The Immune-microenvironment Confers Chemoresistance of ... · Biology of Human Tumors The Immune-microenvironment Confers Chemoresistance of Colorectal Cancer through Macrophage-Derived

Biology of Human Tumors

The Immune-microenvironment ConfersChemoresistance of Colorectal Cancer throughMacrophage-Derived IL6Yuan Yin1, Surui Yao1, Yaling Hu1,2, Yuyang Feng1, Min Li1, Zehua Bian1,Jiwei Zhang1, Yan Qin3, Xiaowei Qi3, Leyuan Zhou4, Bojian Fei5, Jian Zou2,Dong Hua1,6, and Zhaohui Huang1

Abstract

Purpose: Tumor-associatedmacrophages (TAMs) are frequent-ly associatedwith poor prognosis in human cancers.However, theeffects of TAMs in colorectal cancer are contradictory.We thereforeinvestigated the functions, mechanisms, and clinical significanceof TAMs in colorectal cancer.

Experimental Design: We measured the macrophage infil-tration (CD68), P-gp, and Bcl2 expression in colorectal cancertissues using IHC staining. Coculture of TAMs and colorectalcancer cells both in vitro and in vivomodels was used to evaluatethe effects of TAMs on colorectal cancer chemoresistance.Cytokine antibody arrays, ELISA, neutralizing antibody, andluciferase reporter assay were performed to uncover the under-lying mechanism.

Results: TAM infiltration was associated with chemoresistancein patients with colorectal cancer. Colorectal cancer–conditionedmacrophages increased colorectal cancer chemoresistance andreduced drug-induced apoptosis by secreting IL6, which could

be blocked by a neutralizing anti-IL6 antibody. Macrophage-derived IL6 activated the IL6R/STAT3 pathway in colorectalcancer cells, and activated STAT3 transcriptionally inhibited thetumor suppressor miR-204-5p. Rescue experiment confirmedthat miR-204-5p is a functional target mediating the TAM-induced colorectal cancer chemoresistance. miR-155-5p, a keymiRNA regulating C/EBPb, was frequently downregulated inTAMs, resulting in increased C/EBPb expression. C/EBPb tran-scriptionally activated IL6 in TAMs, and TAM-secreted IL6then induced chemoresistance by activating the IL6R/STAT3/miR-204-5p pathway in colorectal cancer cells.

Conclusions: Our data indicate that the maladjusted miR-155-5p/C/EBPb/IL6 signaling in TAMs could induce chemo-resistance in colorectal cancer cells by regulating the IL6R/STAT3/miR-204-5p axis, revealing a new cross-talk betweenimmune cells and tumor cells in colorectal cancer microenviron-ment. Clin Cancer Res; 23(23); 7375–87. �2017 AACR.

IntroductionCancer development and progression are complex processes

that are not only caused by accumulated genetic modifications incancer cells but are also influenced by the surrounding microen-vironment. Cancer cells recruit vasculature and stroma (includingimmune cells, fibroblasts, cytokines, and the extracellular matrixthat surrounds them) to the tumormicroenvironment (TME), andthe activated TME in turn modifies the malignant behaviors of

cancer cells (1). Numerous studies have indicated that infiltratingimmune cells in TME fail to execute antitumor functions butinteract intimately with the tumor cells to promote oncogenesisand progression. The innate immunity cells and cells of theadaptive immune are both involved in the cross-talk (2).Tumor-associated macrophages (TAMs) are one of the mostabundant types of cells in TME, directly affecting tumor progres-sion in many cases (3).

Colorectal cancer is the third most common cancer world-wide (4). Systemic chemotherapy is one of the standard treat-ments for colorectal cancer. However, many patients withcolorectal cancer do not respond to conventional chemother-apy due to drug resistance. Chemoresistance, including inher-ent and acquired drug resistance, is considered to be mediatedby multiple factors, such as drug inactivation, accelerated drugefflux, and alterations in the target cells (5). Recent studies havesuggested that immune cells in TME play important roles inmediating acquired drug resistance and confer resistance tophysiologic mediators of cell death (6, 7). For example, Shreeand colleagues showed that macrophages protect breast cancercells from cell death induced by taxol, etoposide, and doxo-rubicin (8). In advanced non–small cell lung cancer, a highFox3pþ/CD8þ T-cell ratio is associated with a poor responseto platinum-based chemotherapy (9). Ding and colleaguesrevealed that targeting CD4þ T cells constitutes a new type of

1Wuxi Cancer Institute, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu,China. 2Department of Clinical Laboratory Science, Wuxi People's Hospital ofNanjing Medical University, Wuxi, P.R. China. 3Department of Pathology, Affil-iated Hospital of Jiangnan University, Wuxi, Jiangsu, China. 4Department ofRadiation Oncology, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu,China. 5Department of Surgical Oncology, Affiliated Hospital of Jiangnan Uni-versity, Wuxi, Jiangsu, China. 6Department of Medical Oncology, AffiliatedHospital of Jiangnan University, Wuxi, Jiangsu, China.

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

Corresponding Author: Zhaohui Huang, Affiliated Hospital of Jiangnan Univer-sity, 200 Huihe Road, Wuxi, Jiangsu 214062, China. Phone/Fax: 8651-0886-82087; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1283

�2017 American Association for Cancer Research.

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immunotherapy to increase therapeutic efficacy (10). In colo-rectal cancer, the localization and density of immune cells inTME are also associated with response to chemotherapy (11).The presence of high numbers of TAMs was also reported to beassociated with poor clinical outcome in breast, pancreatic,bladder, ovarian, and gastric cancers (12–16). These reportssuggest that immune cells, especially macrophages in TME,may play key roles in modulating biological phenotypes ofcancer cells. In colorectal cancer, contradictory conclusionsabout the effects of TAMs on patient prognosis have beenreported (17–22), and the detailed roles of TAMs in colorectalcancer chemoresistance are largely unclear.

