Fibroblast nemosis arrests growth and induces differentiation of

human leukemia cells

Esko Kankuri1,2*, Olga Babusikova3, Kristina Hlubinova3, Pertteli Salmenper€a1,4, Carla Boccaccio5,Werner Lubitz6, Ari Harjula2 and Jozef Bizik2,3

1Institute of Biomedicine, Pharmacology, University of Helsinki, Finland2Cell Therapy Research Consortium, Third Department of Surgery, Helsinki University, Central Hospital, Finland3Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia4Haartman Institute, University of Helsinki, Finland5Institute for Cancer Research and Treatment, Torino, Italy6Department of Medical/Pharmaceutical Chemistry, University of Vienna, Austria

Interactive signaling between cancer cells and stroma plays an im-portant role in determining tumor development. We recentlyfound tumor cell-derived factors to induce fibroblast clustering,and that the high amounts of hepatocyte growth factor/scatter fac-tor (HGF/SF) produced by these cell–cell contact-activated fibro-blasts enhanced the invasiveness of c-Met-expressing cancer cells.We now observed that leukemia cells lacking c-Met respond tothis novel type of fibroblast activation, nemosis, with growtharrest and differentiation to a dendritic cell-like phenotype. Thiseffect was counteracted by introduction of c-Met expression intothese cells. Moreover, those leukemia cell lines harboring properlyprocessed c-Met showed no effect in response to nemosis. We pro-pose this effect to be mediated by nemosis-derived cytokine sig-nals, and present the potential candidates induced in the nemoticfibroblasts: interleukins-1, -6, -8, -11, leukemia inhibitory factorand granulocyte-macrophage-colony-stimulating factor. Ourresults emphasize the role of activated stromal fibroblasts in con-trolling progression of certain hematologic malignancies in ac-Met expression-dependent manner.' 2007 Wiley-Liss, Inc.

Key words: leukemic cell differentiation; growth arrest; nemosis;HGF/c-Met pathway

Control over cell differentiation and proliferation requires thecomplex interactions of signals to be orchestrated both spatiallyand temporally. The majority of these developmental cues origi-nate from the mesenchyme surrounding the precursor cells. Inmesenchymal tissues, fibroblasts are ubiquitous sentinel cells1,2

that modulate a series of developmental and pathologic conditionsranging from cell differentiation and organogenesis to inflamma-tion and cancer.2,3 Being the major stromal cellular constituents,fibroblasts play a dominant role by controlling differentiation andproliferation of hematopoietic precursors,4 and are a rich source ofseveral factors governing hematopoiesis.5 Proper maturation ofleukocytes requires strict control over the prevailing proliferativeactivity of the immature blasts, and is achievable only by a recip-rocal complex interaction with their surrounding mesenchyme.4–6

Leukemic cells have, however, lost their ability to translate theseregulating signals properly, and prefer an undifferentiated pheno-type with sustained intense proliferation.7–9

The hepatocyte growth factor/scatter factor (HGF/SF)-c-Met-pathway is a major regulator of tumor-stromal interac-tions.10,11 HGF/SF is a stroma-derived paracrine mediator theeffects of which are transmitted via the receptor tyrosine ki-nase c-Met on target cells.12 Stimulation of this pathwayincreases cell proliferation and motility, induces morphogene-sis, and is thus implicated in organogenesis, regeneration,wound healing, tumor cell invasiveness, metastasis and cancerprogression. In addition to its several multifunctional roles asa mitogen, motogen and morphogen,12 HGF/SF is a regulatorof hematopoiesis, as well.13,14 Cancer cells exhibit variableexpression of c-Met thus rendering them differently prone forstimulation by stroma-derived HGF/SF.15 A lack of c-Metexpression or improper processing of the precursor protein in

tumor cells thus makes them more liable to other stimuli, andleads to alternate translation of stromal signals within the tu-mor microenvironment.

In fibroblasts we recently found a novel biological process thatwas triggered by cell–cell contacts.16 The contact-activated cellswere characterized by massive induction of genes such as cyclooxy-genase-2 and HGF/SF.16,17 On the basis of unique features showingexclusively proinflammatory activity of this distinct type of fibro-blast activation by biological means, we designated this processnemosis.17 We subsequently found that exposure to these nemoticfibroblasts dramatically enhanced tumor cell invasiveness. Wedemonstrated this effect to be exclusively mediated by HGF/SF viaa transient phosphorylation of c-Met, detectable only when this re-ceptor underwent proper processing in the tumor cells.17

On the basis of our earlier findings on profuse induction ofHGF/SF, we now analyzed the effect of nemosis on hematologicmalignancies differently expressing the c-Met receptor. We nowreport that when cell lines differed in c-Met expression theyresponded to fibroblast nemosis differently. The c-Met-negativecell lines responded with discernible growth arrest, chemotaxis,and differentiation, whereas the c-Met-positive cells remainedunresponsive. We therefore next investigated the extent of secre-tion of hematopoiesis-associated cytokines from nemotic fibro-blasts, and provide here the first insight into the intracellular path-ways activated by these fibroblast nemosis-derived signals, inaddition to the HGF/SF signal.

