neutrophils increase oral squamous cell carcinoma invasion ......research article neutrophils...

Research Article

Neutrophils Increase Oral Squamous CellCarcinoma Invasion through an Invadopodia-Dependent PathwayJudah E. Glogauer1, Chun X. Sun1, Grace Bradley2, and Marco A.O. Magalhaes1,2

Abstract

Neutrophils have recently been shown to promote invasion andcorrelate with a poor prognosis in different cancers, including headand neck squamous cell carcinomas. In this study, we analyze theeffects of neutrophils in the invasion of oral squamous cell carci-noma (OSCC) using a combination of conditioned media, directand indirect coculture of humanperipheral bloodneutrophils, andUMSCC47 cells (OSCC cell line). Invasion andmatrix degradationwere determined using a modified in vitro invasion assay and aninvadopodia assay, respectively. UMSCC47 and neutrophil cocul-tures or conditioned media from cocultures increased UMSCC47

invasion, invadopodia formation, andmatrix degradation. Furtheranalysis revealed an increase in TNFa and IL8 in supernatants ofcocultures compared with neutrophil or UMSCC47 cultures aloneand that inhibition of TNFa and IL8 significantly decreasedOSCC invasion. Our results show that neutrophils increase theinvasiveness of OSCC through the activation of invadopodiaand matrix degradation, suggesting a paracrine activation loopbetween the two cells. Importantly, the presence of neutrophilsin the oral environment may modulate the clinical behavior ofOSCC. Cancer Immunol Res; 3(11); 1218–26. �2015 AACR.

IntroductionThe overall poor clinical outcome of oral squamous cell car-

cinoma (OSCC) is associated with its late detection, local aggres-siveness, and presence of metastatic disease. The precise mechan-isms underlying metastatic spread, however, are not well under-stood. Neutrophils have an important role in cancer biology (1–3), including tumor progression (4, 5), metastasis (6), and extra-cellular traps (NET)-dependent tumor metastasis (7). Althoughthe role of inflammation and the immune system in the progres-sion of cancers has been studied extensively, only recently has itbeen investigated in the setting of OSCC (8–13). Trellakis andcolleagues (11) have shown that neutrophil infiltration of headand neck squamous cell carcinoma (HNSCC) was correlated withpoor clinical outcome, while Wang and colleagues (14) usedtongue squamous cell carcinomas to show that neutrophil infil-trationwas associatedwith lymphnodemetastasis, higher clinicalstage, and increased chances of tumor recurrence. These findingsraised the possibility of a cross-talk and possible costimulation ofcancer cells and neutrophils. In support of this hypothesis, Dumi-tru and colleagues have shown that neutrophils challenged withpharyngeal squamous cell carcinoma cells (FaDu) showed astrong activation of p38/MAPK, CREB, and p27, leading to anincrease in chemotaxis, cell survival, and secretion of CCL4 and

CXCL8. They also observed an increase in expression of MMP9andCCL4byCD66b-positive cells in human tumor sections (15).In a follow-up publication, Dumitru and colleagues (16) haveshown that neutrophils increased cortactin phosphorylation insquamous cell carcinomas, promoting cancer migration. Thisfinding is particularly important because metastatic spread isassociatedwith the formation of specialized subcellular structuresregulated by cortactin phosphorylation called invadopodia (17,18). Invadopodia are dynamic actin-rich subcellular structureswith matrix-degrading machinery found in cancer cells (19).

Here, we investigate the effects of neutrophils on the invasive-ness of OSCCs using a combination of OSCC cell lines (UMSCC1and UMSCC47) and peripheral blood neutrophils. We provideevidence that neutrophils increase the invasiveness of OSCC,independent of direct contact between the two cells. Coculturewith neutrophils increases invadopodia formation and matrixdegradation in cancer cells. A significant increase in invasion,invadopodia formation, and degradation was also seen in cancercells exposed to conditioned media from neutrophils andUMSCC47 cocultures. Further analyses suggest that the neutro-phil-dependent increase in invasion observed is based on aparacrine loop involving TNFa and IL8. Our findings reveal anovel mechanism in which neutrophils can increase the invasive-ness of OSCC through the activation of invadopodia and matrixdegradation.