In this study, we observed that the macrophage infiltration inhuman colorectal cancer tissues is associated with chemoresis-tance and poor prognosis. Further functional and mechanisticstudies revealed that maladjusted miR-155-5p/C/EBPb/IL6 sig-naling in TAMs induce the chemoresistance in colorectal cancercells by regulating the IL6R/STAT3/miR-204-5p axis, revealing anew cross-talk between immune cells and tumor cells in colorectalcancer TME. These findings might contribute to insight concern-ing TME and the poor response of colorectal cancer cells toconventional chemotherapy.

Materials and MethodsCell culture and treatment

Four human colorectal cancer cell lines (DLD1, HCT-8,HT-29, and LoVo), human embryo intestinal mucosa cellsCCC-HIE-2, human monocyte cell line THP-1, mouse colorec-tal cancer cell line CT26.WT, and macrophage cell lineRAW264.7 were purchased from ATCC. All of the cell lineswere obtained during 2008 to 2014 and cultured following theinstructions recommended by ATCC as we described previously(23). Cells were confirmed to be mycoplasma free and passagedno more than 18 to 25 times after thawing. Cell lines were

characterized by Genewiz Inc. using short tandem repeatmarkers (last tested in 2017). THP-1 cells were treated with200 nmol/L phorbol 12-myristate 13-acetate (Sigma) for24 hours to differentiate into adhered macrophages. In someexperiments, colorectal cancer cells were treated with BSA or25 ng/mL IL6 (Novoprotein) for 3 days; the media werereplaced with serum-free media and the cells were harvested24 hours later.

For coculture experiments, macrophages (5 � 105) wereplaced into the lower chamber in 12-well plate and colorectalcancer cells (5 � 105) were added into the upper chamber of atranswell insert with a 0.4-mm pore size (Millipore). In someexperiments, cocultures were treated with neutralizing anti-bodies to IL6 (anti-IL6; Abcam) at 1:400 following the man-ufacturer's instructions. The colorectal cancer–conditionedmacrophage cells were washed with PBS and added with freshserum-free media. Then, 24 hours later, conditioned media(CM) were collected and filtered with a 0.22-mm filter.

Clinical samplesTumor tissues from 81 patients with colorectal cancer were

obtained from the Affiliated Hospital of Jiangnan University(Wuxi, Jiangsu, China; Supplementary Table S1). These caseswere followed up for more than 5 years. The samples weregathered with informed consent according to the InstitutionalReview Board of Ethical Committee–approved protocol.

IHCIHC staining was performed on 4-mm sections of paraffin-

embedded tissue samples to detect the protein expression levels ofCD68, P-gp, Bcl2, IL6, and RAB22A. In brief, the slides wereincubated in anti-CD68 (1:200, Santa Cruz Biotechnology),anti-P-gp (1:200, Santa Cruz Biotechnology) antibodies, anti-Bcl2 (1:100, Santa Cruz Biotechnology), C/EBPb (1:200, Abcam),anti-IL6 (1:600, Santa Cruz Biotechnology), STAT3 (1:500, CellSignaling Technology), pSTAT3 (1:200, Cell Signaling Techno-logy), and anti-RAB22A (1:200, Proteintech) at 4�C overnight.The subsequent steps were performed using the EnVision FLEXHigh pH 9.0 Visualization System (DAKO). All slides were inde-pendently evaluated by two pathologists without the knowledgeof the patients' clinical information. The staining intensity wasvisually scored and stratified (score 0-3) as described previously:negatively stained0 (�),weakly stained1 (þ),moderately stained2 (þþ), and strongly stained 3 (þþþ; refs. 23–26). Additionalmethods are described in Supplementary Materials andMethods.

RNA isolation and qRT-PCRTotal RNAwas extracted from tissues or cells using RNAiso Plus

(TaKaRa). The concentrations of RNA were determined using aNanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthe-sized using HiFiScript cDNA Synthesis Kit (CWBIO). qRT-PCRanalyses were conducted to quantitate the relative mRNA expres-sion using UltraSYBR Mixture (CWBIO), with b-actin as aninternal control. Stem-loop qRT-PCR assays using TaqManmiRNA probes (Applied Biosystems) were performed to quantifythe levels of mature miRNAs. The primers used are listed inSupplementary Table S2.

Plasmid constructs and siRNAThe human pri-miR-155 sequence was amplified from normal

human genomic DNA and cloned into the lentivirus expression

Translational Relevance

Tumor-associatedmacrophages (TAMs) are frequently asso-ciated with poor prognosis in many types of human cancers.However, the effects of TAMs in colorectal cancer are contra-dictory. We now present data showing that TAM infiltration incolorectal cancer tissues was associated with chemoresistancein patients with colorectal cancer. Macrophages increasedcolorectal cancer resistance to chemotherapeutical agents andreduced drug-induced apoptosis by secreting IL6 and activat-ing the IL6R/STAT3/miR-204-5p pathway in colorectal cancercells. Decreased miR-155-5p expression was observed inTAMs, which was associated with increased activity of theC/EBPb/IL6 signaling, and restoring miR-155-5p expressionin macrophages could reverse the macrophage-induced che-moresistance in colorectal cancer cells. Together, maladjustedmiR-155-5p/C/EBPb/IL6 signaling in TAMs could inducechemoresistance in colorectal cancer cells by regulating theIL6R/STAT3/miR-204-5p axis, revealing a novel cross-talkbetween immune cells and tumor cells in the colorectal cancermicroenvironment, and therapeutic miR-155-5p overexpres-sion in TAMs appears to be a new therapeutic strategy toimprove chemotherapeutic efficacy in colorectal cancer.

Yin et al.

Clin Cancer Res; 23(23) December 1, 2017 Clinical Cancer Research7376




vector pWPXL to generate pWPXL-miR-155. The promoter regionof IL6 containing the potential C/EBPb-binding sites was ampli-fied from human genomic DNA using PrimerSTAR Premix(TaKaRa) and was then cloned into the region directly up-stream of modified firefly luciferase cassette in a pGL3 LuciferaseReporter Vector (p-Luc). Duplex siRNAs were purchased fromGenePharma.