Material and methods

Materials

Antibodies for immunoblotting were rabbit anti-p38 antibody(Ab) (sc-535, Santa Cruz Biotechnology, Santa Cruz, CA), mouseanti-p-p38 Tyr182 monoclonal antibody (MAb) (sc-7973), rabbitanti-JNK Ab (CST-0252, Cell Signaling Technology, Danvers,MA), mouse anti-p-JNK Thr183/Tyr185 MAb (sc-6254), rabbitanti-ERK1/2 Ab (sc-94), mouse anti-p-ERK1/2 Tyr204 MAb (sc-7383), rabbit anti-Akt Ab (CST-9272), rabbit anti-p-Akt Ser473Ab (CST-9271), rabbit anti-JAK1 Ab (sc-7228), rabbit anti-JAK2Ab (sc-294), rabbit anti-JAK3 Ab (sc-513), rabbit anti-TYK2 Ab(sc-169), rabbit anti-cleaved caspase-3 Asp175 Ab (CST-9661),

This article contains supplementary material available via the Internet athttp://www.interscience.wiley.com/jpages/0020-7136/suppmat.Grant sponsor: Finnish government; Grant numbers: EVO/TYH5202,

TYH7201. Grant sponsor: Slovak Academy of Sciences; Grant numbers:VEGA 2/6017/6, 2/4021/6. Grant sponsors: Syd€antutkimuss€a€ati€o, HelsinkiUniversity’s Research Funds.*Correspondence to: Institute of Biomedicine, Pharmacology, Biome-

diucm, P.O. Box 63 (Haartmaninkatu 8), 00014 University of Helsinki,Helsinki, Finland. Fax:1358-9-191-25364.E-mail: esko.kankuri@helsinki.fiReceived 6 June 2007; Accepted after revision 16 August 2007DOI 10.1002/ijc.23179Published online 20 November 2007 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 122, 1243–1252 (2008)' 2007 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

mouse anti-full-length caspase-3 Ab (sc-7272), mouse anti-cas-pase-8 Ab detecting both full length and active fragments (CST-9746), rabbit anti-caspase-9 Ab detecting both full length and 35/37 kDa cleaved fragments (CST-9502), rabbit anti-PARP Ab(CST-9542), rabbit anti-BclxL Ab (sc-7195), goat anti-actin Ab(sc-1615), mouse anti-Bax MAb (sc-7480), mouse anti-Bcl-2MAb (sc-509), goat anti-COX-1 Ab (sc-1752) and goat anti-COX-2 Ab (sc-1746). Indomethacin (I7378) was from Sigma (St. Louis,MO) and the NS-398 (No. 70590) from Cayman Chemical (AnnArbor, MI).

Antibodies for flow cytometry (FACS) from the BeckmanCoulter Company (Miami, FL) were: anti-CD1a-PE (IM1942),anti-CD3-PE (IM1282), anti-CD10-PE (IM1915), anti-CD11a-FITC (IM0860), anti-CD11b-PE (IM2581), anti-CD11c-PE(IM1760), anti-CD13-PE (IM1427), anti-CD14-FITC (IM0645),anti-CD15-FITC (IM1423), anti-CD16-FITC (IM0814), anti-CD28-FITC (IM1236), anti-CD33-PC5 (IM2647), anti-CD34-PC5(IM2648), anti-CD38-FITC (IM0775), anti-CD40-PE (IM1936),anti-CD41-FITC (IM0649), anti-CD45-FITC (IM0782), anti-CD45RA-FITC (IM0584), anti-CD45RO-PE (IM1307), anti-CD49d-FITC (IM1404), anti-CD49e-FITC (IM1854), anti-CD51-FITC (IM1855), anti-CD54-PE (IM1239), anti-CD61-FITC(IM1758), anti-CD80-FITC (IM1853), anti-CD83-PC5 (IM3240),anti-CD86-PE (IM2729), anti-CD117-PC5 (IM2657), anti-CD152-PE (IM2282) and anti-HLA-DR-FITC (IM1638). Theanti-CD68-FITC (GM-4152) was from Caltag Laboratories (Bur-lingame, CA).

Cell cultures

Cultures of foreskin-derived human fibroblasts, HFSF-132,were used from passages 7 to 15 as described.16 KG-1, THP-1, U-937, K562, Jurkat and Raji were from the American Type CultureCollection (ATCC, Manassas, VA). All cells were cultured inRPMI 1640 (Life Technologies, Paisley, Scotland) supplementedwith 10% fetal bovine serum (Life Technologies), 100 Ag/mLstreptomycin and 100 units/mL penicillin.

Spheroid formation was initiated as described by Bizik et al.(2004). Briefly, U-bottom 96-well plates (Costar, Cambridge,MA) were treated with 0.8% LE agarose (BioWhittaker,Rockland, ME) prepared in sterile water to form a thin film of anonadhesive surface. Fibroblasts were detached from culturedishes by trypsin/ EDTA, and a single cell suspension (4 3 104

cells/mL) was prepared in a complete culture medium. To initiatespheroid formation, 250-mL aliquots were seeded into individualwells and the dishes incubated at137�C in a 5% CO2 atmosphere.

