ComplementPathwayIsFrequentlyAlteredinEndometriosis and ... · pelvic inﬂammation in endometriosis and endometriosis-associated ovarian cancer (EAOC). Experimental Design: RNA was

Biology of Human Tumors

Complement Pathway Is Frequently Altered in Endometriosisand Endometriosis-Associated Ovarian Cancer

Swati Suryawanshi1, Xin Huang1, Esther Elishaev2,3, Raluca A. Budiu1, Lixin Zhang1, SungHwan Kim4,Nicole Donnellan3, Gina Mantia-Smaldone5, Tianzhou Ma4, George Tseng4, Ted Lee3, Suketu Mansuria3,Robert P. Edwards1,3, and Anda M. Vlad1

AbstractPurpose: Mechanisms of immune dysregulation associated with advanced tumors are relatively well

understood. Much less is known about the role of immune effectors against cancer precursor lesions.

Endometrioid and clear-cell ovarian tumors partly derive from endometriosis, a commonly diagnosed

chronic inflammatory disease. We performed here a comprehensive immune gene expression analysis of

pelvic inflammation in endometriosis and endometriosis-associated ovarian cancer (EAOC).

Experimental Design: RNA was extracted from 120 paraffin tissue blocks comprising of normal

endometrium (n ¼ 32), benign endometriosis (n ¼ 30), atypical endometriosis (n ¼ 15), and EAOC

(n ¼ 43). Serous tumors (n ¼ 15) were included as nonendometriosis-associated controls. The immune

microenvironment was profiled using Nanostring and the nCounter GX Human Immunology Kit,

comprising probes for a total of 511 immune genes.

Results: One third of the patients with endometriosis revealed a tumor-like inflammation profile,

suggesting that cancer-like immune signaturesmay develop earlier, in patients classified as clinically benign.

Gene expression analyses revealed the complement pathway as most prominently involved in both

endometriosis and EAOC. Complement proteins are abundantly present in epithelial cells in both benign

and malignant lesions. Mechanistic studies in ovarian surface epithelial cells from mice with conditional

(Cre-loxP) mutations show intrinsic production of complement in epithelia and demonstrate an early link

between Kras- and Pten-driven pathways and complement upregulation. Downregulation of complement

in these cells interferes with cell proliferation.

Conclusions: These findings reveal new characteristics of inflammation in precursor lesions

and point to previously unknown roles of complement in endometriosis and EAOC. Clin Cancer Res;

20(23); 6163–74. �2014 AACR.

IntroductionThe immunological makeup of the host can protect

against cancer development via complex mechanisms com-

monly referred to as "immune surveillance" (1). For certaintumors, transition to malignancy occurs via precursorlesions, where complex changes in immune surveillancelead to chronic inflammation (2). While tumor–immunecell interactions have been long studied and are now welldeciphered, much less known are the roles of immuneeffectors against cancer precursor lesions in general andovarian cancer precursors in particular. Ovarian epithelialtumors are highly heterogeneous and may arise from dif-ferent premalignant lesions, according to their histology(3). High-grade serous ovarian tumors represent the mostcommon histologic subtype and, to some extent, they areconsidered to originate in the fallopian tubes (4, 5). Endo-metrioid, clear-cell, and low-grade serous tumors mayderive, at least in part, from endometriosis, a chronicinflammatory disease that can affect approximately 10%to 15% of women in the reproductive age group and 20%to 50% of women with infertility (6, 7). Endometriosisconsists of endometrial-like tissue (comprising glandularepithelia and stromal cells) outside the uterine cavity and isoften treated through a combination of hormone therapyand surgical removal of lesions (8).

1Department of Obstetrics, Gynecology and Reproductive Sciences, Uni-versity of Pittsburgh School of Medicine and Magee Women's ResearchInstitute, Pittsburgh, Pennsylvania. 2Department of Pathology, Universityof Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 3MageeWomen's Hospital of Pittsburgh, University of Pittsburgh MedicalCenter, Pittsburgh Pennsylvania. 4Department of Statistics University ofPittsburgh, Graduate School of Public Health, Pittsburgh Pennsylvania.5Section of Gynecologic Oncology, Department of Surgical Oncology, FoxChase Cancer Center, Philadelphia, Pennsylvania.

