Journal of Cellular Biochemistry 103:1–8 (2008)

ARTICLES

Co-Inoculation of Prostate Cancer Cells WithU937 Enhances Tumor Growth andAngiogenesis In Vivo

Matthew Craig,1 Chi Ying,1 and Robert D. Loberg1,2*1Department of Urology, University of Michigan, Ann Arbor, Michigan2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan

Abstract Tumor-associated macrophages (TAMs) have been implicated in promoting tumor growth anddevelopment. Here we present evidence that demonstrates that co-inoculation of male athymic nude mice with PC-3prostate cancer cells and U937 promonocytic cells enhances tumor growth and increases tumor angiogenesis. Maleathymic nude mice were co-inoculated with PC-3 and U937 cells (control or IL-4 stimulated) and tumor growthwas monitored over time. Immunohistochemical analysis of tumor specimens was performed for proliferationmarkers (e.g., Ki67) and the effects of IL-4 stimulation on U937 cells were analyzed for chemokine expression. Thepresence of U937 cells increased the rate of tumor growth in vivo and stimulated increased microvascular density withinthe tumor bed. Stimulation of U937 cells with IL-4 resulted in a significant increase in several pro-angiogenic andpro-tumor chemokines (e.g., CCL2). Co-inoculation increases prostate cancer growth via upregulation of chemokines thatinduce angiogenesis within the tumor. J. Cell. Biochem. 103: 1–8, 2008. � 2007 Wiley-Liss, Inc.

Key words: tumor associated macrophage; interleukin 4; chemokine

The role of tumor-associated macrophages(TAMs) in prostate cancer metastasis has notbeen well investigated. Macrophages can be‘‘polarized’’ towards one of two phenotypicallydifferent subsets of macrophages: TH1 (alsoknown as M1 macrophage) or TH2 (also knownas M2 macrophage) [Sica et al., 2006]. M1macrophages are known to produce pro-inflam-matory cytokines and play an active role in celldestruction while M2 macrophages primarilyscavenge debris and promote angiogenesis andwound repair [Sica et al., 2006]. Recently, theM2 macrophage population is thought to bephenotypically similar to the TAM populationthat promotes tumor growth and development.Recently, we have reported the role of CCL2(monocyte chemoattractant protein 1; MCP-1)in prostate cancer growth in vitro [Loberg et al.,

2006a]. CCL2, as well as CCL7 and CCL8, is animportant chemokine that is known to regulatemonoctye/macrophage trafficking and has beenreported to be present in several solid tumorbeds [Negus et al., 1997; Kelly et al., 1998; VanDamme et al., 2004]. The mechanism thatinduces the biological change from a tumordestructive macrophage to a tumor promotingTAM is not understood. Several hypotheseshave been suggested including alternative cyto-kine release from the tumor cells that induces aphenotypic switch in the macrophage popula-tion. Interleukin 4 (IL-4) has been implicated asan important cytokine inducing the differentia-tion of classical macrophages into alternativemacrophages [Hart et al., 1999]. The role of IL-4in regulating the differentiation of monocytes/macrophages into TAMs in prostate cancer hasnot been reported. Here we present evidencethat demonstrates that co-inoculation of maleathymic nude mice with PC-3 prostate cancercells and U937 promonocytic cells enhancetumor growth and increases tumor angiogen-esis. Further, stimulation of U937 cells withIL-4 prior to inoculation enhanced tumorgrowth in vivo and stimulated angiogenesiswithin the tumor bed.

� 2007 Wiley-Liss, Inc.

Grant sponsor: NIH; Grant number: 5P50CA069568.

*Correspondence to: Robert D. Loberg, Ph.D., University ofMichigan, 7312 CCGC, 1500 E. Medical Center Dr., AnnArbor, MI 48109-0946. E-mail: rloberg@umich.edu

Received 15 January 2007; Accepted 20 March 2007

DOI 10.1002/jcb.21379

MATERIALS AND METHODS

Reagents

Human Recombinant Interlukin-4 (hrIL-4)was obtained from R&D systems; Drabkin’sReagent, containing Potassium Ferricyanide,Potassium Cyanide, and Sodium Bicarbonate,was obtained from RICAA Chemical Company.