For the coculture and nemosis stimulation experiments, the leu-kemia cells were cultured for various time-periods with 24-hr-pre-formed fibroblast spheroids at a 1:1 leukemia cells:fibroblast ratio.After incubation, the leukemia cells were separated from multicel-lular spheroids by gravity based on the much higher density ofmulticellular aggregates as compared to leukemic cells/mono-cytes. The remaining supernatant contained leukemic cellswhereas the spheroids remained in the pellet. For the estimation ofgrowth curves, cell numbers were evaluated by cell-counting inB€urker chambers. For immunoblotting, FACS, and adherence test-ing, the residual spheroids were removed from cocultures bygravitational differential sedimentation.

Morphology of leukemic cells 96 hr after coculturing was eval-uated by phase contrast microscopy. The leukemic cells’ adher-ence was estimated after 96 hr of coculturing with fibroblast sphe-roids. Thereafter aliquots of cell lines were seeded onto standardcell-culture dishes for 24 hr. The cultures were washed, and adher-ent cells were harvested by trypsinization, were counted, and thepercentage of these adherent cells was calculated.

Chemotaxis of leukemic cells was performed in agarose-treated6-well plates as cocultures of 24-hr-preformed fibroblast spheroidswith the naı̈ve leukemia cell lines. We calculated with an oculargrid the number of leukemic cells located at a distance from the

spheroid double its own diameter, and measured these cells around15 spheroids per well.

Lentiviral vector transduction

293FT cells were transfected together with pRRLsinPPT.CMV.MCS.METwt.Wpreplasmid18 and second-generation pack-aging vectors pHCMV-G and pCMVD8.91 using Lipofectamine2000 (Invitrogen, Carlsbad, CA). After 72 h, the supernatant wascollected, the viral particles were concentrated using ultracentri-fugation (26,000g for 1.5 h), and were resuspended in PBS.GFP-expression lentivirus (pLV-PGK/GFP) used as control wasa kind gift from professor Seppo Yl€a-Herttuala (AIV-Institute,Kuopio, Finland). Virus stocks were stored in 270�C until trans-duction.

THP-1 cells were seeded (5 3 105 cells/well) into a 6-well plateand transduced with 1:20 dilution of virus concentrate and titer of1.43 107 in the presence of polybrene (8 lg/mL). After 16 h, virus-containing medium was removed, cell were washed and resus-pended in normal growth medium for experimentation.

Cell cycle analysis

For DNA histograms and cell cycle analyses, leukemic cellswere cocultured for 96 hr with 24-hr-preformed fibroblast sphe-roids, and separated from fibroblast clusters by sedimentation.These cells were then washed with PBS and fixed in 1% parafor-maldehyde, were treated with RNAse (100 lg/mL), their DNAwas stained with propidium iodine (50 lg/mL), and they were ana-lyzed by FACS.

Immunoblotting

Cell samples were lysed directly in SDS-PAGE sample-loadingbuffer: 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 20% glycerol,5% b-mercaptoethanol and 0.005% bromophenol blue, supple-mented with Complete Mini-protease inhibitor mixture tablets(Roche, Mannheim, Germany) and boiled for 5 min. Lysates werecentrifuged at 14,000 rpm for 15 min to sediment particulate-in-soluble material. These samples were separated in SDS-PAGE(gradient of polyacrylamide 5–15%, 3.5% stacking gel). The pro-teins were transferred electrophoretically from the gel to a nitro-cellulose membrane (Schleicher & Schuell, Dassel, Germany),with transfer efficiency verified by Ponceau-S staining. Afterblocking of the membrane with 2.5% low-fat dry milk in TBS, 20mmol/L Tris-HCl, 150 mmol/L NaCl and 0.1% Tween 20 at pH7.5, it was incubated with specific primary antibodies, followed byan alkaline phosphatase-conjugated secondary antibody (Promega,Madison, WI). Protein bands were visualized according to manu-facturer’s recommendations.

Flow cytometry

For flow cytometric analysis, the leukemia cells cocultured forindicated time points and after differential sedimentation toremove spheroids were incubated on ice with antigen-specific anti-bodies or with isotype-matched antibodies as controls, and fixed in1% paraformaldehyde. FACS analysis was done by an EPICSALTRA flow cytometer with the EXPO32 analysis program (bothfrom Beckman Coulter, Fullerton, CA).

Measurements of cytokine concentrations byenzyme-linked immunoassays

Fibroblast spheroid-conditioned medium was collected at 96 hrafter initiation of spheroid formation from the 96-well plates. Con-centrations of IL-1b, IL-6, IL-8, IL-11, GM-CSF, LIF, oncostatinM and TNF-a were quantified by commercial ELISA kits andreagents according to manufacturers’ instructions. The human IL-1b, human IL-11, human LIF, human TNF-a and human oncosta-tin M ELISAs were from R&D Systems (Minneapolis, MN), thehuman IL-6 and human IL-8 ELISAs were from the Central Labo-ratory of the Netherlands Red Cross (CLB, Amsterdam). Cytokinequantification in the nemotic fibroblast-conditioned medium for

1244 KANKURI ET AL.

human IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, interferon-g (IFN-g) and TNF-a was carried out with the Bio-Plex HumanCytokine Th1/Th2 Panel (Bio-Rad Laboratories, Hercules, CA,catalogue number 171-A11081), by the Luminex 100 System(Luminex, Austin, TX).