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

S. Suryawanshi and X. Huang contributed equally to this article.

Corresponding Author: Anda M. Vlad, Department of Obstetrics, Gyne-cology and Reproductive Sciences, University of Pittsburgh, School ofMedicine and Magee Women's Research Institute, Room B403, 204 CraftAvenue, Pittsburgh PA 15213. Phone: 412-641-2985; Fax: 412-641-6156;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1338

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 6163

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Published OnlineFirst October 7, 2014; DOI: 10.1158/1078-0432.CCR-14-1338

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Although endometriosis remains largely benign, malig-nant transformation may occur in up to 1% of cases, mostcommonly from ovarian lesions (7, 9, 10). Gene profilingstudies recently revealed that clear-cell and low-gradeendometrioid carcinomas share a similar gene expressionpattern, consistent with a common origin. As a prevailingtrait, inactivating mutations of ARID1A, a chromatin-remodeling gene, have been found in 49% of clear-cellcarcinomas and 30% of endometrioid ovarian cancers(11–13).

It is estimated that 60% to 80% of all endometriosis-associated ovarian cancers (EAOC) occur in the presence ofatypical endometriosis (AE), often found in direct conti-nuity with the tumor, suggesting AE as the transitioningentity from benign lesions to malignant variants. The inter-mediate, "atypical" lesions, are defined by several histologiccriteria, including large nuclei with moderate to markedpleomorphism, increased nuclear-to-cytoplasmic ratio, cel-lular crowding, stratification, or tufting (14, 15).

EAOC are believed to be a culmination of a multifacetedcomplex of pathogenic factors (16). In conjunction withendocrine imbalance and oxidative stress, immune dysre-gulation is a major factor that contributes to disease path-ogenesis (17). Inwomenwith endometriosis, the peritonealfluid often shows increased levels of proinflammatorymediators like TNFa, IL1b, and IL6 (18). In addition, thepatients show dysfunctional macrophages, depressed kill-ing capacity of NK cells, increased accumulation of regula-tory T suppressor cells all of which may favor chronicinflammation and promote the initiation and progressionof EAOC (18, 19).Notably, the vastmajority of studies havepreviously focused on profiling immunity in patients withendometriosis by either measuring cellular phenotypes or

soluble mediators in the peritoneal fluid or peripheralblood (20).

Here, we focus on the tissue immune microenviron-ment and provide the first comprehensive immune tran-scriptome profiling of tissues from 120 cases comprisingnormal controls, benign endometriosis, AE, and EAOCcases. With a broad collection of 511 immune geneprobes, we identified gene expression changes associatedwith each disease state. One of the immune pathwaysenriched for differentially expressed genes in all diseasecategories was the complement pathway. Complementproteins were significantly upregulated in epithelial cells.Using a novel, ovarian surface epithelium (OSE)-derivedcell line from genetically engineered mice with condi-tional mutations that trigger EAOC, we demonstrate thatactivation of Kras and Pten tumor-driving pathways leadsto upregulation of complement in epithelial cells. Theseresults reveal for the first time the link between tumor-initiating events and immune surveillance via comple-ment, and point to this pathway as a potential target fortherapy and early prevention in EAOC.

Materials and MethodsEthics statement

This research study protocol was approved by the Insti-tutional Review Board (IRB) at the University of Pittsburgh,(Pittsburgh, PA).

PatientsWe accessed n ¼ 120 formalin-fixed paraffin-embedded

(FFPE) tissue samples, through the Health ScienceTissue Databank at the University of Pittsburgh MedicalCenter (UPMC, Pittsburgh, PA). The FFPE tissue blockscomprised of normal endometrium (n ¼ 32, equally dis-tributed between secretory and proliferative stage), benignendometriosis (n¼ 30) consisting of ovarian endometriosis(n¼11), and extra ovarian endometriosis cases (n¼19), AE(n ¼ 15), clear-cell tumors (n ¼ 12), and endometrioidovarian cancer (n ¼ 16). Serous tumors (n ¼ 15) wereincluded as nonendometriosis-associated controls.