Lentivirus production and transductionThe pWPXL-GFP or pWPXL-miR-155 plasmid was cotrans-

fected into HEK-293T cells along with the packaging plasmidpsPAX2 and the envelope plasmid pMD2G using Lipofecta-mine 2000 (Invitrogen) as we described previously (27). Virusparticles were harvested 48 hours after cotransfection and wereindividually used to infect THP-1 cells. The cells were thenharvested at 3 days after infection for Western blotting andqRT-PCR validation.

Tumor formation in mouseCT26.WT cells were subcutaneously injected into the left arm-

pits of male BALB/c mouse at 5 weeks of age with or withoutRAW264.7 cells. Daily bolus doses of 5-fluorouracil (5-FU) at 25mg/kg were given intraperitoneally for two 4-day cycles 10 daysafter the injection. The mice were sacrificed after a period of 21days and examined for the growth of subcutaneous tumors. Allanimal care and handling procedures were performed in accor-dance with the NIH's Guide for the Care and Use of LaboratoryAnimals and were approved by the Ethical Committee of Affili-ated Hospital of Jiangnan University.

Assessment of drug sensitivity and apoptosisColorectal cancer cells were treated with 5-FU or oxaliplatin

(LOHP; range, 0–100 mg/mL), and cell inhibition was thenassessed by a CCK-8 assay (Dojindo). The IC50 was calculated.For the apoptosis analysis, colorectal cancer cellswere treatedwith5 mg/mL 5-FU or LOHP for 48 hours. These cells were thenharvested and subjected to apoptosis analysis using anAnnexin V-FITC and Propidium Iodide Labeling Kit (CWBIO).

Analysis of cytokine profile in culture mediumAHuman Cytokine Antibody Arrays Kit (RayBiotech) was used

to detect cytokines in the culture medium according to themanufacturer's instructions. Briefly, the arrays were incubatedwith 100 mL of medium, biotin-conjugated antibodies, and aStreptavidin-Fluor–linked secondary antibody one by one for 2hours. The glass chip was scanned with a laser scanner (Axon,Genepix) using a Cy3-compatible (green; 532 nm) laser. Quan-titative array analysis was performed using GenePixPro6.0(Axon).

ELISAELISA development reagents (duo-set kit) for human IL6 and

mouse IL6 were purchased from ExCell Bio, and the assay wasperformed according to the manufacturer's instructions. Absor-bance was measured using a microplate reader (HIDEX).

Western blottingProtein extracts were probed with antibodies against human

phospho-STAT3 (Tyr705) (1:1,500, CST), STAT3 (1:1,000, Pro-teintech), IL6R (1:500, Santa Cruz Biotechnology), RAB22A

(1:1,000, Proteintech), P-gp (1:1,000, Santa Cruz Biotechnology)antibodies, Bcl2 (1:1,000, Santa Cruz Biotechnology), C/EBPb(1:1,000, Abcam), or b-actin (1:2,000, Abcam). Peroxidase-con-jugated anti-mouse or rabbit antibody (The Jackson Laboratory)was used as a secondary antibody and the antigen–antibodyreactionwas visualized by an enhanced chemiluminescence assay(CWBIO).

Luciferase reporter assayTHP-1–differentiated macrophages were cultured in 96-well

plates and cotransfected with 50 nmol/L of miR-155-5p mimic,CEBPB siRNA (siCEBPB), 50 ng of luciferase reporter, and 10 ngof pRL-CMV Renilla luciferase reporter using Lipofectamine2000 (Invitrogen). Forty-eight hours after transfection, theluciferase activities were assayed using a Luciferase Assay Kit(Promega).

FISH and immunofluorescence stainingmiRCURY LNA Detection probe for hsa-miR-155-5p

obtained from TSINGKE labeled with FAM (488 nm, green)at the 50 and 30 terminus. In brief, preliminary hybrid liquid wasadded to cover slides at 37�C for 1 hour. Then, a total of 100 mLdiluted probe was added to each slide, and slides were kept inthe hybridization chamber and incubated in oven at 37�Covernight. Posthybridization washes were performed and coun-terstained with DAPI. For immunofluorescence staining, serialsections of the same specimens were incubated with CD68antibodies labeled with Cy3 (red, eBioscience). All images werecaptured on an Olympus fluorescence microscope equippedwith vision software.

Statistical analysesThe data were expressed as the mean � SEM and were

subjected to the Student t test or the Mann–Whitney U testunless otherwise specified (c2 test or a Spearman correlation).Cox proportional hazards regression analysis was used to cal-culate HRs and the 95% confidence intervals. A value of P < 0.05was considered statistically significant. The SPSS 16.0 packagewas used for the statistical analyses.

ResultsInfiltration of macrophage in human colorectal cancertissues

In our previous colorectal cancer cohort studies, differentoverall survival (OS) times have been observed in stage IIIpatients who received surgery and standardized chemotherapy(23, 24). Patients with poor survival seemed to be resistant tothe chemotherapy. We supposed that the TME, especially theinfiltration of macrophages, may be associated with the che-moresistance and prognosis of colorectal cancer. To assess po-tential effects of macrophage infiltration on the chemoresis-tance of colorectal cancer, we assessed the status of TAMs incolorectal cancer tissues. First, we selected 65 patients withstage III colorectal cancer who received radical operations andwere treated with 5-FU, L-OHP, and leucovorin from thecolorectal cancer cohort reported previously (23, 24). Thirty-seven living patients with an OS longer than 60 months werelabeled as chemotherapy sensitive (sensitive group), and 28deceased patients with OS shorter than 24 months wereregarded as chemotherapy resistant (resistant group). IHC

Macrophages Induce Chemoresistance in Colorectal Cancer

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Yin et al.