Materials and MethodsCell lines

UMSCC1 and UMSCC47 cells were from Dr. Thomas Carey(University of Michigan, Ann Arbor, MI). The UMSCC47 cell lineis derived from a 53-year-oldmanwith ametastatic lateral tonguesquamous cell carcinoma (T3N1M0). UMSCC47 has a wild-typeTP53 gene and is positive for HPV16 (20). UMSCC1 is derivedfrom a man with a nonmetastatic floor of mouth squamous cellcarcinoma (21). Both UMSCC1 and UMSCC47 were recently

1Matrix Dynamics group, Faculty of Dentistry, University of Toronto,Toronto, Ontario, Canada. 2Oral Pathology and Oral Medicine, Facultyof Dentistry, University of Toronto, Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Marco A.O. Magalhaes, University of Toronto, 511-124Edward Street, Toronto, ON, M6G1G6, Canada. Phone: 416-979-4920 ext. 4485;Fax: 416-979-4938; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0017

�2015 American Association for Cancer Research.

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shown to represent a unique genetic background and to be free ofcontamination (20). All cell cultures and experiments were per-formed in DMEM supplemented with 10% FBS, 100 nmol/Lnonessential amino acids (Sigma), penicillin, and streptomycin(100 mg/mL). Peripheral blood neutrophils were isolated andpurified from healthy donors as described before (22). Neutrophilviability by Trypanblue exclusion after 24hours of incubationwithcancer cells was 64.2% (�0.04%). The UMSCC1 and UMSCC47viability rateswere 86.4% (�0.04%) and 83.7% (�0.08%), respec-tively, after 24 hours of incubation with neutrophils.

Antibodies and inhibitorsCortactin antibody (Ab3333; mouse monoclonal) was from

Abcam. Tks5 antibody (M-300, rabbit polyclonal) was fromSantaCruz Biotechnology. Rabbit P421 Cortactin antibody was pur-chased from Cell Signaling Technology. Secondary antibodies(goat anti-mouse Alexa Fluor-555 and goat anti-rabbit AlexaFluor-647) were from Life Technologies. Anti-IL8 (ab18672) andTNFa (ab8348) antibodies were from Abcam. GM6001 inhibitorwas from Sigma (M5939), and MMP9 inhibitor I was fromMillipore (1177749-58-4).

Neutrophil conditioned mediaNeutrophil conditioned media (CMN) was collected from a

24-hour cultures of neutrophils at a concentration of 400,000cells/mL. Conditioned media from neutrophils and UMSCC47cocultures (CMC47) was collected from cultures of 800,000neutrophils and 200,000 UMSCC47 in 2 mL of DMEM for 24hours. CMSEP (conditioned media from separated cultures) wascollected from100,000UMSCC47orUMSCC1 cells cultured on a3.0-mm pore Transwell insert, physically separated from 400,000neutrophils in the bottom chamber (total of 1 mL of media). Forall the experiments, the media was centrifuged at 400 � g for5 minutes to remove cells and stored at �80�C until use.

Matrix degradation and invadopodia analysisFifty thousand UMSCC47 cells were plated on Alexa Fluor-488

gelatin matrix-coated Mattek dishes (10 mm) and incubated for24 hours as described previously (23, 24). Where indicated,50,000 (1:1) or 200,000 (4:1) neutrophils were added to theculture. Before fixation, the samples weremonitored using a ZeissPrimo Vert DIC microscope equipped with an AxioCam ERccamera. The cells were fixed in 3.7%PFA for 20minutes, followedby immunostaining. The specimens were blocked in 1% BSA, 1%FBS for 1 hour, and incubated with primary antibodies forCortactin (1:300) and Tks5 (1:50) for 1 hour. After three washes,secondary antibodies anti-mouse Alexa Fluor-555 (1:300) andanti-rabbit Alexa Fluor-647 (1:300)were added to the cells, whichwere incubated for 1 hour at room temperature. Matrix degrada-tion and invadopodia formation were analyzed under spinningdisk confocal microscopy (Quorum spinning disk confocal, LeicaDMIRE2). The number of invadopodia was calculated as thenumber of colocalizing cortactin and Tks5 spots divided by thecell area. The degradation area was calculated as described before(19). Image analysis was completed using the Volocity 6.3 andImageJ 1.46.