Results

c-Met expression in leukemia cells, their growthcharacteristics and cell cycle analysis

In analysis of leukemia cell lines for their expression of theHGF/SF receptor c-Met, expression of the properly processedform appeared on U-937, Jurkat, Raji and K562 cell lines but noton THP-1 or KG-1 cells (Figure 1a). All these cells were cocul-tured with nemotic fibroblasts for their growth characteristics. Thecell lines THP-1 and KG-1 lacking c-Met responded with discerni-ble growth arrest, whereas the cell lines expressing c-Met showedno significant alterations in their proliferation rates. For subse-quent experiments on stimulation with nemotic fibroblasts, wechose the c-Met-negative THP-1 and KG-1 cell lines, and thec-Met-positive U-937 cell line as control.

Cell cycle analysis of KG-1, THP-1 and U-937 cells

Nemotic fibroblasts induced a dramatic growth inhibition ofthe cells lacking c-Met, whereas growth of the c-Met-positive celllines showed only marginal, if any, inhibition (Figure 1b).

Attenuation of growth by nemosis-derived signals was evident at72 hr for the KG-1 cells, but was already evident at 48 hr in theTHP-1 cell line, suggesting enhanced sensitivity to nemosis ofthe latter cells. In the control U-937 cells only a modest anddelayed effect on proliferation was apparent after 96 hr of incuba-tion. The nemosis-arrested proliferation of the responder cell linespersisted throughout the study. With the control cells reachingtheir growth plateau at 168 hr, nemosis inhibited the proliferationof the cell lines by 67% for KG-1, 83% for THP-1 and 6% forU-937 cells.

Figure 1c shows the leukemic cell lines’ cell-cycle phase distri-bution as evaluated by DNA histograms. When treated with nemo-sis, 30.2% of the KG-1 and 31.3% of the THP-1 cell populationsaccumulated in the G0G1 phase. This shift away from the M andS phases indicates cell-cycle arrest associated with differentiation.The U-937 cells retained their cell-cycle characteristics, with nopopulation shifts in response to fibroblast nemosis. Moreover,treatment of clustering fibroblasts with the nonsteroidal anti-inflammatory cylooxygenase-inhibitors, NS-398 and indometha-cin, to prevent prostaglandin production16 had no effect on inhibi-tion of proliferation by nemosis (data not shown).

c-Met expression and dependence of nemosis response

Stimulation of the monocytoid leukemic cells by nemosis didnot alter their expression levels of c-Met (Fig. 2a). To show de-pendency of the nemosis effect on the HGF/SF-c-Met-pathway,

FIGURE 1 – c-Met expression in leukemia cell lines, and growth characteristics of selected cell lines subjected to nemosis. (a) Expression ofthe c-Met receptor in leukemia cell lines KG-1, THP-1, U-937, Jurkat, Raji and K562. Arrow indicates position of the properly processed formof c-Met (145 kDa). (b) Proliferation kinetics of leukemia cell lines KG-1, THP-1 and U-937 with and without stimulation by nemotic fibroblastspheroids in coculture. *p < 0.05, **p < 0.01, ***p < 0.001 between treatments at indicated time-points. (c) DNA histogram data and percent-age of cells as divided into cell cycle G0G1, G2 and S phases of leukemia cell lines KG-1, THP-1 and U-937 with (closed bars) and without(open bars) stimulation by nemotic fibroblast spheroids in coculture for 96 hr.

1245NEMOSIS-INDUCED LEUKEMIA CELL DIFFERENTIATION

we introduced wild type human c-Met expression into THP-1 cellsusing a lentiviral vector, and nemosis response in terms of growtharrest to GFP-transduced control cells. Receptor expression wasevident only in the c-Met-transduced cells. Increased expressionof c-Met persisted throughout the 5 days of experimentation (Fig.2b) for growth curve analysis of cells subjected to fibroblastnemosis. Although the intensity of c-Met expression declined dur-ing the experiment, the c-Met-transduced cells resisted growtharrest whereas the GFP-transduced cells entered growth arrest(Fig. 2c). The nemosis-uninfluenced GFP and c-Met expressingcells exhibited similar growth characteristics (Fig. 2c).

Morphological and functional characteristics ofleukemic cells in response to nemosis

Cell cycle arrest at the G0G1 phase is associated withinduction of differentiation.19 In KG-1 and THP-1 cells,nemosis led to an increased proportion of adherent cells by19.8 and 31.6% (Fig. 3a). These cells showed morphologicalfeatures of a dendritic-cell-like phenotype with cell elongationand formation of stellate pseudopodia (Fig. 3b), but inresponse to nemosis, the U-937 cells changed neither theirmorphology nor their pattern of adherence (Fig. 3b). Inductionof an adherent phenotype in the nemosis-responsive cell lines

was associated with increased expression of intercellular adhe-sion molecule-1 (ICAM-1) (Fig. 3c).