staining of CD68 (the marker of human macrophage) wasperformed to measure macrophage infiltration (CD68þ) inthese tumor tissues. Stronger macrophage infiltration (CD68scores 2 or 3) was frequently observed in the resistant groupcompared with the sensitive group and was associated withpoor survival (P < 0.05, Fig. 1A; Supplementary Table S3;Supplementary Fig. S1A). Second, to confirm the correlationof macrophage infiltration to drug resistance, we measured theexpression of MDR1 (P-gp), a key marker of multidrug resis-tance (MDR), and the apoptosis maker BCL2 (Bcl2) in thesetumor tissues. The results of qRT-PCR revealed significantupregulation of MDR1 and BCL2 in the resistant group com-pared with the sensitive group (Supplementary Fig. S1B). IHCstaining of P-gp and Bcl2 proteins showed the same trend (Fig.1B and C; Supplementary Table S4 and S5). In addition, aschemotherapy usually induces acquired chemoresistance ofcancer cells, the surviving cancer cells after chemotherapy oftendisplay MDR phenotypes (28). Therefore, we further assessedthe macrophage infiltration and P-gp/Bcl2 expression intumors from 16 patients with colorectal cancer receivingneoadjuvant chemotherapy (NCT, neoadjuvant group). Asexpected, these tumors did show stronger macrophage infil-tration and the expression of P-gp and Bcl2 than the Sensitivetumors (Fig. 1A–C). Moreover, the expression levels of CD68,P-gp, and Bcl2 are relatively higher in the postresection speci-mens compared with their corresponding untreated biopsies(Supplementary Fig. S1C). Taken together, these results sug-gest that TAMs are associated with chemoresistance and poorsurvival in colorectal cancer.

Colorectal cancer–conditioned macrophages induce in vitroand in vivo chemoresistance in colorectal cancer

To further confirm the relationship between TAMs anddrug resistance in colorectal cancer, a tumor formation assay ina BalB/C mouse model was performed. The mouse macrophagecells RAW264.7 preincubated with mouse colorectal cancer cellsCT26.WT for 3 days were used to mimic TAMs in human colo-rectal cancer tissues. As shown in Fig. 1D, CT26.WT cells (controlgroup) or CT26.WT mixed with RAW264.7 (pre-cocultured; 1:1;coinjection group) were injected into flanks of BalB/C mice. Tendays after injection, daily bolus doses of 5-FU at 25 mg/kg wereadministered intraperitoneally to each mouse for two 4-daycycles. The tumors of the coinjection mice were more resistantto 5-FU therapy than those of the control mice (Fig. 1D), indi-cating that TAMs promote in vivo chemoresistance in colorectalcancer.

To mimic the effects of TAMs on drug resistance in vitro, thehuman monocyte cell line THP-1 was induced into macrophagesand then coculturedwith different colorectal cancer cell lines (HT-

29, LoVo, DLD1, or HCT-8) for 3 days. Subsequently, thesecolorectal cancer cells were subjected to drug sensitivity andapoptosis analysis. The results showed that the IC50s of coculturedcolorectal cancer cells were significantly higher than those of theircorresponding control cells (P < 0.01; Fig. 1E; Supplementary Fig.S2A), and coculture with macrophages significantly increased theresistance to chemotherapeutic agents (5-FU and LOHP) andreduced drug-induced apoptosis in colorectal cancer cells (Fig.1F; Supplementary Fig. S2B). Taken together, these results suggestthat TAMs strongly increase resistance of colorectal cancer cells tochemotherapeutic drugs.

Colorectal cancer–conditioned macrophages induce thechemoresistance of colorectal cancer by secreting IL6

To explore how TAMs increase the in vitro chemoresistance ofcolorectal cancer cells, we treated colorectal cancer cells withCM from the colorectal cancer–conditioned macrophages. AfterCM treatment, the colorectal cancer cells exhibited increasedchemoresistance compared with the control cells (Supplemen-tary Fig. S3A), suggesting that some secreted factors in TAMscould affect colorectal cancer cells. Given the key signalingtransduction role of cytokines between different types of cellsin TME, we speculated that TAMs promote chemoresistance bysecreting certain cytokines. Therefore, we measured the cyto-kine profiles of CM from macrophages cocultured with colo-rectal cancer cells (HT-29) or embryo intestinal mucosa cells(CCC-HIE-2), and three cytokines (IL6, IL1b, and MCP2) weresignificantly altered in the CM from the macrophages cocul-tured with HT-29 compared with the macrophage control(Fig. 2A). Of the three cytokines, IL6 emerged as the mostprominently upregulated and abundant cytokine. Therefore,IL6 was selected for subsequent analyses. An ELISA assayfurther confirmed the increase of IL6 in the CMs from macro-phages cocultured with four different colorectal cancer cells(Fig. 2B), and increased IL6 staining was also observed in thedrug-resistant tumors and the tumor tissues receiving NCT(Fig. 2C; Supplementary Table S6).

To evaluate whether IL6 is critical for chemoresistance incolorectal cancer, an exogenous recombinant IL6 was added inthe culture medium of several colorectal cancer cell lines. Theresults showed that IL6 significantly increased the resistance ofthese colorectal cancer cells to chemotherapeutic drugs (5-FU andLOHP) and reduced drug-induced apoptosis (Fig. 2D and E;Supplementary Fig. S3B and S3C). In addition, the mRNA levelsof IL6R, MDR1, and BCL2 were also upregulated in IL6-treatedcolorectal cancer cells (Fig. 2F).