RNA extraction and NanostringThe RNeasy FFPE Kit (Qiagen) was used to isolate total

RNA from FFPE tissues. The nCounter GX Human Immu-nology Kit (Nanostring) was used to profile 511 immunegenes. Genes in this kit belong to several pathways such asinflammatory disease, cell to cell signaling, cellular devel-opment, cell death, and hematologic system development.Additional details are included in Supplementary Materialsand Methods.

Immunohistochemistry and immunocytochemistryImmunohistochemical studies were performed in FFPE

tissue with the following antibodies: anti-human C7,C3, C4b, MASP1, CFH, CFD, membrane attack complex(MAC). Detailed protocols, clone identification, antibodydilutions, and image acquisition are included in Supple-mentary Materials and Methods.

Translational RelevanceEndometrioid and clear-cell ovarian cancers are partly

derived from endometriosis, a chronic inflammatorydisease that often shows signs of local invasion, treat-ment resistance, and recurrence.Majority of studies havepreviously focused on profiling immunity in endome-triosis patients by either measuring cellular phenotypesor solublemediators in the peritoneal fluid or peripheralblood. We performed here a comprehensive tissueimmune gene expression analysis in lesions of endome-triosis and endometriosis-associated ovarian cancer(EAOC). Some of the patients with endometriosisrevealed a cancer-like inflammation profile, suggestingthat cancer-like immune signatures may develop earlier,in patients with lesions that are classified as clinicallybenign. Gene expression analyses revealed the comple-ment pathway as most prominently involved in bothendometriosis and EAOC. Our studies provide a novelinsight into immune dysregulation in endometriosisand EAOC and reveal complement as a potential targetfor prevention and treatment.

Suryawanshi et al.

Clin Cancer Res; 20(23) December 1, 2014 Clinical Cancer Research6164




Reverse transcriptase quantitative PCRTwo micrograms of purified RNA was used for reverse

transcription using the iScript cDNA Synthesis Kit(Bio-Rad). Quantitative reverse transcriptase PCR (qRT-PCR) was carried out with the CFX96 Touch Real-TimePCR Detection System (Bio-Rad) using the SsoAdvancedSSO SYBR Green Supermix (Bio-Rad). Detailed protocolsare provided in Supplementary Materials and Methods.

Establishment of the murine MUC1þ/�KrasG12D/þ

PtenloxP/loxP (MKP) OSE cell lineMKPOSE cells were derived in Anda Vlad’s laboratory at

MageeWomen’s Research Institute, from primary OSE cellsisolated from healthy MKP mice (21) via gentle trypsiniza-tion of six ovaries collected at necropsy from three healthyfemale mice, according to previously published protocols(22) and as further detailed in SupplementaryMaterials andMethods. Authentication was performed through genomicPCR of MKPOSE cells for the presence of loxP cassettes atthe (murine-specific) Kras and Pten loci before and afterAdCre infection, as shown in Supplementary Fig. S2. Thisverification approach was performed for each experimentdescribed herein.

Complement-mediated cell lysisA total of 5�105AdCre infected anduninfectedMKPOSE

cells were incubated with 90 mL mouse ascites and 5 mL ofanti-MUC1 antibody (50 mg/mL) and 15 mL of DMEMmedium. Either reagents alone or cells in serum-free medi-um served as controls. As positive controls for lysed cells, weincubatedMKPOSE cells with 0.2%Triton for 15minutes at37�C. Number of live and dead cells were measured afterstaining with either 7-Aminoactinomycin-D (7-AAD) orpropidium iodide (BD Biosciences), via flow cytometry.Similarly treated, normal mouse splenocytes served as con-trol for complement-induced cell lysis.