Increased adherence of KG-1 and THP-1 cells by nemosis sug-gests that the clustered fibroblasts can also produce factors affect-ing cell motility and chemotaxis. We therefore evaluated the che-motactic responses of KG-1, THP-1 and U-937 cells in coculturewith the nemotic fibroblasts. Comparison of the responses of theanalyzed cell lines showed that both KG-1 and THP-1 were che-motactically drawn towards the fibroblast clusters undergoingnemosis, whereas the U-937 cells were unresponsive (Fig. 3d).Compared to the U-937 cells, nemosis attracted the KG-1 andTHP-1 cells to accumulate in the vicinity of the fibroblast clustersat an 11- and a 22-fold enhanced density.

Since monocyte maturation and differentiation are associatedwith decreased antigen uptake through macropinocytosis,20 westudied, as a parameter of differentiation on a functional scale, theeffect of nemosis on pinocytotic activity. The ability of the leuke-mia cell lines to repel FITC-labeled dextran was assessed at 24and 96 hr of coincubation of leukemia cells with nemotic sphe-roids. After stimulation of the cell lines with nemosis followed byincubation with FITC-dextran, flow cytometry revealed a distinctinhibition of FITC-dextran uptake in nemosis-treated KG-1 (meanintensity change 211.92) and THP-1 cells (mean intensity change

FIGURE 2 – Lentiviral vector transductionof c-Met into THP-1 cells. (a) Expression ofc-Met in the leukemia cell lines KG-1, THP-1 and U-937 stimulated for 96 hr by nemoticfibroblast spheroids (1) and without stimula-tion (2). (b) c-Met expression kinetics inGFP-control and c-Met-transduced THP-1cells at indicated time points. (c) Proliferationkinetics of THP-1 cells transduced with GFP-control or c-Met lentiviral vector with orwithout stimulation by nemotic fibroblastspheroids in coculture. *p < 0.05, **p < 0.01,***p < 0.001 between treatments at indicatedtime-points.

1246 KANKURI ET AL.

23.86), with the U397 cells showing an increase of 0.91 in inten-sity (data not shown). These data are in accordance with morpho-logical characterization showing induction of adherence and thepresence of dendritic cell-like pseudopodia on the nemosis-responsive KG-1 and THP-1 cells.

Changes in surface antigen expression ofnemosis-stimulated cell lines

The phenotypic characterization of nemosis-treated cells wascarried out by FACS analysis of the cell-surface antigens, asshown in the Supplementary Table, comparing control antigen in-tensity to that of nemosis-treated cells. From this analysis, a clearinduction pattern of 5 cell-surface markers emerged. Interestingly,and in accordance with the other cell responses, these antigenswere induced only in KG-1 and THP-1 cells. No induction of thesesurface antigens was evident in U-937 cells, which responded withan overall expressional down-regulation.

In the nemosis-responsive cell lines we identified induction ofthe following: dendritic cell marker CD11c, the leukocyte com-mon antigen CD45RA, the adhesion molecule CD54, the dendriticcell-associated T-cell costimulatory molecule CD86, and themembrane peptidase CD13. The time-dependence of CD86 induc-tion (Fig. 4) showed response kinetics similar to the growth arrestin KG-1 and THP-1 cells, with the latter cell line reacting morepromptly also by this parameter. No effect on the induction ofCD86 occurred when, prior to coculture with the leukemia cells,fibroblast clusters were formed in the presence of NS-398 or indo-methacin (data not shown).

Further population analysis was carried out based on differentialexpression of CD45 in various lineages and differentiationstages.21 By gating on CD45 new subpopulations emerged innemosis-treated KG-1 and THP-1 cells (Fig. 5), but no changesoccurred in response to nemosis in the subpopulation characteris-tics of U-937 cells. The emerging populations were positive for allthe nemosis-induced markers, especially for CD11c and CD13, incontrast to a nonresponsive similar CD45-positive population ofU-937 cells. Nemosis increased CD11c and CD86 positivity in aCD45-positive population with low SSC values of KG-1 andTHP-1 cells. Considered together, these results suggest that inresponsive cell lines nemosis induces expression of antigens

FIGURE 3 – Adherence, morphology and chemotactic response ofthe leukemic cells to nemosis. (a) Percentage of cells from the totalcell population adhering to culture dish after coculture stimulation bynemotic fibroblasts as compared to unstimulated cells of leukemia celllines KG-1, THP-1 and U-937. ***p < 0.001 compared to respectivecontrol cells. (b) Morphology of adherent cells with or without stimu-lation by nemotic fibroblast spheroids. Cell elongation and presenceof pseudopodia evident in stimulated KG-1 and THP-1 cells whereas,after stimulation, U-937 cells retain their phenotype. (c) Expression ofintercellular adhesion molecule-1 (ICAM-1) in KG-1, THP-1 and U-937 cells with (1) or without (2) stimulation by nemotic fibroblastspheroids in coculture. Increased ICAM-1 expression of the nemosis-responsive cell lines KG-1 and THP-1 associated with increased ad-herence to the cell culture dish. (d) Quantification and morphology ofleukemia-cell (KG-1, THP-1, U-937) chemotactic movement towardsfibroblast clusters in coculture. Increased chemotactic accumulation ofKG-1 and THP-1 cells visible around a nemotic spheroid, whereasU-937 cells are unresponsive. ***p < 0.001 compared to U-397 cells.