To further investigate whether colorectal cancer–conditionedmacrophages affected chemoresistance in colorectal cancerthrough IL6, the drug sensitivity and apoptosis were assessed

Figure 1.Macrophage infiltration is associated with chemoresistance in colorectal cancer. A–C, IHC staining was performed on tumor tissues from 65 patients with stage IIIcolorectal cancer and 16 patients receiving neoadjuvant chemotherapy (neoadjuvant group) to detect the expression levels of CD68 (A), P-gp (B), and Bcl2protein (C). Of the 65 patients, 37 living patients with good prognosis labeled as chemotherapy sensitive (sensitive group) and 28 deceased patients withpoor prognosis were regarded as chemotherapy resistant (resistant group). D, A tumor formation assay in a BalB/C mouse model. A total of 106 CT26.WT cells(control group, n ¼ 8) or CT26.WT mixed with 106 RAW264.7 (pre-cocultured; 1:1; coinjection group, n ¼ 8) were injected subcutaneously into the right flankof each nude mouse. Ten days after the injection, daily bolus dose of 5-FU at 25 mg/kg was given intraperitoneally for two 4-day cycles. E, CCK-8 assayswere performed to calculate the IC50 of LoVo or HT-29 cells cocultured with macrophages, and corresponding colorectal cancer cells without the coculturewere used as controls. IC50s were labeled on the curve. LOHP, oxaliplatin. F, LoVo or HT-29 cells cocultured with macrophages were treated with 5 mg/mL 5-FUor LOHP for 48 hours. The cells were then harvested and subjected to apoptosis analysis. � , P < 0.05; �� , P < 0.01.






in colorectal cancer cells cocultured with macrophages and ananti-IL6 neutralizing antibody. The results showed that theaddition of neutralizing anti-IL6 antibody to the coculturesystem suppressed the macrophage-induced chemoresistancein colorectal cancer cells (Fig. 3A and B; Supplementary Fig.S4A and S4B). In addition, the macrophage-induced upregula-tion of IL6R, MDR1, and BCL2 in colorectal cancer cells wasalso inhibited by the anti-IL6 antibody (Fig. 3C). Collectively,these data indicate that colorectal cancer–conditioned macro-phages induce chemoresistance in colorectal cancer cells bysecreting IL6.

Colorectal cancer–conditioned macrophages modulatecolorectal cancer chemoresistance by inhibiting miR-204-5p

To decipher themolecular mechanism thatmediates the effectsof macrophage-secreted IL6 on the chemoresistance of colorectalcancer, IL6R and STAT3, key mediators of IL6 signaling, wereanalyzed in colorectal cancer cells. The results showed that IL6Rexpression and STAT3 activation (Tyr705 phosphorylation) wereconsiderably enhanced in colorectal cancer cells cocultured withmacrophages, which could be blocked by a neutralizing anti-IL6antibody (Fig. 4A). We further detected the levels of STAT3 andpSTAT3 in colorectal cancer tissues and revealed that the pSTAT3

Figure 2.

Macrophages modulate the colorectal cancer chemoresistance through IL6. A, Cytokine array analysis on the conditioned media (CM) from the macrophagescocultured with CCC-HIE-2 or HT-29. The bottom left corner is a table summarizing the relative signal intensity of the indicated cytokines. B, IL6 levelswere detected in the CM of macrophages cocultured with various colorectal cancer cells by ELISA assay. C, IHC staining of IL6 was performed onclinical colorectal cancer tissues described in Fig. 1. D, CCK-8 assays were performed to evaluate the effect of IL6 on the sensitivity (IC50) of LoVo orHT29 cells to 5-FU or LOHP. BSA was used as control. IC50s were labeled on the curve. E, The effect of IL6 on the 5-FU or LOHP-induced apoptosis incolorectal cancer cells. LoVo or HT-29 cells were treated with 5 mg/mL 5-FU or LOHP for 48 hours. F, Relative mRNA expression of IL6R, BCL2, and MDR1in colorectal cancer cells treated with IL6. � , P < 0.05; �� , P < 0.01.

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staining is stronger in the resistant and neoadjuvant groups thanin the sensitive group (Fig. 4B). Activated Stat3 can bind to threeputative STAT-binding sites located in the TRPM3/miR-204 pro-moter region, leading to the transcriptional repression ofmiR-204(29, 30). Moreover, our previous study revealed that miR-204-5pincreases the sensitivity of colorectal cancer cells to 5-FU andLOHP (23, 24). We speculated that miR-204-5p may be thekey functional target of macrophage-secreted IL6 in colorectalcancer cells. The qRT-PCR results did reveal that miR-204-5p wassignificantly downregulated in the Resistant tumors and thetumors receiving NCT (Fig. 4C). The downregulation of miR-204-5p was also observed in colorectal cancer cells coculturedwith macrophages as well as in IL6-treated colorectal cancer cellscompared with the control cells (Fig. 4D).

We previously reported that miR-204-5p enhances the chemo-therapeutic sensitivity in colorectal cancer cells by inhibitingRAB22A and Bcl2 (23, 24). As expected, in contrast to thedecreased expression of miR-204-5p in the drug-resistant tumorsand IL6-treated colorectal cancer cells, colorectal cancer–condi-tioned macrophages or IL6 could induce RAB22A and Bcl2upregulation in colorectal cancer cells, which could be reversedby IL6 blocking or ectopic miR-204-5p expression (Fig. 4E and F).IHC staining of RAB22A performed on clinical colorectal cancertissues further confirmed these in vitro results (Fig. 4B; Supple-mentary Table S7). We further observed that ectopic miR-204-5pexpression could reverse macrophage-induced chemoresistancein colorectal cancer cells (Fig. 4G; Supplementary Fig. S5), sug-gesting that macrophage/IL6 could induce MDR via miR-204-5p.

Figure 3.

Colorectal cancer–conditioned macrophages induce colorectal cancer chemoresistance by secreting IL6. A, CCK-8 assays were performed to calculatethe IC50 of LoVo or HT-29 cells cocultured with macrophages and treated with an anti-IL6 neutralizing antibody. IC50s were labeled on the curve.B, LoVo or HT-29 cells cocultured with macrophages and an anti-IL6 neutralizing antibody were treated with 5 mg/mL 5-FU or LOHP for 48 hours. Thecells were then harvested and subjected to apoptosis analysis. C, Relative mRNA expression of IL6R, BCL2, and MDR1 in colorectal cancer cells coculturedwith macrophages and treated with an anti-IL6 neutralizing antibody. �, P < 0.05; �� , P < 0.01.