Western blot analysisTwo lakh UMSCC47 cells were plated on 6-cm dishes with or

without 800,000 neutrophils for 24 hours. Specimens werelysed at 4�C with Laemmli buffer, sonicated for 5 seconds,

boiled for 10 minutes, and subjected to 8% SDS–PAGE. Mem-branes were blocked and immunoblotted with mouse Cortactin(1:3,000) and rabbit P421 cortactin (1:1,000) in 5% BSA Tris-buffered saline-Tween (TBS-T) at 4�C overnight. Membraneswere washed three times for 10 minutes with TBS-T. All Westernblot analyses were done using the secondary antibodies fromLI-COR (mouse-680 and rabbit-800) and read using the LI-COR Odyssey infrared imaging system. LI-COR Image Studio3.1.4 was used for densitometry analysis. Results are expressedas the ratio between phosphorylated cortactin and total cor-tactin in the same blot.

Transwell invasion assayTranswell assays were performed as described previously (19).

Briefly, 8.0-mm Matrigel-coated Transwell supports from BectonDickson Canada (BD) were used to evaluate cell invasion. Fiftythousand UMSCC47 or UMSCC1 cells were suspended in 500 mLof 10%FBS/DMEMand seeded in theupper chamber. Thebottomchamber was filled with 1mL of 10%FBS/DMEMwith or without1 nmol/L EGF. In experiments with indirect coculture, 50,000(1:1) or 200,000 (4:1) human peripheral blood neutrophils wereadded to the bottom chamber. In direct coculture experiments,neutrophils were added to the upper chamber mixed with thecancer cells. Membranes were equilibrated at room temperaturefor 10 minutes before cells were added and the cells allowed toinvade for 24 hours followed by fixation in 3.7% PFA. Whereindicated, 5 mg/mL of anti-TNFa or 10 mg/mL of anti-IL8 anti-bodies were added to the media 30 minutes before the experi-ment. Intact membranes were stained with Alexa Fluor-488 Phal-loidin (Life technologies) for 30 minutes and visualized in aNikon Eclipse TE300 epifluorescence microscope. The cells at theupper side of the membrane were removed using a Q-tip. Cellinvasionwas calculated as the average cell coverage area inmm2onthe underside of the membrane compared with the UMSCC47control. Results were based on analysis of 20 fields (�20) infive independent experiments. The MMP inhibitor GM6001(25 mmol/L) and MMP9 inhibitor (1 mmol/L) were added asindicated.

ELISAELISA kits forMMP9, EGF, and TNFawere purchased from Life

technologies (Novex) and the human ELISArray kit was custommade fromQIAGEN and included IL6, IL8, IL12, GM-CSF, TNFa,and TGFb. For the ELISA experiments, 200,000 UMSCC47 cellswere cultured in 2 mL of culture media. Where indicated, neu-trophils were added at a 1:1 ratio (200,000) or 4:1 ratio(800,000). The cells were cultured for 24 hours and the super-natants were collected and centrifuged at 400� g for 5minutes toremove cells and stored at �80�C until use. The ELISA experi-ments followed the manufacturer's protocol for each cytokinetested.

Statistical analysisIn experiments containing multiple group analysis, ANOVA

was performed associated with Tukey tests. Where indicated,statistical analysis was calculated using the unpaired, two-tailedStudent t test. Statistical significance was defined as P < 0.05. Forall figures �, P < 0.05; ��, P < 0.01; and ��, P < 0.001. Error barsrepresent the standard error of the mean (SEM).

Neutrophils Increase Oral Carcinoma Invasion

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ResultsNeutrophils increase the invasiveness of OSCC