FIGURE 4 – Time-dependence of CD86 surface antigen expression.CD86 expression evaluated by FACS at indicated time points for leu-kemia cell lines KG-1, THP-1 and U-937 in coculture with nemoticfibroblast spheroids.

1247NEMOSIS-INDUCED LEUKEMIA CELL DIFFERENTIATION

involved in antigen presentation and T-cell stimulation, alongwith dendritic cell characteristics.

Cytokine production in nemotic fibroblasts

We previously reported that nemotic fibroblasts are an amplesource of the c-Met ligand HGF/SF.17 As present data indicatesgrowth arrest and differentiation of leukemia cells in response tofibroblast nemosis to be overcome by HGF/SF-c-Met-signaling, weevaluated a pattern of cytokines known to be associated with modu-lation of chemotaxis and leukemia cell proliferation. We foundinduced release of interleukin(IL)-1b, IL-6, IL-8, IL-11, granulo-cyte-macrophage colony-stimulating factor (GM-CSF), and leukemiainhibitory factor (LIF) from nemotic spheroids compared to the cor-responding monolayer cultures at 96 hr from culture initiation (Fig.6). The levels of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon-g,oncostatin M and TNF-a remained low or undetectable, and wereunaffected by cell culture arrangement. The cytokines most abun-dantly produced by the nemotic fibroblasts were IL-6 and IL-8, withfold-inductions (mean production in spheroids) of 3.7 (25.4 ng/mL)and of 8.0 (134.2 ng/mL) as compared to the corresponding mono-layer cultures. Production of IL-1b and LIF was induced 18.3 and3.8 fold in the nemotic fibroblasts, which also produced GM-CSFundetectable from monolayer cultures (Fig. 6). Our results thus sug-gest that nemosis is a fundamental source of an array of proinflam-matory cytokines and growth factors regulating monocyte functions.

Apoptosis-related intracellular changes in the leukemiacell lines by nemosis

Inhibition of tumor cell growth is usually accompanied byinduction of apoptosis. We therefore evaluated the known apopto-

tic pathways in the growth-arrest and differentiation responses ofTHP-1 and KG-1 cells to nemosis. In Figure 7a, expression of sev-eral apoptosis-associated proteins revealed that the cleaved, activeform of the universal apoptosis executor, caspase-3, occurs inresponse to nemosis only in the nemosis-responsive cell lines.That the unresponsive U-937 showed no effect suggests activationof apoptosis in the nemosis-responsive cells. No changes in theexpression of the active cleaved forms of the initiator caspases -8and -9 were detectable in any of the cell lines, but expression ofthe full-length caspase-8 was induced by nemosis in the THP-1and KG-1 cells. In this case, as well, the U-937 cells showed noeffect. The increases in caspase-3 cleavage and caspase-8 expres-sion were not, however, reflected in the expression of the apopto-sis-regulating Bcl-xL, Bcl-2, or Bax proteins, suggesting theirapoptosis-unrelated mechanism of action.

Because of the evident cleavage and increased expression of theactive form of caspase-3 caused by nemosis in THP-1 and KG-1cells, we evaluated the extent of poly-ADP-ribose-polymerase(PARP) cleavage associated with DNA damage, apoptosis andcaspase-3 activity.22 Figure 7a shows the expression pattern offull-length PARP (p116) and its cleaved inactive form p89 in leu-kemia cell lines subjected to nemosis.

Effects of nemosis on leukemia cells’ intracellularsignaling cascades

We evaluated the involvement of MAPKs c-jun N-terminalkinase (JNK), extracellular signal-regulated kinases p44/p42(ERK1/2) and p38 in leukemia cell responsiveness to nemosis(Fig. 7b). In all the naı̈ve leukemia cell lines, we found constitu-tive phosphorylation of these kinases, reflecting their mitotically

FIGURE 5 – Cell population analysis of leukemia cell lines KG-1, THP-1 and U-937 based on expression of CD45. Subpopulation analysisbased on CD45 expression and divided into dual-labeling of cells for expression of indicated surface antigens (CD11c, CD86, CD54, CD13).Top figures show expression of CD45 at baseline and with cells stimulated with nemotic fibroblasts in coculture. On the basis of CD45 expres-sion, untreated U-937 cells show 2 populations with high and low granularity and show no change after treatment, whereas in KG-1 and THP-1cells a new emerging population with high granularity is evident after stimulation with nemotic fibroblast spheroids. In these nemosis-responsivecell lines, expression of CD11c and CD86 is enhanced also in the cell population with low granularity.