siRNA transfectionCommercially available ON-TARGETplus siRNA con-

sisting of mixture of four mRNA regions directed againstmouse C7 or mouse Gapdh were used (Thermo ScientificDharmacon RNAi Technologies). Transfection methodswere according to the manufacturer’s instructions andare further detailed in Supplementary Materials andMethods.

nCounter data preprocessingExperiments were compliant with nCounter mRNA

Expression Assay (http://www.genetics.pitt.edu/forms/nCounter_Gene_Expression_Data_Analysis_Guidelines.pdf) and the complete datasets are available in the GeneExpression Omnibus database under accession numberGSE57545. To minimize the impact of detection anom-alies, we normalized the dataset to the positive controlcount values (with sum of positive control counts).Subsequently, we applied negative control normaliza-tion (negative control mean plus two SDs). Finally, wefiltered out samples with a positive control normaliza-

tion factor outside the recommended range of 0.3 to 3 orwith an estimated background >3 SDs from the mean.

Statistical analysesTo identify differentially expressed genes between any

twopatient categories, we used the generalized linearmodelwith negative binomial distribution family for countresponse data. Once negative binomial models were fittedand dispersion estimates were obtained, we performedtesting procedures for determining differential expressionusing an exact test with the quantile-adjusted conditionalmaximum likelihood (qCML) method. For a paired group,we evaluated the average effect of disease type over involv-ing patients. All statistical programming was implementedin R, using the edgeR package (23).

For hierarchical clustering, we first filtered out featureswith means or SDs under the 50th quantile of all thefeatures. The filtering procedure left 100 expressed (largemeans) and informative (large SDs) features, based onwhich hierarchical clustering with complete linkage wasthen applied.

ResultsImmune gene expression profiles can distinguishdifferent disease categories

The clinical demographics of all patients and controlsused in this study are summarized in Table 1. Most of theindividuals self-identified as Caucasians, regardless of dis-ease category. The majority of patients in control group(18/32, 56%) underwent hysterectomy due to leiomyomas.The remaining of cases were treated due to ovarian cysts(7 cases), adenomyosis, pelvic organ prolapse, and endo-metrial polyps (2 cases of each). One patient underwentprophylactic procedure due to risk for Lynch syndrome.

Patients with endometriosis were younger than patientswith ovarian cancer, as expected (P ¼ 4.635e�11). Gravid-ity and parity scores were also lower in patients withendometriosis and EAOC as compared with healthy indi-viduals (P < 0.05), in line with findings pointing to hor-monal imbalances that accompany these conditions (24).Althoughdisease stagingwasnot consistently performed forthe endometriosis cases in this retrospective study, weclassified the subjects according to the type of lesions, usingpredefined criteria (25–27). Of all endometriosis cases, 9cases (47%) were classified as "peritoneal" disease and 9 as"deeply infiltrating lesions" (DIE). One case presented withabdominal wall disease. The vast majority (13/15 cases,87%) of all AE lesions presented in the context of ovarianendometrioma. The remaining two cases had peritoneallesions.

To profile the immune microenvironment in endome-triosis and EAOC, we extracted mRNA from formalin-fixedand paraffin-embedded (FFPE) tissue and performedNano-stringmeasurements of 511 immune gene transcripts, usingthe nCounter GX Human Immunology Kit. Multidimen-sional scaling, constructed using 65% cut-off thresholdfor differentially expressed genes, shows a good level ofseparation of control individuals, endometriosis, and