We have analyzed the invasiveness of UMSCC47 andUMSCC1cells in the presence of human neutrophils using a Transwellinvasion assay. Both UMSCC1 and UMSCC47 cells invadedthrough Matrigel, although significantly more UMSCC47 cellsinvaded compared with nonmetastatic UMSCC1 cells (Supple-mentary Fig. S1A). Figure 1A illustrates the experimental designfor the coculture invasion experiment. As seen in Fig. 1B and C,neutrophils increase the invasion ofUMSCC47 cells and the effectismore significantwith ahigh ratio (4:1)of neutrophils comparedwith a low ratio (1:1). UMSCC47 invasion is completely blockedby the MMP inhibitor GM6001, while the inhibition of MMP9only partially blocks invasion. The experimentwas repeated in thepresence of 1 nmol/L of EGF as a chemoattractant in the bottomchamber, and similar results were observed (Fig. 1D). UMSCC47viability after coculture was 83.7% (� 0.08%), Neutrophils via-bility was 64.2% (� 0.04%) after 24 hours of coculture, support-ing earlier reports showing increased survival of TAN (25, 26).Together, these results show that direct coculture with neutrophilsincreases the invasiveness of cancer cells in the absence of anyother stimulation.

Neutrophil-mediated increase in invasiveness does not requiredirect contact

In order to evaluate whether the increase in invasivenessrequired direct contact between cancer cells and neutrophils,we have used an adapted invasion assay. UMSCC47 cells were

cultured in the upper chamber, whereas neutrophils werecultured in the lower chamber, thereby physically separatingthe neutrophils from the cancer cells (indirect coculture;Fig. 2A). Figure 2B shows that neutrophils significantlyincrease the invasiveness of UMSCC47 cells without directcontact. As seen in Supplementary Fig. S1B, UMSCC1 cellsalso increased invasion in the presence of neutrophils. Theseobservations suggest that soluble secreted cues rather thandirect contacts are necessary for the increase in invasion.GM6001 completely inhibits invasion, while MMP9 inhibitorpartially blocks invasion.

Evidence of a feedback loop between UMSCC47 cells andneutrophils

We have used several variations of conditioned media toevaluate the possible feedback loop established between neutro-phils and cancer cells, including conditioned media from neu-trophils and UMSCC47 cells (CMC47), conditioned media fromna€�ve neutrophils only (CMN), and conditioned media fromneutrophils andUMSCC47 orUMSCC1 cells physically separatedby a Transwell membrane (CMSEP47 and CMSEP1). The condi-tioned media was added to the lower chambers of the invasionassay, as shown in Fig. 3A. CMC significantly increased theinvasiveness of UMSCC47 cells (Fig. 3B). This is in contrast toconditioned media from na€�ve neutrophils (CMN), which failedto increase theUMSCC47 invasiveness. Figure 3C shows that bothCMSEP1 andCMSEP47 increased the invasion ofUMSCC47 cells.Similar results were also observed with UMSCC1 cells (Fig. 3D).

Figure 1.Neutrophils increase UMSCC47invasion. A, experimental design. Theresults are calculated on the basis ofthe area (mm2) occupied by cancercells that invaded through theMatrigel(seen under the membrane). B,representative images of theunderside of the Transwell membrane.C, 50,000 UMSCC47 cells were platedin the upper chamber and 50,000 (1:1ratio) or 200,000 (4:1) neutrophilswere added. Where indicated,GM6001 (25 mmol/L) and MMP9(1 mmol/L) inhibitors were added.D, UMSCC47 invasion in the presenceof an EGF gradient (n ¼ 6, ANOVAP < 0.0001; Tukey test � , P < 0.05;��, P < 0.01; ��, P < 0.001).

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The results show that the neutrophil-mediated increase in inva-siveness depends on a costimulatory pathway between neutro-phils and cancer that does not direct contact between them,suggesting a feedback loop involving secreted cues by cancer cellsand neutrophils.

Neutrophils increase the number of invadopodia in UMSCC47cells

Invadopodia are specialized subcellular structures used bycancer cells to invade surrounding tissues (17). We analyzed thenumber of invadopodia in UMSCC47 cells in the presence of