1248 KANKURI ET AL.

active phenotype. In the KG-1 and THP-1 cell lines, whichresponded to nemosis with growth arrest and differentiation, phos-phorylation of p38 MAPK was dramatically quenched, whereasno changes in p38 MAPK phosphorylation in response to nemosisoccurred in the U-937 cell line. A similar but less pronounced in-hibition of phosphorylation was evident in ERK1/2, with the U-937 cells again left unresponsive to nemosis. The level of phos-phorylation and expression of JNK in all cell lines remained rela-tively unchanged. In the KG-1 and THP-1 cells induced toundergo differentiation by fibroblast nemosis, expression of JAK1and JAK3 was increased. In the nonresponsive U-937 cell line,expression of JAK1 was downregulated, with no visible expres-sion of JAK3 (Fig. 7b). Phenotypic differences between U-937and the nemosis-responsive KG-1 and THP-1 cells were reflectedin TYK2 as well as in JAK3 expression. JAK2 expression corre-lated neither with growth arrest nor with differentiation (Fig. 7b).

Discussion

Nemotic fibroblasts, activated by cell–cell contacts, induce agrowth inhibitory and differentiating response in leukemia cellsspecifically lacking c-Met. This effect is not seen in cells express-ing c-Met, and can be counteracted by introducing c-Met to the re-sponsive cells suggesting that HGF/SF-c-Met signaling can be uti-lized by leukemic cells to evade stromal growth control signals.The nonreactivity in terms of cell function, morphology and anti-gen expression of c-Met expressing leukemia cells suggests alsothat alternate mechanisms for motility, chemotaxis and cell differ-entiation are used by monocytes in contrast to epithelial cells, anddemonstrates an alternate strategy for utilization of c-Met in tumorcells derived thereof. Moreover, we show that in nemosis, a noveltype of stromal cell biological reactivity, production of inflamma-tion-, cell growth- and differentiation-associated cytokines IL-1b,IL-6, IL-8, IL-11, LIF and GM-CSF is induced. It must be stressed

FIGURE 6 – Hematopoiesis-associated cytokine release from fibroblast spheroid clusters and monolayers. Analysis of cytokine (interleukin IL-1, IL-6, IL-8, IL-11, granulocyte-macrophage colony-stimulating factor, GM-CSF and leukemia inhibitory factor, LIF) production from fibro-blasts cultured as spheroids or as monolayers at the corresponding time (96 hr) after spheroid formation. ***p < 0.001 between groups.

1249NEMOSIS-INDUCED LEUKEMIA CELL DIFFERENTIATION

that neither in this study nor in our previous or ongoing work havewe found any induction of anti-inflammatory cytokines in nemo-sis. This suggests that fibroblast cell-cell contacts activate a bio-logically diverse, yet directed, paracrine signaling cascade. Fur-thermore, our results suggest that translation of stroma-derivednemosis signaling with massive induction of signaling molecules

within a given cell microenvironment is largely determined by thephenotype and receptor-expression profile of the target cells.

Tumor cells, showing an imbalance between cell survival anddeath, prefer a growth promoting undifferentiated phenotype. Weshow that leukemic cells responding with growth arrest alsounderwent differentiation when subjected to fibroblast nemosis.Using FACS-gating on CD45, the leukocyte common antigen, wefound in the nemosis-responder cell lines emerging new popula-tions with enhanced expression of CD11c and CD86. CD54 andCD13 cell-surface antigens were also increased in the emerginghigh-granular population suggesting that the leukemic cells influ-enced with nemosis gained a phenotype reminiscent of antigen-presenting cells.

Expression of the CD11c, a marker for myeloid dendritic cells23

associated with differentiation and maturation,24 was coinducedwith CD86 in the nemosis-responders. Similar to CD11c, expres-sion of CD86 on leukemia cells is associated with a dendritic cell-like phenotype.25 In addition to CD11c, increased adherence ofthe growth-arrested cells is explained by increased expression ofCD54 (ICAM-1), which mediates adhesive interactions, for exam-ple by binding to the b2 subfamily integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1).26 Similar to CD11c, also ICAM-1is required for leukocyte migration and, like CD86, ICAM-1 bind-ing functions as a costimulatory signal for the activation of T cellsin antigen presentation.27

We extended our evaluation of nemosis-induced leukemia cellgrowth arrest and differentiation to the molecular intracellular levelby evaluating the involvement of the major pathways associatedwith growth arrest and differentiation. We found intensive cleavageof the executor caspase-3, in contrast to unchanged expression ofBcl-2, Bcl-2XL and Bax, accompanied by no changes in cleavageof the initiator caspases-8 and -9. In contrast to the executor cas-pases requiring proteolytic cleavage for activation, the initiators arealso activated without cleavage by oligomerization or dimeriza-tion.28 Interestingly, caspases 3 and 8 also regulate differentia-tion.29 Furthermore, the fact that cell numbers after nemosis treat-ment remained unchanged implies that the nemosis-responsive phe-notype favors differentiation over apoptotic death. Surprisingly, noincreased proteolytic processing of PARP, a caspase-3 substrate,22

occurred; this suggests for caspases-3 and -8 an apoptosis-unrelateddifferentiating function. PARP is associated with DNA repair, withcell proliferation and differentiation, and with transcriptional regu-lation.22 We never found increases in protein levels of Bax, Bcl-2and Bcl-2XL to be associated with reduced PARP expression, sug-gesting an apoptosis-unrelated and differentiation-associated mech-anism for PARP downregulation.30 PARP expression and activitymay also reflect the changes in mitotic rate of leukemic cells; aftercells undergo growth arrest, for example in response to nemosis,they have less need for PARP expression and activity in the nucleusto protect the fragile opened DNA of mitosis.31