Complement in Endometriosis and Endometriosis-Associated Ovarian Cancer

www.aacrjournals.org Clin Cancer Res; 20(23) December 1, 2014 6165




EAOC patients (Fig. 1A). Unsupervised clustering of 100filtered genes (as described in statistical analyses section inMaterials andMethods) also distinguished the three diseasecategories well (Fig. 1C). Twenty-two of 28 (79%, afterremoval of outliers as described in Materials and Methods)healthy control samples and 22 of 28 (79%) of cancer casescluster within their disease category and separate from eachother (Fig. 1C clusters 1 and 2, respectively). Endometriosisshows a mixed immune gene profile with some (n ¼ 11,37% of cases) clustering with healthy controls (cluster 1)while others (n¼ 10, 33%) display profiles more similar tocancer (cluster 2). The remaining 30% were also moresimilar to cancer, albeit in the most distant cluster 3. Theseresults suggest that in some patients with endometriosis,endometriosis-associated inflammation carries features typ-ically associated with cancer immune environments. Inaddition, similar comparisons using the AE cohort showthat the vast majority (n¼ 13, 85%) of cases in this diseasecategory have a cancer-like immune environment as theyhomogeneously cluster closer to the EAOC cases than to thecontrol group (Fig. 1B andD), providing further support forAE as a tumor precursor lesion.

Differential expression of immune genes thatdifferentiate between controls, endometriosis, andEAOC reveal complement pathway

Analyses of differentially expressed genes using a falsediscovery rate (FDR) of 5%, and log2 fold change inexpression�1 identified 39 immune genes that distinguishnormal from endometriosis, 73 immune genes that differ-entiate between endometriosis and EAOC, and 99 immune

genes that are different in EAOC compared with controls(Fig. 2A). Analyses of AE reveal 95 and 75 differentiallyexpressed genes when compared with controls and EAOC,respectively (Fig. 2A). The complete list of all differentiallyexpressed genes and their P values is shown in Supplemen-tary Table S1. Analysis of the differentially expressed genelists comparing endometriosis with control or EAOCrevealed that a total of 74 genes were present in at leasttwo of the three signatures (Fig. 2B). Nine of the 74 geneswere present in all three datasets and of these 9, 5 genesbelong to the complement pathway: complement factors 7,D, B, H (C7, CFD, CFB, CFH), and mannose-associatedserine protease 1 (MASP1; Fig. 2B). Two other complementgenes encoding for complement factors 3 and 4a (C3, C4A)were also revealed in other two-way intersections such asnormal versus endometriosis, endometriosis versus EAOCand normal versus EAOC (Supplementary Table S1). Inaddition, C3, C7, and CFH remain dysregulated in AE(Supplementary Fig. S1) and when analyzed together, adifferentiating pattern of the 7 complement genes (C3,C4A,C7, CFH, CFD, CFB, and MASP1) was detected among allfour disease categories (control, endometriosis, AE, andEAOC; Fig. 2C). Notably, changes in expression for mostof the complement genes displayed a consistent trend, fromlow levels seen in control endometrium to higher expres-sion in endometriosis, AE, and EAOC (Fig. 2D). In contrast,MASP1, which encodes for mannan-binding lectin serineprotease 1, follows the opposite pattern, suggesting thatlectin pathway of complement activation may be notinvolved in endometriosis and EAOC-associated inflamma-tion. In addition, Ingenuity Pathway Analyses confirms that

Table 1. Demographic and clinical characteristics of patients

Characteristic

Normal,n ¼ 32(26.7%)

Benignendo,

n ¼ 30 (25%)

Atypicalendo,

n ¼ 15 (12.5%)

EAOCa,n ¼ 28(23.3%)