Cell Culture

U-937 cells are a promonocytic cell linepurchased from ATCC and can be induced toterminal monocytic differentiation phorbolesters [Sundstrom and Nilsson, 1976]. U-937cells and PC-3 prostate cancer cell lines weremaintained in RPMI 1,640þ 10% fetal bovineserum (FBS). HDMVEC cells were maintainedin complete endothelial cell growth mediumþ10% FBS. U937 cells were grown in suspensionand were split for passaging while PC-3 cellsand HDMVEC cells were passaged by trypsini-zation using 1� TrypsinþEDTA (InvitrogenCorp., Carlsbad, CA), resuspended in appro-priate growth medium, and used within 10 pas-sages of each other for consistency. Peripheralblood mononuclear cells (PBMCs) were isolatedfrom peripheral blood from a 33-year-oldhealthy male. Monocytes were isolated usingCD14þ magnetic bead selection (Miltenyi, Inc.)IL-4 stimulated U937 cells (U937IL-4) wereincubated in SF RPMIþ IL-4 at a concentrationof 10 ng/ml for 72 h. All cell lines were incubatedat 378C in 5% CO2.

Xenograft Model of Tumorigenesis

Xenograft tumors were established as pre-viously described [Loberg et al., 2006b]. Briefly,male athymic mice (5–6 weeks) were injectedsubcutaneously with 1� 105 PC-3 cells with orwithout 1� 104 U937 cells or 1� 104 U937IL-4

cells in 200 ml Matrigel (BD Biosciences, Inc.). Asecond group of male athymic mice (5–6 weeks)were injected subcutaneously with 1� 105 PC-3cells with or without 1� 104 PBMCs or 1� 104

PBMCsIL-4 cells in 200 ml Matrigel (BD Bio-sciences, Inc.). Four mice were assigned toeach group. Tumor volumes were calculated bycaliper measurement performed weekly tomonitor and track tumor growth (tumorvolume¼L�W�W� 0.56). Tumors were har-vested once the tumor reached the predefinedcritical mass of 1,000 mm3.

Histology

Xenograft tumors were harvested and placedin fresh 10% formalin. Tumors were paraffinembedded and 5 mm sections were cut andplaced on glass slides. Hematoxylin and eosinstain was performed per the manufacturer’sinstructions (Sigma, Inc.). Identification ofmacrophages was accomplished by labelingwith an anti-CD68 antibody, identification ofneovascularization was accomplished by label-ing with an anti-CD31 antibody, cell prolifera-tion was identified using a Ki-67 antibody, andapoptosis was identified using Apoptag. Tissuesections were incubated for 10 min in Citratebuffer, pH6.0 and microwaved. Samples werethen incubated with anti-CD68 (DakoCytoma-tion, Inc.; 1:1,000), anti-CD31 (DakoCytoma-tion, Inc.; 1:50), Ki67 (DakoCytomation, Inc.;1:50), or Apoptag (Chemicon, Inc.) for 30 minand detected with LSABþdetection/DAB (3,30-Diaminobenzidine; Sigma, Inc.) for 5 min.Slides were dipped in hematoxylin for 1 s as acounterstain.

Microvascular Density

CD31 stained slides were viewed at 100�magnification, representative images weretaken, and microvessels were counted. Indivi-dual vessels were counted and the data ispresented as the averages for each group andexpressed as number of vessels per 100�objective field.

Hemoglobin Assay

Xenograft tumors were harvested, weighed,and incubated in 1 ml of Drabkin’s Reagent andleft rocking at 48C for 1 week. Drabkin’sReagent converts the total hemoglobin in asample to its cyanoderivative, cyanmethemo-globin which can be used to quantify totalhemoglobin content in a sample. Absorbancewas read at 450 nm, and total hemoglobinvalues (mg/ml per 1 g of tissue) were calculatedfrom a hemoglobin curve prepared from astandard.