FIGURE 7 – Immunoblot analysis of apoptosis-related and intracel-lular signaling proteins in leukemia cells. (a) Expression of apoptosis-related molecules in leukemia cell lines KG-1, THP-1 and U-937 with(1) or without (2) stimulation by nemotic fibroblast spheroids in co-culture for 96 hr. In nemosis-responsive cell lines KG-1 and THP-1,activation-associated cleavage of caspase-3 and -8 is evident. Nocleavage products of caspase-9 and reduced expression of the cleavedform of poly(ADP-ribose)polymerase (PARP) are visible. Phenotypedifferences between nemosis-responders and the nemosis-unrespon-sive cell line U-937 evident in expression levels of the pro-apoptoticBax protein. (b) Expression of phosphorylated and total levels of mito-gen-activated protein kinases (MAPK) p38, JNK, ERK1/2 and the Aktkinase in comparison to expression of the differentiation-associatedJanus-kinase family JAK1, JAK2, JAK3 and TYK2 in leukemia celllines KG-1, THP-1 and U-937, stimulated (1) or unstimulated (2)with nemotic fibroblast spheroids. Increased dephosphorylation of p38and ERK1/2 is evident in the nemosis-responsive cell lines KG-1 andTHP-1 together with increased expression of JAK1 and JAK3. Thenemosis-unresponsive cell line U-937 showed no expressional differ-ences for these proteins.

1250 KANKURI ET AL.

The MAPK cascades involving JNK, ERKs and p38s are tightlyassociated with cell proliferation, differentiation and death.32 De-spite no changes in JNK expression or phosphorylation, the phos-phorylation of p38 MAPK–and to a minor extent also that ofERK1/2–was significantly suppressed in cells responding to nemo-sis with differentiation and growth arrest. Active p38 preventsJurkat T-cell apoptosis,33 and inhibits differentiation of promyelo-cytic cells.34 This is in agreement with our data showing that inhi-bition of p38 MAPK phosphorylation occurs only in those cellsresponding to nemosis with differentation and growth arrest. Itthus suggests that the highly proliferating phenotype is associatedwith active p38 MAPK and may indicate an altered or constitu-tively active p38 pathway in these cells.

Signal transduction by the Janus protein tyrosine kinase family(JAK) members (JAK1, JAK2, JAK3 and TYK2) is intimatelyassociated with monocyte and leukemia cell differentiation.35 Wefound induction of the JAK kinases JAK1 and JAK3 in the nemo-sis-responsive cell lines. This is concordant with the prevailingview of expressional control of JAK136 and JAK337 in hematopoi-etic differentiation. Induction of the JAKs in the nemosis-respon-sive cell lines suggests involvement of IL-6 and GM-CSF in medi-ating the effect of differentiation. The detailed intracellular mech-anisms involved require further investigations currently ongoingin our laboratory.

Fibroblast nemosis is a unique novel type of inherent stromalactivation that leads to production of a distinct set of paracrinemediators guiding differentiation and growth in a target cell pheno-type-dependent manner. Through direct effects on differentiationof hematological tumor cells, in a way dependent on HGF/SF-c-

Met pathway, nemosis may influence responses of the immune sys-tem to malignancy (Fig. 8). Nemosis, activated by the biologicalmeans of fibroblast cell–cell contacts, represents a novel type ofstromal reactivity with signals showing proinflammatory, growthand differentiation functions. Differentiation of leukemic cells intothe dendritic cell lineage can stimulate anti-leukemic actions of T-cells38,39; such differentiation can be suggested as immunother-apy.40 Our results present the first in vitro evidence that homotypicstromal cell–cell interactions leading to nemosis can provide suffi-cient signaling to modulate and restrain neoplastic growth.

Acknowledgements

The authors thank Mona Schoultz for her diligent expert assis-tance on flow cytometry, Lahja Eurajoki and Alena Kadnarova fortheir skillful technical assistance, and Libusa Stevulova for prepar-ing the figures on flow cytometry. We thank Jana Jakubikova forperforming the Luminex analysis and Carol Norris for author edit-ing the language.

Note Added in Proof

Jozef Bizik, Esko Kankuri, Pertteli Salmenper€a, and AnttiVaheri contributed to the discovery of specific gene expressionand data on cytokine mRNA. Whereas this data is not presented inthe manuscript, it is fully acknowledged as an important source ofinformation to this paper. This data on gene expression and cyto-kine mRNA and protein profiling have now been submitted forpublication (Pertteli Salmenper€a et al., submitted; Anna Enzerinket al., submitted).

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