Figure 2.Increased invasion does not requiredirect contact between neutrophilsand UMSCC47. A total of 50,000UMSCC47 cells were plated in theupper chamber and 50,000 (1:1 ratio)or 200,000 (4:1 ratio) neutrophilswere added to the bottom chamber asdescribed in Materials and Methods.Where indicated, GM6001 (25 mmol/L)and MMP9 (1 mmol/L) inhibitors wereadded. No chemoattractant wasadded to the bottom chamber. Theresults are calculated on the basis ofthe area (mm2) occupied by cancercells that invaded through the Matrigel(seen under the membrane; n ¼ 4,ANOVA P < 0.0001; Tukey test� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Figure 3.Conditioned media from neutrophilsand UMSCC cells increase invasion. A,experimental design. B, 50,000UMSCC47 cells were plated in theupper chamber and conditionedmedia from na€�ve neutrophils (CMN)or UMSCC47 cells and neutrophils(CMC47) were added to the bottomchamber. C, 50,000 UMSCC47 cells(C) or UMSCC1 cells (D) were plated inthe upper chamber, and culture mediafrom UMSCC47 cells and neutrophilscultured separately (CMSEP47), orUMSCC1 cells and neutrophils culturedseparately (CMSEP1), were added tothe bottom chamber. In all theexperiments, cells were cultured for24 hours. The results are calculated onthe basis of the area (mm2) occupiedby cancer cells that invaded throughthe Matrigel (seen under themembrane). Where indicated,GM6001 (25 mmol/L) was added(n¼ 5, ANOVA P < 0.0001; Tukey test� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






neutrophils. As shown in Fig. 4A,UMSCC47 cells shownumerousinvadopodia, identifiedbyTks5 and cortactin colocalization (19).A corresponding dot-like degradation of the matrix is seen inassociation with invadopodia. In neutrophil cultures or neutro-phil and cancer cocultures, an ill-defined superficial degradationarea was seen, corresponding to areas of neutrophil contact withthe matrix (Supplementary Fig. S2A and S2B). Figure 4B showsthat neutrophils significantly increase the number of invadop-

odia and the effect is more pronounced in the 4:1-ratio groupcompared with the 1:1-ratio group. Conditioned media experi-ments showed that CMC47 significantly increased the number ofinvadopodia in contrast to CMN (Fig. 4B). The size of the indi-vidual invadopodium was not changed by the presence of neu-trophils (Fig. 4C). Cortactin phosphorylation is a key regulatorystep of invadopodium maturation. Maturation of invadopodiarequires the phosphorylation of cortactin at residues 421 and 466

Figure 4.Neutrophils increase invadopodiaformation. A, representative images ofUMSCC47 cells plated on a gelatinmatrix. Cells were plated on AlexaFluor-488 gelatin-coated Mattekdishes and incubated for 24 hoursfollowed by immunostaining forcortactin and Tks5. Right, thedegradation of gelatin. Neutrophilsand inhibitors were added asdescribed in Materials andMethods. B,neutrophils increase the formation ofinvadopodia. Where indicated,neutrophils (1:1 and 4:1) andconditioned media (CMN and CMC47)were added to the culture. Thenumber of invadopodia wascalculated as colocalized Tks5 andcortactin spots per field divided by thecell coverage area (The results arenormalized to the 1:1 ratio group). Theaverage size of each invadopodia wascalculated, and results are shown inpanel C. D, 200,000 UMSCC47 cellswere plated on a 6-cm dish andcultured for 24 hours. Whereindicated, 800,000 (4:1) neutrophilswere added to the culture. Cortactinphosphorylationwas calculated as theratio between phosphocortactin andtotal cortactin densitometry signal inthe same blot. Each dot representsone independent experimentnormalized to control (n¼ 4, ANOVA;Tukey tests �� , P < 0.001; Westernblot analysis; n ¼ 5; t tests P < 0.017).

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by a kinase cascade including Src and Arg tyrosine kinases down-stream of EGFR (23). A recent report shows that neutrophilsinduce cortactin phosphorylation in oropharyngeal carcinomacells (16). Considering the current evidence and our observationthat neutrophils increase the number of invadopodia, we testedthe effects of neutrophils on cortactin phosphorylation. As seenin Fig. 4D, neutrophils induced a small increase in the phosphor-ylation of cortactin in UMSCC47 cells compared with control.These findings suggest that the observed increase in invasivenesscould be mediated by an increase in the number of invadopodia.

Neutrophils increase UMSCC47 matrix degradationTumor invasion is a multistep process that requires tumor cells

todegrade the basementmembrane and remodel the surroundingconnective tissue. We have analyzed the ability of UMSCC47 cellsto degrade matrix in the presence of neutrophils or conditionedmedia. Fig. 5 shows that the presence of neutrophils increasedUMSCC47 degradation, particularly at a 1:4 ratio of neutrophilsand cancer cells. Also, similar to the results seen in the invasionexperiments, conditioned media from UMSCC47 and neutro-phils (CMC) increased matrix degradation while conditionedmedia from na€�ve neutrophils (CMN) failed to increasedegradation.