SOC,n ¼ 15(12.5%) Pb

Age at presentation (years), mean (SD) 46.5 (6) 40.1 (10.9) 48.01 (6.5) 54.8 (11.6) 65.4 (12.4) 1.135e-09Gravidity, median (IQR)c 2 (1) 1 (2.5) 1 (2) 1.5 (2.25) 2 (2) 0.016Parity, median (IQR)c 2 (2) 0.5 (1.25) 1 (1.75) 0.5 (2) 2 (2) 0.002Body mass index (kg/m2), mean (SD) 30.8 (9.9) 28 (7.3) 31.5 (12.7) 28.7 (8.1) 28.4 (6.6) 0.8Race (white), n (%) 28 (93%) 28 (93%) 15 (100%) 27 (100%) 13 (93%) 0.5History of tobacco use, n (%) 9 (33%) 10 (36%) 3 (21%) 9 (36%) 6 (50%) 0.7History of alcohol use, n (%) 14 (54%) 13 (46%) 8 (57%) 11 (46%) 4 (33%) 0.7Preoperative CA125 level (U/mL) 27 79.1 18.8 736.3 1,920 0.005History of diabetes, n (%) 0 (0%) 1 (3%) 1 (7%) 5 (22%) 2 (17%) 0.035History of hypertension, n (%) 10 (35%) 5 (17%) 6 (46%) 12 (50%) 9 (75%) 0.007Stage of diseased, n (%)Early N/A N/A N/A 15 (53%) 3 (20%) 0.08Late N/A N/A N/A 12 (42%) 11 (73%)

NOTE: The values in bold are statistically significant.Abbreviations: Endo, endometriosis; SOC, serous ovarian cancer; N/A, not applicable.aEAOC group comprises endometrioid (n ¼ 16, 57%) and clear-cell (n ¼ 12, 43%) tumors.bANOVA or Kruskal—Wallis test for continuous variables; c2 or Cochran—Mantel–Haenszel c2 test for categorical variables.cIQR, interquartile range (25th percentile, 75th percentile).dTumor stage was available in 27 (96%) and 14 (93%) of EAOC and SOC cases, respectively.

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complement is the most significantly dysregulated immunepathway in endometriosis andEAOC(Supplementary TableS2). Importantly, complement was not among the toppathways in serous ovarian cancer, further demonstratingheterogeneity amongovarian cancer histotypes and suggest-ing that complement involvementmay be specific to EAOC.

Humoral immunity is also included in the top net-works in endometriosis and EAOC (SupplementaryTable S3A and S3B), but not in serous ovarian cancer(Supplementary Table S3D) although both EAOC andserous cancer seems to trigger changes predominantly incellular immunity.

A ControlEEAOC

ControlAEEAOC

B

)2()1( (3)C

(1) (2)

D

Figure 1. Global gene expressionacross disease categories. A andB, multidimensional scaling plotshowing separation of diseasecategories with for differentiallyexpressed genes filtered with 50-quantile cut off of mean and SD.EAOC cases are shown in red andcontrols ingreen. Endometriosis (E)and atypical endometriosis (AE)are shown in blue (A and B,respectively). C and D,unsupervised clustering of 100filtered genes. EAOC cases arelisted in red and controls in green; Eand AE are listed in blue (C and D,respectively).






A

Controls E AE EAOCC

DE genes = 39 DE genes = 73 DE genes = 99EControls EAOCControlsE EAOC

D

Ctrl. Vs. IDE

Foldchange

Ctrl. vs. EOACFold

change

E. vs. EAOCFold

change(P )(P )(P )

C7 23.7(3.39E–61)

7.84(2.56E–26)

0.33(9.30E–05)

CFB 2.8(3.36E–08)

7.38(1.66E–26)

2.62(1.88E–09)

CFD 2.59(2.73E–08)

0.39(0.000178)

0.15(2.57E–21)

CFH 7.17(2.49E–28)

3.19(2.56E–11)

0.44(0.001844)

MASP1 0.37(6.84E–11)

0.036(6.66E–41)

0.096(5.48E–16)

GNLY 0.35(2.89E–10)

0.036(3.79E–5)

0.1(7.67E–23)

IL2RB 0.39(2.37E–09)

0.17(4.27E–19)

0.44(0.000221)

LTF 9.9(5.61E–16)

31.46(4.32E–37)

3.18(3.65E–08)

PIGR 0.32(2.12E–13)

2.34(2.30E–06)

7.22(8.49E–22)

Controls AE AE

Controls

Benign E

AE

EAOC

C7 CFB

CFD

MASP1

CFH

DE genes = 95 DE genes = 75

E vs. EAOC

Normal Controlsvs. E

9

17

740

5 3218

B

EAOC

C4A

CFH

CFB

C3

C7

CFD

MASP1

vs. EAOC

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Taken together, these results suggest that gene expressionof several complement components is high and that com-plement activation may be a common biologic processinvolved in immune surveillance in endometriosis andEAOC (Supplementary Table S3C and S3D). Furthermore,presence of humoral immunity points towards comple-ment as a possible mediator of epithelium–immune stomainteractions, potentially via antibody-dependent, comple-ment-mediated immunity.