In addition, ectopic miR-204-5p expression could not block themacrophage-induced upregulation of P-gp in colorectal cancercells, indicating a miR-204-5p–independent manner (Fig. 4F).Taken together, these results imply that TAM-secreted IL6 mod-ulates colorectal cancer chemoresistance partly by inhibitingmiR-204-5p signaling.

miR-155-5p inhibits the macrophage-induced colorectalcancer chemoresistance by influencing IL6 secretion inmacrophages

We previously demonstrated that miR-155, an miRNA down-regulated in TAMs and M2 macrophages, is a critical moleculecontrolling the macrophage phenotypic switch, and miR-155–modified TAMs can be reprogrammed and regain antitumorcapacity (31). Interestingly, we observed that miR-155-5p levelswere markedly lower in the macrophages cocultured with colo-rectal cancer cells than in their corresponding control macro-phages (Fig. 5A). Functional analysis revealed that ectopic miR-155 expression (Fig. 5B) significantly reverses colorectal cancerchemoresistance induced by these colorectal cancer–conditionedmacrophages, suggesting a key role of miR-155 in the TAM-induced chemoresistance in colorectal cancer (Fig. 5C; Supple-mentary Fig. S6A).

We have previously reported that miR-155-5p promotes theM1 polarization of macrophages by suppressing the C/EBPb(CAAT/enhancer-binding protein b) signaling cascade (31), andC/EBP-binding motifs have been identified in the promoterof IL6 (32). We speculated that decreased miR-155-5p signalingresult in increased IL6 production in TAMs. As expected,

colorectal cancer–conditioned macrophages increased C/EBPbexpression, which could be blocked by miR-155-5p over-expression (Fig. 5D). A negative association was also observedbetween miR-155-5p and C/EBPb expression in TAMs (Fig. 5E).Moreover, restoring miR-155-5p expression significantly in-hibited IL6 expression and secretion in colorectal cancer–conditioned macrophages (Fig. 5F). To further validate theregulation of the miR-155-5p/C/EBPb signaling on the IL6expression in macrophages, the promoter region of IL6 contain-ing the C/EBP-binding motifs was cloned into a luciferasereporter plasmid, and luciferase assays showed that transfectionof miR-155-5p or siCEBPB could inhibit the IL6 promoter-derived luciferase expression in macrophages (Fig. 5G; Supple-mentary Fig. S6B), indicating that miR-155-5p inhibits IL6expression by targeting C/EBPb in macrophages. Consequently,the miR-204-5p expression was significantly increased, whereasIL6R, MDR1, and BCL2 were downregulated in colorectal cancercells cocultured with miR-155–overexpressing macrophages(Fig. 5H). Moreover, we used peripheral blood monocytes(PBM) from healthy donors and TAMs isolated from humancolorectal cancer tissues to confirm the miR-155-5p/C/EBPb/IL6 axis. LPS and IFNg were used to induce inflammatorymacrophages from PBMs (IPBMs). The results were in accor-dance with those in colorectal cancer–cocultured macrophages/TAMs (C/EBPb and IL6 are upregulated and miR-155-5p isdownregulated in TAMs). Interestingly, miR-155-5p is alsoupregulated in LPS-induced IPBM, suggesting that LPS mayregulate macrophage activation via a mechanism different fromthat in tumor microenvironment (Supplementary Fig. S7).

Figure 4.

Macrophages modulate colorectal cancer chemoresistance through inhibiting miR-204-5p. A, Western blot analyses of STAT3, p-STAT3, and IL6R incolorectal cancer cells cocultured with macrophages and treated with an anti-IL6 neutralizing antibody. B, IHC staining of STAT3, p-STAT3, andRAB22A in clinical colorectal cancer tissues described in Fig. 1. C and D, Relative miR-204-5p expression in clinical colorectal cancer tissues (C)described in Fig. 1 and in colorectal cancer cells cocultured with macrophages and treated with IL6 (D). E, Western blot analysis of P-gp, Bcl2, and RAB22A incolorectal cancer cells treated with IL6, or in colorectal cancer cells cocultured with macrophages and treated with an anti-IL6 neutralizing antibody.F, Ectopic miR-204-5p expression inhibited RAB22A and Bcl2 expression in colorectal cancer cells cocultured with macrophages. G, CCK-8 assayswere performed to evaluate the effect of miR-204-5p on the drug sensitivity of LoVo or HT29 cells cocultured with macrophages. IC50s were labeledon the curve.

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Collectively, these data indicate that decreased miR-155-5pexpression in TAMs results in C/EBPb overexpression, whichactivated IL6 transcription and led to increased expression andsecretion of IL6.

miR-155-5p overexpression reverses the TAM-inducedcolorectal cancer chemoresistance in mice

To further test the regulation of miR-155 on the TAM-inducedcolorectal cancer chemoresistance in vivo, we stably overexpressedmiR-155 in mouse macrophages (RAW264.7-miR-155) usinglentivirus transduction (Fig. 6A). Consistent with the results inhuman cell lines, both C/EBPb and IL6 levels were significantlydecreased in RAW264.7-miR-155 cells compared with the controlcells (Fig. 6A). Consequently, the expression of IL6R, BCL2, andMDR1 were downregulated, and miR-204-5p expression wasincreased in colorectal cancer cells (CT26.WT) coincubated withRAW264.7-miR-155 macrophages compared with the controlcells (Fig. 6B). Then, CT26.WT cells mixed with RAW264.7-GFPor RAW264.7-miR-155 (1:1) were injected into the flanks of BalB/C mice and treated with 5-FU. As shown in Fig. 6C and D, thexenograft tumors of CT26.WT/RAW264.7-miR-155 were moresensitive to the 5-FU therapy than the CT26.WT/RAW264.7 con-trol. IHC staining in these tumors also confirmed the inhibitoryeffects of miR-155 overexpression in macrophages on the expres-sion of P-gp, Bcl2, and RAB22A in colorectal cancer cells (Fig. 6E).Taken together, these data demonstrate that stably overexpressingmiR-155 in TAMs could reverse the TAM-induced colorectalcancer chemoresistance.