Conditioned Media

Five lakhs cells per well were plated andallowed to adhere overnight. The following day,media was aspirated off and 1 ml of fresh serumfree media was added. Conditioned media wascollected either 24 or 72 h later.

2 Craig et al.

Endothelial Tube Formation Assay

In vitro tube formation was performed aspreviously described [Zhou et al., 2004]. Growthfactor reduced Matrigel was diluted with coldserum-free medium to a concentration of 10 mg/ml. Fifty microliters of the solution was added toeach well of a 96-well plate and allowed to form agel at 378C for 30 min. HDMVEC cells (150,000cells/ml) in SF RPMI, RPMIþ 10% FBS, U937CM, or PC-3 CM were added to each well andincubated overnight at 378C in 5% CO2. Underthese conditions, EC will form delicate networksof tubes that are detectable within 2–3 h and arefully developed after 8–12 h. After incubationthe wells were washed, and the Matrigel and itsendothelial tubes were fixed with 3% parafor-maldehyde.

Proliferation Assay

Cells were seeded at a density of 1� 105 cells/ml for PC-3 cells and 5� 105 cells/ml for U937cells in a 96 well plate in RPMIþ 10% FBS.Twenty-four hours after seeding, media waschanged to either serum free RPMI, RPMIþ10% FBS, or conditioned media from either PC-3 or U937 cells. Cell growth was determined 72 hlater using the WST-1 assay (Pierce Biotech,Inc.) following the manufacturer’s instructions.

Cytokine/Chemokine Analysis By Bio-Plex Assay

Cytokines and chemokines were measuredusing a human 27-plex cytokine panel (Bio-Rad,Hercules, CA) following the manufacturer’sinstructions. Briefly, cytokine/chemokine stan-dards were prepared in the same fresh mediumthat was used for culturing the cells and wereassayed in duplicate. To each well, beads coatedwith antibody to each chemokine or cytokinewere mixed with either standards, samples, or ablank in a final volume of 100 ml and incubatedfor 30 min with continuous shaking (300 rpm) atroom temperature. After washing, biotinylatedantibodies were added for 30 min with con-tinuous shaking followed by streptavidin–phycoerythrin (PE) for 10 min. The fluorescenceintensity of the beads was measured usingthe Bio-Plex 200 system and Bio-Plex Manager4.1 software with five-parametric-curve fittingwas used for data analysis.

Real Time RT-PCR

Total RNA was isolated from cell lines usingTrizol (Invitrogen Corp.) following the manu-

facturer’s specifications. Purified RNA (5 mg)was converted to cDNA using Super Script IIreverse transcriptase (Invitrogen Corp.) follow-ing the manufacturer’s instructions and usedfor gene expression analysis by real time PCRusing an ABI Prism 7900 HT thermocycler.Primers and probes were purchased from Appli-ed Biosystems, Inc. and used with TaqMan1

Universal PCR Master Mix, No AmpErase1

UNG. GAPDH was used as an internal controlto normalize and compare each sample. Cycleconditions for real time PCR were 958C (15 s),608C (1 min), 728C (1 min) for 40 cycles.Threshold cycle number for each sample wasnormalized to GAPDH for that sample andexpressed on a log scale relative to GAPDHexpression.

Statistical Analyses

Data was analyzed with GraphPad Prizmsoftware. A One-way-ANOVA analysis was usedwith Bonferroni’s post-hoc analysis for compar-ison between multiple groups. A Students t-testwas used for comparison between two groups.Significance was defined as a P value <0.05.