TNFa and IL8 are increased in supernatants of neutrophil andUMSCC47 cocultures

Paracrine stimulation of cancer and immune cells has beenreported in several malignancies, including breast adenocarcino-ma (27).We investigated the possible role of secreted cytokines inthe communication between UMSCC47 and neutrophils. Figure6A shows that cocultures of UMSCC47 and neutrophils at a 4:1ratio increased the secretion of TNFa in the culture media com-pared with neutrophils or UMSCC47 alone. Both UMSCC47 andneutrophils secrete MMP9, but there were no significant additiveeffects in the coculture groups (Fig. 6B). There was no significant

change in the EGF concentration among the experimentalgroups (Fig. 6C). Figure 6D–G show the results from an ELISArrayexperiment, where groups are tested for multiple cytokinessimultaneously. Figure 6D shows that both neutrophils andcancer cells secrete IL8 in the supernatant. When these cells arecultured together, there is a sharp increase in the concentrationof IL8 in the media, suggesting a costimulatory mechanism.UMSCC47 cells also secrete other cytokines, including TGFb (Fig.6E), IL6 (Fig. 6F), and GM-CSF (Fig. 6G). Importantly, TGFb hasbeen associated with a neutrophil protumor phenotype (2).

Neutralization of TNFa and IL8 inhibits neutrophil-inducedincrease in invasion

On the basis of the ELISA results showing an increase in TNFaand IL8 in cocultures, we have used neutralizing antibodiesagainst both cytokines. As seen in Fig. 7A, inhibition of TNFa orIL8 decreased UMSCC47 invasion in the presence of neutrophils.Neutralization of both TNFa and IL8 completely inhibited theobserved neutrophil-induced increase in invasion (Fig. 7B). In theabsence of neutrophils, neutralization of TNFa and/or IL8 had nosignificant effects on UMSCC47 invasion.

DiscussionAn understanding of the mechanisms of invasion and meta-

stasis is essential for improvement of the clinical outcome ofOSCC. Here, we provide evidence that neutrophils in the cancermicroenvironment increase the invasiveness of OSCC. This find-ing is extremely relevant because the oral cavity has a constantpopulation of neutrophils in the saliva and crevicular fluids,recruited as part of the surveillance of pathogens entering throughthe digestive tract and modulation of oral biofilms. In specificinflammatory states, including periodontal disease, there is anincrease in the population of oral neutrophils, and they showdifferent genetic expression compared with those from healthycontrols (28). Because the increase in invasiveness was indepen-dent of direct contact between cancer cells and neutrophils, aneutrophil-rich microenvironment might predispose cancer cellsto a more aggressive/invasive behavior. In that regard, oral neu-trophils may be used as prognostic markers for OSCC, and futurestudies will evaluate this hypothesis. Considering the differencesbetween oral and peripheral blood neutrophils, future studies areneeded to evaluate the effects of oral neutrophils on cancerinvasion compared with the findings reported here.

Both the UMSCC1 and UMSCC47 cell lines used in this studyshowed an increase in invasion in the presence of neutrophils, butthe nonmetastaticUMSCC1 cellswere not used in further analysesbecause there were very few cells invading (Supplementary Fig.S1A). Importantly, these differencesmay also represent variationsin the pathogenesis of the disease because UMSCC47 are HPVþ

cells while UMSCC1 are HPV� carcinoma cell lines. HPVþ oraland oropharyngeal cancers show differences in clinical presenta-tion, including younger age, higher N stage at presentation(lymph node metastasis), and overall improved survival com-pared with HPV� tumors (29). The differences seen in in vitroinvasion in this studymay represent the differences in the biologicand clinical differences betweennonmetastaticUMSCC1 cells andmetastatic UMSCC47 cells. Further studies are needed to clarifyHPVþ and HPV� OSCC behavior in detail.