DiscussionRecent data revealed that the malignant behaviors of cancer

cells are regulated by other types of cells in TME, especiallyimmune cells. MDR is a major reason for chemotherapy failure,resulting in tumor metastasis and relapse. In this study, we foundthat increased macrophage infiltration in colorectal cancer tissueswas significantly associated with increased chemoresistance andpoor prognosis. Further studies revealed that TAM-derived IL6induces chemoresistance in colorectal cancer cells by partly reg-ulating the IL6R/STAT3/miR-204-5p pathway, uncovering a newcross-talk between TAMs and colorectal cancer cells.

Clinical observations have shown that high macrophage infil-tration is generally associated with poor prognosis in humancancer, including breast, gastric, prostatic, pancreatic, ovarian, andcervical carcinomas (12–16). Interestingly, contradictory resultshave also been reported in several types of human cancers,including colorectal cancer (17–22). In colorectal cancer, anincreased infiltration of TAMs was associated with lymph nodemetastases and tumor progression (19, 21), and M2 macrophageinfiltration was associated with poor prognosis in colorectalcancer (20, 22). However, increased macrophage infiltration wasalso reported to correlate with improved survival in colorectalcancer (17, 18).Gutmacrophages are highly plastic cells, and theirphenotype is dependent on the local microenvironment in colo-rectal cancer (33). It is difficult to establish a standard assay tomeasure TAMs indifferent tumors,whichmaypartly explain theseinconsistencies in colorectal cancer. Despite the inconsistentresults concerning the prognostic role of TAMs in colorectalcancer, recent data showed that TAMs exert tumor-promotingfunctions by regulating cell growth, metastasis, and drug resis-tance in colorectal cancer (34–38). Because the phenotypes of

TAMs are highly plastic and associated with tumor stage and theirlocation in TME (33, 39, 40), we measured the total macrophageinfiltration other than M1 or M2 macrophage in representativepatients with colorectal cancer to investigate its clinical signifi-cance. We observed that increased macrophage infiltration isassociated with poor survival and increased expression of P-gpand Bcl2 in colorectal cancer cells, suggesting the key role of TAMson chemoresistance.

Chemotherapy is a standard treatment for most patients withcolorectal cancer; however, many patients did not respond tochemotherapeutic agents due to chemoresistance. Chemoresis-tance is usually due to two types of factors: intrinsic factors withinthe colorectal cancer cells themselves and extrinsic factors in theTME (5). TAMs could mediate resistance to antitumor drugs invarious ways. It has been reported that TAMs suppress CD8þ Tlymphocyte activity and then promote chemoresistance to pacli-taxel and metastasis in mammary tumors (41). Angst and col-leagues showed that mononuclear cell–derived IL1b conferschemoresistance in pancreatic cancer cells by upregulating cyclo-oxygenase-2 (42). These studies highlight the key roles of macro-phages in chemoresistance. Macrophages constitute one of themajor populations of cells that reside in the colorectal cancer TME(43). As increasedmacrophage infiltration is associatedwith poorsurvival in our study, in vitro and in vivo experiments furtherconfirmed the phenomenon observed in clinical colorectal cancertissues, demonstrating the key regulatory role of TAMs on theMDR of colorectal cancer. Consistent with our results, a recentstudy showed that macrophages could induce 5-FU resistance incolorectal cancer by releasing putrescine (35).

To explore the interaction between TAMs and colorectalcancer cells, we treated colorectal cancer cells with CM fromTAMs. Consistent with the changes in the expression of MDR orapoptosis-related genes, the TAM-derived CM-stimulated colo-rectal cancer cells exhibited drug-resistant behaviors, suggestingthat TAMs secrete a factor that affects the chemoresistance ofcolorectal cancer cells. Given the key role of cytokines in cell–cell interactions, we screened the changes of the secretorycytokine profile in the CM from colorectal cancer–conditionedmacrophages and identified IL6 as the most significantly upre-gulated cytokine. Subsequent functional assays confirmed thatIL6 is accountable for the TAM-induced chemoresistance incolorectal cancer.

As a key proinflammatory factor, IL6 is also implicated in theregulation of tumorigenesis, progression, and MDR (44–47). IL6can induce MDR in human cancer cells by complicated mechan-isms, such as increasing expression of MDR-related genes (MDR1and GSTP1) and apoptosis-inhibitory proteins (Bcl-2, Bcl-xL, andXIAP), and activation of AKT, ERK, MAPK, and STAT3 signaling(47, 48). Here, we revealed that by binding with its specific ligandIL6R, IL6 subsequently phosphorylates STAT3 and leads to che-moresistance in colorectal cancer. We had previously reportedthat miR-204-5p could significantly inhibit cell growth, metasta-sis, and chemoresistance in colorectal cancer cells (23, 24). In thisstudy, we further validated that miR-204-5p expression in TAM-conditioned colorectal cancer cells was significantly inhibited bymacrophage-secreted IL6, whereas RAB22A and BCL2, confirmedtarget genes of miR-204-5p, were highly expressed in these colo-rectal cancer cells. Mechanistic studies revealed that activatedSTAT3 by macrophage-derived IL6 transcriptionally inhibitedthe expression of miR-204-5p. These data uncovered a newmechanism mediating decreased expression of miR-204-5p






Yin et al.





in colorectal cancer andprovide anewmechanismbywhichTAMsinduce chemoresistance in colorectal cancer. Interestingly,a recent study reported that miR-204 targets IL6R andsensitizes epithelial ovarian cancer cells to cisplatin, whichpartly explained the upregulation of IL6R in colorectal cancercells cocultured with macrophages or treated with IL6 in ourstudy (49).