RESULTS

The affects of monoctyes on prostate cancergrowth were assessed in vivo using a xenographmouse model. PBMCs from a normal, healthyadult male and the U937 premonocytic cell linewere used. Athymic nude mice were subcuta-neously injected with PC-3 prostate cancer cellsalone or in combination with monocytes ormonocytes pre-stimulated with IL-4 (10 ng/mlfor 72 h). Average weekly tumor growth for eachgroup was determined by weekly caliper mea-surement for co-innoculation with PBMCs(Fig. 1A) or U937 cells (Fig. 1B). As a control, agroup of mice received xenograft implants ofPBMCs only or U937 cells only (data notshown). Injection of 1� 104 PBMCs or U937cells into athymic mice failed to develop intopalpable subcutaneous tumors after 10 weeks.Co-innoculation of athymic nude mice with PC-3þU937IL-4 cells and PC-3þU937 cells signifi-cantly decreased the time to critical tumor mass(1,000 mm3) compared to PC-3 alone (mediantime in weeks—PC-3þU937IL-4: 6.5 weeks, PC-3þU937: 7 weeks, PC-3: 12 weeks; P< 0.05)(data not shown). The presence of macrophagesin the xenograft tumor bed was confirmed byCD68þ staining (Fig. 1C–H). Interestingly, the

Macrophages Enhance Prostate Cancer Growth 3

PC-3 only xenograft contained CD68þ cellssuggesting host (mouse) macrophage infiltra-tion into the tumor bed after inoculation(Fig. 1C).

The presence of TAMs in tumor growth andprogression has previously been associated withincreased tumor angiogenesis [Crowther et al.,2001]. Thus, the xenograft tumors were har-vested after each mouse reached the criticaltumor mass, and the affects of co-inoculationwith U937 cells on angiogenesis in the tumorenvironment were analyzed using both theDrabkin’s hemoglobin assay and immunohisto-logical staining for CD31 and microvasculardensity (MVD). MVD analysis revealed a sig-nificant increase in vascularization in PC-3þU937 and PC-3þU937IL-4 tumors comparedto PC-3 only tumors and IL-4 pretreated PC-3tumors (PC-3þU937: 292.0� 143.8, PC-3þU937IL-4: 380.3� 106.6, PC-3IL4: 106� 40.9,and PC-3: 85.5� 38.0; mean�SD, P< 0.01)(Fig. 2A). Similarly, analysis of hemoglobincontent using the Drabkin’s assay revealed thatco-innoculation of PC-3 cells with U937 cellsincreased tumor hemoglobin concentration.Specifically, PC-3 tumors had an average con-centration (mg/ml per 1 g of tissue) of1.44� 1.00, PC-3IL4 2.046� 0.76, PC-3þU9377.92� 3.54, and PC-3þU937IL-4 7.37� 4.73(mean�SD) (Fig. 2B). To further assess therole of secreted factors by U937 cells in combi-nation with PC-3 prostate cancer cells on

enhanced tumor angiogenesis, conditionedmedia from U937 or PC-3 cells was used for anin vitro tube formation assay. HDMVEC cellswere incubated overnight on reduced growthfactor MatrigelTM and allowed to form a branch-ing network of capillary-like tubes. Incubationwith U937IL-4 CM revealed a significantincrease (P< 0.01) in tube formation as com-pared to U937 CM, PC-3 CM, and SF media(Fig. 2C).

To address the hypothesis that macrophageswork in concert with prostate cancer cells topromote growth and neo-angiogenesis of thetumor, U937 conditioned media was analyzedfor cytokine/chemokine expression. PC-3 condi-tioned media is shown as a control for thecytokines/chemokines that would be present inthe in vivo co-innoculation experiments. Of thetwenty-seven cytokines/chemokines analyzedU937 cells produce IL-8, IL-9, IL-10, IP-10,MCP-1 (CCL2), RANTES (CCL5), and VEGFat >1,000 pg/ml (IL-8: 1,896� 348.2, IL-9:1,340� 358.0, IL-10: 1,595� 409.5, IP-10:2,254� 689.3, MCP-1 (CCL2): 4,970� 1,607,RANTES(CCL5): 1,560� 465.4, and VEGF:5,061� 819.9 pg/ml; mean�SD) (Fig. 3A). Afterstimulation of U937 cells with IL-4 (10 ng/ml)for 72 h, conditioned media was analyzed forcytokine/chemokine expression. IL-1ra, IL-8,IL-9, IL-10, IP-10, MCP-1 (CCL2), RANTES(CCL5), and VEGF were expressed at >1,000pg/ml and significantly elevated compared to