Here, we show for the first time that neutrophils increase thenumber of invadopodia and matrix degradation by oral cancer

Figure 5.Neutrophils and CMC47 increase UMSCC47 matrix degradation. UMSCC47cells were plated on Alexa Fluor-488 gelatin-coated Mattek dishes andincubated for 24 hours. Where indicated, protease inhibitors (GM6001 andMMP9 inhibitor), neutrophils (1:1 and 4:1), and conditioned media (CMN andCMC47) were added to the culture. Degradation was measured as theaverage area of detectable loss of fluorescence per field normalized to the 1:1ratio group. Results are based on four independent experiments (ANOVAP < 0.001; Tukey test � , P < 0.05).






cells. Invadopodia have been identified in numerous invasivecancer cell lines (30), and evidence shows that invadopodia areassociated with invasion and metastasis (31, 32). These special-ized subcellular structures are activated by cortactin phosphory-lation downstream of EGFR stimulation through the activation ofSrc and Arg kinases and combine focal degradation of the ECMwith an actin-dependent protrusion (19, 23).Cortactin is requiredfor invadopodia formation (33), and it converges different path-ways leading to cancer invasion. The increase in cortactin phos-phorylation reported here is similar to earlier observations byDumitru and colleagues (16) in oropharyngeal cancers. Thechanges in matrix degradation induced by neutrophils are notas robust as the overall changes in invasion observed in theTranswell assays or invadopodia formation. This finding has twopossible explanations: The thin matrix degradation assay usedhere analyzes one aspect of invadopodia activation, which is the

localized recruitment of active MMPs to invadopodia. Becausethis is a thinmatrix assay, it is very sensitive but lacks the ability ofanalyzing three-dimensional characteristics or stability of theinvadopodia. The other explanation is that the function of inva-dopodia in invasion also includes actin-dependent protrusiveforces, elongation, and adhesion, which could also be affectedby neutrophils in our model. Thus, the degradation results mustbe analyzed in conjunction with the increase in number ofinvadopodia and cortactin phosphorylation, which collectivelyshows that neutrophils promote an invasive phenotype. It is alsolikely that the effects of neutrophils in OSCC involve not onlyincreased invadopodia formation and matrix degradation butalso changes in protein expression, motility, and cytoskeletonremodeling beyond invadopodia.

Our results point to a new paracrine activation loop betweenOSCC and neutrophils involving TNFa and IL8. A significant

Figure 6.Neutrophil and UMSCC47 cocultureslead to increased secretion of TNFaand IL8. A total of 200,000 UMSCC47cells were plated on a 6-well plate andcultured for 24 hours. Whereindicated, 200,000 (1:1) or 800,000(4:1) neutrophils were added to theculture. The "media-only" group hadno cells in culture. A, concentration ofTNFa in the supernatant (n ¼ 3,ANOVA P < 0.0162; Tukey test�� , P < 0.001). B, concentration ofMMP9 in culture supernatants.C, concentration of EGF in culturesupernatants. D, quantification of IL8in the supernatant using QIAGENELISArray, results are expressed aspercentage of positive control.E, quantification of TGFb in thesupernatant. F, quantification of IL6 inthe supernatant. G, quantification ofGM-CSF in the supernatant.

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increase was observed in both TNFa and IL8 concentration insupernatants of neutrophil and UMSCC47 cocultures. In addi-tion, neutralization experiments inhibited neutrophil-inducedincreases in invasion but did not affect UMSCCbaseline invasion.TNFa has been shown to induce activation of EGFR through theprocessing enzyme ADAM17 (TACE) by releasing EGF ligands(34) Takamune and colleagues (35) usedOSCC cell lines to showthat TNFa meditated the maturation of ADAM17 resulting inincreased invasion. ADAM17 is involved in the cleavage andshedding of TNFa, EGFR ligands, and other proteins with atransmembrane ectodomain (36). ADAM17 is upregulated indifferent malignancies, including breast, ovarian, and colon can-cers (37), and has been shown to increase expression of EGFR innon–small lung cancer cells (36). IL8 is a potent neutrophilchemoattractant and primer (38). We propose that the increasedIL8 secretion recruits and primes neutrophils, which, in turn,secrete TNFa, leading to the activation of cancer cells throughADAM and EGFR ligands. In addition, the secretion of TGFb byUMSCC47 contributes to a protumor neutrophilic phenotype asreported previously (39). These findings support our experimentsshowing that conditioned media from na€�ve neutrophils did notincrease invasion, while conditioned media from cancer cells andneutrophils cultured directly or indirectly (CMC47, CMSEP47and CMSEP1) increased invasion. This is the first direct evidenceof neutrophils and OSCC establishing reciprocal stimulationneeded to increase the invasion of OSCC. Further studies areneeded to clarify the specific mechanism underlying the neutro-phil effects on the cancer cells, includingwhichother pathways areactivated in addition to the activationof invadopodia shownhere.