MDR1 is often upregulated in chemoresistant tumor cells,and colorectal cancer–conditioned macrophages could induceMDR1 expression by secreting IL6 in a miR-204-5p–indepen-dent manner, suggesting the complicated multidimensionalregulatory mechanisms of MDR by TAM-derived IL6. Consis-tent with our results, Wang and colleagues reported that auto-crine production of IL6 confers chemoresistance in ovariancancer cells by activating several pathways and genes, includingMDR1 and BCL2 (48). Autocrine production of IL6 could also

confer MDR in breast cancer cells by inducing C/EBP andMDR1expression (50). On the basis of our data and previous studies,we propose a working model for TAM-induced chemoresis-tance in colorectal cancer (Fig. 6F). The detailed mechanism bywhich TAMs induce MDR1 expression in colorectal cancershould be investigated in future work.

TAMs are an important area of research for the effectivetreatment of tumors. Our previous study demonstrated thatmiR-155 is a critical molecule controlling the macrophagephenotypic switch and that miR-155–modified TAMs can bereprogrammed and regain tumor-killing capacity. In this study,we revealed that miR-155-5p was frequently downregulated incolorectal cancer–associated macrophages, which resulted inincreased C/EBPb expression. C/EBPb transcriptionally activat-ed IL6 in TAMs and finally induced chemoresistance by acti-vating the IL6R/STAT3/miR-204-5p pathway in colorectal

Figure 6.

Stably overexpressing miR-155 could reverse the macrophage-induced colorectal cancer chemoresistance. A, Relative expression of miR-155-5p,IL6, and C/EBPb in the miR-155–overexpressing RAW264.7 cells. B, Relative expression of miR-204-5p, IL6R, MDR1, and BCL2 in CT26 cellscocultured with the miR-155–overexpressing RAW264.7 or the control cells. C and D, miR-155–overexpressing macrophages sensitize 5-FUtherapy in colorectal cancer in a mouse model. A total of 106 CT26.WT cells (control group, n ¼ 8) or CT26.WT mixed with 106 RAW264.7(pre-cocultured; 1:1; coinjection group, n ¼ 8) were injected subcutaneously into the right flank of each nude mouse. Ten days after the injection,daily bolus dose of 5-FU at 25 mg/kg were given intraperitoneally for two 4-day cycles. E, H&E and IHC staining were performed on the implantedtumors to detect the expression levels of RAB22A, P-gp, and Bcl2 protein. F, A working model for the TAM-induced chemoresistance incolorectal cancer. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Figure 5.miR-155-5p reversed the macrophage-induced colorectal cancer chemoresistance by inhibiting IL6 secretion in macrophages. A, Relative miR-155-5pexpression in macrophages cocultured with colorectal cancer cells. B, Relative expression of miR-155-5p in macrophages stably expressing miR-155.C, CCK-8 assays were performed to calculate the IC50 of LoVo or HT-29 cells cocultured with the miR-155 overexpressing macrophages or the controlmacrophages. IC50s were labeled on the curve. D, The protein expression of C/EBPb in miR-155–overexpressing macrophages and in macrophagescocultured with colorectal cancer cells. E, The expression of miR-155-5p, CD68, and C/EBPb was detected in colorectal cancer samples described in Fig. 1using the in situ hybridization assay, immunofluorescence, and IHC staining, respectively. F, Relative miR-155-5p and IL6 expressions were detected inthe miR-155–overexpressing TAMs by qRT-PCR. G, Dual reporter assay was performed in macrophages transfected with miR-155-5p mimic andrecombinant plasmids containing the IL6 promoter reporter constructs. H, Relative expression of miR-204-5p, IL6R, MDR1, and BCL2 in colorectal cancercells cocultured with the miR-155–overexpressing macrophages or the control macrophages. �� , P < 0.01.






cancer cells. Restoration of the expression of miR-155-5p inTAMs reversed the TAM-induced chemoresistance in colorectalcancer cells. Thus, restoring miR-155-5p expression in TAMsmay provide a new therapeutic strategy to reverse MDR incolorectal cancer at the TME level.

In conclusion, we revealed a new cross-talk betweenimmune cells and tumor cells in the colorectal cancer micro-environment. The maladjusted miR-155-5p/C/EBPb/IL6 sig-naling in TAMs could induce chemoresistance in colorectalcancer cells by regulating the IL6R/STAT3/miR-204-5p axis.TAM-targeted regulation may represent a promising therapeu-tic strategy to improve chemotherapeutic efficacy in colorectalcancer.

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

Authors' ContributionsConception and design: Y. Yin, Z. HuangDevelopment of methodology: Y. Yin, Z. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Yin, S. Yao, Y. Hu, Y. Feng, M. Li, Y. Qin, X. Qi,L. Zhou, B. Fei, Z. Huang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Yin, Z. Bian, L. Zhou, Z. HuangWriting, review, and/or revision of the manuscript: Y. Yin, J. Zou, D. Hua,Z. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Zou, Z. HuangStudy supervision: Z. HuangOther (i.e., evaluating scores of the IHC staining): J. Zhang

Grant SupportThis study was partially supported by grants from the National Natural

Science Foundation of China (81672328, 81772636, 81301784, and81272299), Natural Science Foundation of Jiangsu Province (BK20151108and BK20150004), Fundamental Research Funds for the Central Universities(NOJUSRP51619B and JUSRP51710A), Medical Key Professionals Programof Jiangsu Province, Medical Innovation Team Program of Wuxi, andHospital Management Centre of Wuxi (YGZXZ1401).

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

ReceivedMay 4, 2017; revised August 16, 2017; accepted September 11, 2017;published OnlineFirst September 19, 2017.

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2017;23:7375-7387. Published OnlineFirst September 19, 2017.Clin Cancer Res Yuan Yin, Surui Yao, Yaling Hu, et al. Colorectal Cancer through Macrophage-Derived IL6The Immune-microenvironment Confers Chemoresistance of

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