Fig. 1. Co-inoculation of athymic mice with PC-3 and monocytes. A: PC-3 co-inoculated with normalPBMCs. Tumor volumes were monitored weekly by caliper measurement in PC-3 (-*-), PC-3IL4 (-&-), PC-3þPBMC (-*-) and PC-3þ PBMC7IL4 (-&-). B: PC-3 co-inoculated with U937 cells. Tumor volumes weremonitored weekly by caliper measurement in PC-3 (-&-), PC-3þU937 (-*-) and PC-3þU937IL4 (-&-).C–H: Tumor sections from PC-3 co-inoculation with U937 cells were analyzed by IHC for the presence ofmacrophages (CD68) and morphology (H&E).

4 Craig et al.

U937 cell conditioned media alone (IL-1ra:1,253.45� 122.92, IL-8: 3,352.07� 75.14, IL-9:2,294.06� 170.51, IL-10: 2,547.23� 90.86, IP-10: 3,592.7� 82.87, MCP-1 (CCL2): 7,693.27�37.31, RANTES(CCL5): 2,595.69� 189.42, andVEGF: 8,527.14� 615.28 pg/ml; mean�SD,P< 0.05) (Fig. 3A). PC-3 cells produce high

levels of IL-6, G-CSF, and VEGF and low levelsof CCL2. Based on these findings, we confirmedMCP-1 (CCL2) expression in the xeongrafttumors from PC-3 only tumors as potentialmediators of the increased angiogenesis andtumor growth and compare them to PC-3þU937 and PC-3þU937IL-4 tumors by quantita-tive real time PCR (Fig. 3B). CCL2 mRNAexpression was found to be significantly ele-vated in PC-3þU937 and PC-3þU937IL-4 com-pared to PC3 only or PC-3IL4 tumors (P< 0.01).

Confirmation of the in vivo prostate cancercell proliferation was seen both by histologyand by an in vitro proliferation assay. Stainingof xenografts with Ki67, a cell proliferationmarker, revealed increased cell proliferation inPC-3þU937 and PC-3þU937IL-4 tumors (PC-3<PC-3þU937<PC-3þU937IL-4) (Fig. 4A–C).There was no significant difference observed inthe amount of apoptosis as detected by Apoptagimmunohistochemical staining (Fig. 4D–F).Further, to determine if factors secreted byU937 cells influenced PC-3 cell growth in vitro,PC-3 cells were incubated in the presence ofU937 CM for 72 h. PC-3 cells incubated in U937CM (either unstimulated or IL-4 stimulated)demonstrated a significant increase in pro-liferation compared to the serum free mediacontrol (SFM: 0.94� 0.16, IL4: 0.89� 0.11,U937 CM: 1.644� 0.25, U937IL-4 CM: 1.633�0.32, P< 0.01) (Fig. 4G). Additionally, toaddress the question of prostate cancer cell-dependent regulation of macrophage cell pro-liferation, U937 cells were incubated in PC-3CM. PC-3 CM induced a significant increase inU937 cell proliferation compared to the serumfree control cells (SFM: 0.47� 0.01, PC3 CM:0.75� 0.12, P< 0.001) (Fig. 4H).

DISCUSSION

In order to investigate the role of macrophageinfiltration in prostate cancer growth, weestablished a xenograft model where PC-3prostate cancer cells were co-inoculated witheither normal PBMCs or the U937 premonocyticcell line. U937 cells were originally isolatedfrom the pleural effusion of a patient withhistiocytic lymphoma but are not terminallydifferentiated. The initial experiment (Fig. 1)compared the response of PC-3 prostate cancercells to co-inoculation with normal PBMCs andU937 cells. PC-3 prostate cancer cells respondedsimilarly to normal PBMCs and U937 cells.

