eur J Immunol 2003;33:815–24.

14. Martens JH, Kzhyshkowska J, Falkowski-Hansen M, Schledzewski K,Gratchev A, Mansmann U, et al. Differential expression of a genesignature for scavenger/lectin receptors by endothelial cells andmacrophages in human lymph node sinuses, the primary sites ofregional metastasis. J Pathol 2006;208:574–89.

15. Irjala H, Alanen K, Grenman R, Heikkila P, Joensuu H, Jalkanen S.Mannose receptor (MR) and common lymphatic endothelial andvascular endothelial receptor (CLEVER)-1 direct the binding ofcancer cells to the lymph vessel endothelium. Cancer Res 2003;63:4671–6.

16. Salmi M, Koskinen K, Henttinen T, Elima K, Jalkanen S. CLEVER-1mediates lymphocyte transmigration through vascular and lymphaticendothelium. Blood 2004;104:3849–57.

17. Karikoski M, Irjala H, Maksimow M, Miiluniemi M, Granfors K, Her-nesniemi S, et al. Clever-1/Stabilin-1 regulates lymphocyte migrationwithin lymphatics and leukocyte entrance to sites of inflammation. EurJ Immunol 2009;39:3477–87.

18. Ålgars A, Irjala H, Vaittinen S, Huhtinen H, Sundstr€om J, Salmi M, et al.Type and location of tumor-infiltrating macrophages and lymphaticvessels predict survival of colorectal cancer patients. Int J Cancer2012;131:864–73.

19. Holmdahl R, Malissen B. The need for littermate controls. Eur JImmunol 2012;42:45–7.

20. Lewandoski M, Meyers EN, Martin GR. Analysis of Fgf8 gene functionin vertebrate development. Cold Spring Harb Symp Quant Biol1997;62:159–68.

21. Koni PA, Joshi SK, Temann UA, Olson D, Burkly L, Flavell RA.Conditional vascular cell adhesion molecule 1 deletion in mice:impaired lymphocyte migration to bone marrow. J Exp Med 2001;193:741–54.

22. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditionalgene targeting inmacrophages and granulocytes using LysMcremice.Transgenic Res 1999;8:265–77.

23. Marttila-Ichihara F, Turja R, Miiluniemi M, Karikoski M, Maksimow M,Niemela J, et al. Macrophage mannose receptor on lymphatics con-trols cell trafficking. Blood 2008;112:64–72.

24. Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, PattariniL, et al. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistanttumors without affecting healthy vessels. Cell 2007;131:463–75.

25. Schledzewski K, Falkowski M, Moldenhauer G, Metharom P, Kzhysh-kowska J, Ganss R, et al. Lymphatic endothelium-specific hyaluronanreceptor LYVE-1 is expressed by stabilin-1þ, F4/80þ, CD11bþmacro-phages in malignant tumours and wound healing tissue in vivo and inbone marrow cultures in vitro: implications for the assessment oflymphangiogenesis. J Pathol 2006;209:67–77.

26. Marttila-Ichihara F, Auvinen K, Elima K, Jalkanen S, Salmi M. Vascularadhesion protein-1 enhances tumor growth by supporting recruitmentof Gr-1þCD11bþ myeloid cells into tumors. Cancer Res 2009;69:7875–83.

27. Jalkanen ST, Butcher EC. In vitro analysis of the homing properties ofhuman lymphocytes: developmental regulation of functional receptorsfor high endothelial venules. Blood 1985;66:577–82.

28. Salmi M, Jalkanen S. Human leukocyte subpopulations from inflamedgut bind to joint vasculature using distinct sets of adhesionmolecules.J Immunol 2001;166:4650–7.

29. Gordon S. Alternative activation of macrophages. Nat Rev Immunol2003;3:23–35.

30. Palani S, MaksimowM,Miiluniemi M, Auvinen K, Jalkanen S, Salmi M.Stabilin-1/CLEVER-1, a type2macrophagemarker, is an adhesion andscavenging molecule on human placental macrophages. Eur J Immu-nol 2011;41:2052–63.

31. Biswas SK, Allavena P, Mantovani A. Tumor-associated macro-phages: functional diversity, clinical significance, and open questions.Semin Immunopathol 2013;35:585–600.

32. PortaC, Larghi P,RimoldiM,TotaroMG,AllavenaP,Mantovani A, et al.Cellular and molecular pathways linking inflammation and cancer.Immunobiology 2009;214:761–77.

33. Movahedi K, Laoui D, Gysemans C, Baeten M, Stange G, Van denBossche J, et al. Different tumor microenvironments contain function-ally distinct subsets of macrophages derived from Ly6C(high) mono-cytes. Cancer Res 2010;70:5728–39.

34. Savage ND, de Boer T, Walburg KV, Joosten SA, van Meijgaarden K,Geluk A, et al. Human anti-inflammatory macrophages induce Foxp3þ

GITRþCD25þ regulatory Tcells,which suppressviamembrane-boundTGFbeta-1. J Immunol 2008;181:2220–6.

35. TurkMJ, Guevara-Patino JA, RizzutoGA, EngelhornME, Sakaguchi S,Houghton AN. Concomitant tumor immunity to a poorly immunogenicmelanoma is prevented by regulatory T cells. J Exp Med 2004;200:771–82.

36. Bouwhuis MG, Ten Hagen TL, Suciu S, Eggermont AM. Autoim-munity and treatment outcome in melanoma. Curr Opin Oncol2011;23:170–6.


Karikoski et al.




2014;20:6452-6464. Published OnlineFirst October 15, 2014.Clin Cancer Res Marika Karikoski, Fumiko Marttila-Ichihara, Kati Elima, et al. Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis

Updated version

10.1158/1078-0432.CCR-14-1236doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2014/10/16/1078-0432.CCR-14-1236.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/20/24/6452.full#ref-list-1

This article cites 36 articles, 17 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/20/24/6452.full#related-urls

This article has been cited by 6 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/20/24/6452To 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-14-1236

http://clincancerres.aacrjournals.org/content/suppl/2014/10/16/1078-0432.CCR-14-1236.DC1

http://clincancerres.aacrjournals.org/content/20/24/6452.full#ref-list-1

http://clincancerres.aacrjournals.org/content/20/24/6452.full#related-urls

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

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/20/24/6452


Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis · Clever-1/Stabilin-1, also known as...

Documents

Transcript of Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis · Clever-1/Stabilin-1, also known as...