In our experiments, MMP9 inhibition partially blocked theinvasion and matrix degradation. Considering that both cellsproduce and secrete MMP9, there are two possible explanationsfor this. The first explanation is that neutrophil-secreted MMP9directly remodels the matrix and facilitates invasion. This isunlikely because neutrophils did not invade through Matrigel orattach to thematrix.Neutrophil-associated degradation presentedas a shallow, round area corresponding to the outline of neu-trophils (Supplementary Fig. S2B). Neutrophils are washed awayduring fixation and therefore cannot be seen in the fixed samples.In addition, cancer cells migrate during the 24 hours of theexperiment and can be seen close to or on top of neutrophildegraded areas. The degradation "dots" caused by cancer cells arestill visible under the neutrophil-mediated degradation, confirm-

ing the superficial nature of neutrophil degradation. Efficientinvasion requires coordinated degradation as seen in the dot-likepattern typical of invadopodia-induced degradation. The secondand more realistic possibility is that both UMSCC47-derivedMMP9 at the invadopodia and neutrophil-secreted MMP9 canbe used by cancer cells to promote invasion and focal matrixdegradation. MMP9 can also indirectly signal to cancer cells bymetabolizing other molecules, including TGFb, IL1b, and TNFa(40). In our model, MMP9 inhibition partially blocked invasionand degradation but did not significantly change the concentra-tion of TNFa or EGF in the supernatant (data not shown). Furtherstudies will help clarify the role of MMP9 in oral cancer invasion.

In summary, our findings describe a novel mechanism linkingneutrophils to OSCC invasion. This may represent a feedbackloop between these two cells and a link between inflammatorystates characterized by neutrophil infiltration and poor prognosisof squamous cell carcinoma.

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

Authors' ContributionsConception and design: M.A.O. MagalhaesDevelopment of methodology: C.X. Sun, M.A.O. MagalhaesAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.E. Glogauer, C.X. Sun, G. Bradley, M.A.O.MagalhaesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.E. Glogauer, C.X. Sun, M.A.O. MagalhaesWriting, review, and/or revision of the manuscript: J.E. Glogauer, G. Bradley,M.A.O. MagalhaesStudy supervision: M.A.O. Magalhaes

Grant SupportThis work is supported by the Department of Oral Pathology and Oral

Medicine of the Faculty of Dentistry, University of Toronto, a Dental ResearchInstitute grant to G. Bradley. M.A.O. Magalhaes is supported by the JaventheySoobiah Scholarship and the Heidi Sternbach Scholarship, Postgraduate Med-ical Education, Faculty of Medicine.

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

Received January 16, 2015; revised May 22, 2015; accepted June 16, 2015;published OnlineFirst June 25, 2015.

Figure 7.IL8 and TNFa are required forneutrophil-induced UMSCC47increase in invasion. A, 50,000UMSCC47 cells were plated in theupper chamber and 200,000 (4:1ratio) neutrophils were added to thebottom chamber. B, UMSCC47invasion in the absence of neutrophils.Where indicated, anti-IL8 (10 ng/mL)andanti-TNF (5 ng/mL)were added tothe media 30 minutes before theexperiment. Cell invasion wascalculated as the average cellcoverage area under the membraneafter 24 hours of incubation (n ¼ 3,ANOVA, P < 0.0162; Tukey test�� , P < 0.01).






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2015;3:1218-1226. Published OnlineFirst June 25, 2015.Cancer Immunol Res Judah E. Glogauer, Chun X. Sun, Grace Bradley, et al. through an Invadopodia-Dependent PathwayNeutrophils Increase Oral Squamous Cell Carcinoma Invasion

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