Fig. 2. The presence of U937 cells induces tumor angiogenesisin vivo. Tumor vasculature was identified by IHC analysis ofCD31 positive staining (A–C). D: Microvascular density wascounted per 100� objective field from three independent fieldsper tumor. Data is displayed as mean� SD and significance wasdetermined with a *P<0.001 and #P<0.05. E: Hemaglobincontent was measured in tumor specimens using the Drabkin’sreagent and displayed as the amount of hemoglobin (mg/ml) pergram of tissue for each tumor (mean� SD, *P< 0.001 and#P<0.05). F: Tube formation assay on growth factor reducedmatrigel using HDMVEC cells was preformed with condition-ed media from PC-3, PC-3IL4, U937, and U937IL4 conditionedmedia. The number of endothelial sprouts were counted per100� objective field from three independent areas and repeatedin triplicate. Data is displayed as themean� SD and significancewas determined with a *P< 0.001 and #P<0.01.

Macrophages Enhance Prostate Cancer Growth 5

Co-inoculation of PC-3 and PBMCs or U937cells resulted in a significant increase in tumorgrowth in vivo. Prestimulation of monocytesprior to xenograft implantation resulted in afurther increase in tumor growth compared tounstimulated monocytes. Logrank analysis of

time to critical tumor mass (tumors >1,000 mm3)revealed a significant trend (w2¼ 3.842, P<0.05) in tumors co-inoculated with U937 cells.These data support the hypothesis that thepresence of tumor infiltrating monocytes/macrophages promotes prostate cancer growth.

Fig. 3. Stimulation of U937 cells with IL4 induces chemokine expression. A: Analysis of chemokineexpression fromPC-3cells (dashedbars),U937 (openbars), andU937cells stimulatedwith IL-4 (10ng/ml for72 h) (closed bars) was preformed using a multi-plex assay platform. IL-1ra, IL-8, IL-9, MCP1 (CCL2), andVEGF were significantly upregulated (P<0.05) after IL-4 stimulation. B: CCL2 mRNA expression in tumorspecimens isolated from PC-3, PC-3þU937, or PC-3-U937IL4 was confirmed by quantitative real time PCRand normalized to GAPDH levels using the D�CT method.

Fig. 4. The presence of U937 cells stimulated PC-3 growth. The effects of U937 cells on PC-3 cellproliferation were analyzed by IHC for Ki67 staining (A–C) and apoptosis using Apoptag (D–F). G: In vitroanalysis of PC-3 proliferationwas determined byWST-1 assay using IL4 (10 ng/ml), conditionedmedia fromU937 cells or U937IL4 cells. Serum free media (SFM) or serum media (10% FBS) was used as controls(*P<0.01). H: In vitro analysis of U937 proliferation was determined by WST-1 assay using conditionedmedia from PC-3 cells (*P<0.01).

6 Craig et al.

The difference between tumor destructivemacrophages and tumor-promoting macro-phages is not clearly understood; however,several reports have suggested that stimulationof recruited monoctyes with IL-4 induces atumor-promoting phenotype and results inTAM formation [Mantovani et al., 1992]. Priorto co-inoculation, we pre-stimulated U937 cellswith IL-4 (10 ng/ml) for 72 h. Surprisingly, nodifference in overall tumor growth and time tocritical tumor mass was observed betweenuntreated U937 cells and IL-4 treated U937cells. There was a significant increase inactively proliferating cells as evident by Ki67immunohistochemical analysis in xenograftscomprised of PC-3þU937 cells compared toPC-3 cells alone. The presence of TAMs invarious neoplastic tissue has been implicatedin stimulating increased tumor angiogenesis,and this effect on increased tumor vascularity isa prominent feature of tumor-promoting macro-phages. Thus, we assessed the vascular contentof the xenograft tumors. There was a significantincrease in microvascular density (MVD) intumors from PC-3þU937 cells compared to PC-3 cells alone. Further, pre-stimulation of U937cells with IL-4 did not significantly increase theamount of neovascularization found within thexenograft tumors. Interestingly, when condi-tioned media from PC-3 cells, U937 cells, orU937 cells pre-stimulated with IL-4 was usedin an in vitro tube formation assay to mimicneoangiogenesis, a significant increase in thenumber of sprouts (tube formation) was identi-fied in the U937IL-4 conditioned media. Thesedata suggest that in an in vitro model IL-4stimulated U937 cells induce angiogenesis;however, in an in vivo model the role of IL-4may be lost due to additional factors (includingendogenous IL-4) present in the xenografts.

In order to understand the role of IL-4stimulated U937 cells and their potential forinfluencing prostate cancer growth and induct-ing angiogenesis within the tumor bed, wecollected 24 h conditioned media from U937cells and U937IL-4 cells and analyzed cytokine/chemokine expression using the Luminex tech-nology. U937 cell conditioned media containedhigh levels (>1,000 pg/ml) of several cytokines(IL-8, IL-9, IL-10, IP-10, MCP1 (CCL2),RANTES (CCL5), and VEGF). Prior stimula-tion of U937 cells with IL-4 significantlyincreased the levels of IL-1ra, IL-8, IL-9,MCP1 (CCL2), and VEGF. The role of CCL2 in

regulating the migration and proliferation ofcancer epithelial cells has been suggested inbreast cancer and multiple myeloma [Valkovicet al., 1998; Ohta et al., 2002; Vanderkerkenet al., 2002; Lebrecht et al., 2004]. Recently wehave reported that CCL2 is elevated in theprostate cancer tumor microenvironment ofmetastatic lesions [Loberg et al., 2006a] and toinfluence prostate cancer growth in vitro[Loberg et al., 2006a; Lu et al., 2006] howeverit remains unclear as to the mechanism ofincreased CCL2 expression. Currently the roleof monocytes on tumor cell proliferation isunclear but our data demonstrates that mono-cytes, and specifically monocytes stimulatedwith IL-4, increase the neovascularization ofprostate cancer tumors and that CCL2 plays asignificant role in regulating monocyte infiltra-tion and vascularization of prostate neoplasias.In addition, these data suggest that stimulationof U937 cells with IL-4 induces a TAM pheno-type and upregulates several cytokines andchemokines (specifically CCL2) that are impor-tant in prostate cancer tumor growth and thatstimulate angiogenesis within the tumor bed.

CONCLUSION

Although relatively few studies have char-acterized the differential expression of chemo-kines and chemokine receptors found onprostate cancer cells and in the prostate tumormicroenvironment, it is apparent that chemo-kines secreted by infiltrating macrophages playa pivotal role in regulating prostate cancer cellgrowth and metastasis. It is unclear what thepredominant chemokine(s) are in the regulationof prostate cancer growth and metastasis, and ifthey are most influential as direct mediators oftumor cell growth or if their prominent role isregulating the microenvironment within whichthe tumor cells thrive (i.e., angiogenesis, infil-tration of tumor-promoting immune cells, etc)[Lissbrant et al., 2000; Lewis and Pollard,2006]. Lin and Pollard [2004] demonstrated inbreast cancer a positive correlation betweenpoor prognosis and the degree of TAM infilt-ration [Lin and Pollard, 2004]. These datacoupled with our preliminary data demons-trating increased tumor growth with the co-inoculation of macrophages in prostate cancersuggest an important role for chemokines andtheir receptors in prostate cancer metastasisand progression. Further investigations into

Macrophages Enhance Prostate Cancer Growth 7

the mechanisms of chemotaxis and migrationvia chemokine signaling would lead to newtherapeutic strategies for cancer.

ACKNOWLEDGMENTS

This study was supported by SPORE CareerDevelopment Award and NIH (5P50CA069568).

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