Complement proteins are abundantly present inepithelial cells endometriosis and EAOCUsing immunohistochemistry (IHC), we confirmed pro-

tein expression for all five complement factors (C7, CFD,CFB, CFH, MASP1) differentially expressed in endometri-osis, control, and EAOC (Fig. 2B). Complement factor 7(C7) is highly expressed in endometriosis and ovariancancer while normal endometrium shows little to nomRNA expression (Fig. 2D). Accordingly, expression ofC7 protein, a member of the membrane attack complex(MAC), is minimal in eutopic (control) endometrial glandsand stroma, whereas epithelial cells in endometriosis, AE,and EAOC show a clearly increased protein production(Fig. 3A). Scoring of C7 protein shows that at least for thiscomplement component, its expression levels are in linewith the C7 gene expression data (Fig. 2D).Expression of other complement proteins such as CFB,

CFD, CFH, and MASP1 was confirmed by IHC (Fig. 3C).Similar to C7, most of the complement proteins werepresent in epithelial cells lining endometriotic glands orendometrioma and in epithelial tumor cells (in EAOC).Staining pattern reveals complement proteins are distrib-uted both intracellularly and on the cell surface, suggestingan endogenous production and consumption in epitheliallesions of endometriosis and cancer.

Complement genes in a murine model for EAOC thatmirrors expression seen in human tumorsOn the basis of the above ex vivo findings that comple-

ment proteins are abundant in epithelial cells in endome-triosis, premalignant, and malignant lesions, we proposednext to investigate in vitro the link between the complementpathway and early carcinogenic events in ovarian epithelialcells. To accomplish this, we employed ovarian surfaceepithelial cells derived from triple transgenic mice thatprogress to human mucin 1 (MUC1)- expressing endome-trioid ovarian cancer that closelymirrors the human disease(21). The mice heterozygously express human MUC1 asa transgene, and simultaneously carry the conditional

LoxP-Stop-LoxP K-rasG12D oncoallele and the floxedPtenloxP/loxP gene (28). In this Cre-LoxP in vivo system, injec-tion of Cre recombinase encoding adenovirus (AdCre)under the ovarian bursa of female MKP mice triggers pro-gression to endometrioid ovarian tumors in about 7 to 8weeks (21). The mouse tumors show similarly increasedepithelial cell expression of complement proteins (Fig. 4A),further demonstrating that the MUC1KrasPten (MKP)mouse model, which replicates with high fidelity the his-topathology of human EAOC (21, 28), may also provide anuseful preclinical tool for exploring the complement biol-ogy in the ovarian tumor microenvironment.

Conditional activation of tumor driving pathwaysleads to complement gene upregulation

Using conditional mice with a Kras-activating mutationandPtendeletion,we studiednext howengagement of theseclassical oncogenic and tumor suppressor pathway, respec-tively, affects complement activation in primary epithelialcells. To accomplish this, we generated a novel cell line(MKPOSE) from primary OSE cells of healthyMUC1KrasP-ten mice (21), using previously established protocols (22).At baseline, Kras and Pten/Pi3K pathways are unaffected inthese mice (21, 28). However, as with our in vivo experi-ment, in vitro exposure of theMKPOSE cells to AdCre floxesout loxP sites and triggers Pten deletion and oncogenic Krasactivation (Supplementary Fig. S2A), similarly to levels seenin vivo in tumors (21).

We postulated that MKPOSE cells exposed to AdCrecreate a surrogate in vitro system for early transformationand tumor formation and offer an experimental setting inwhich we can monitor early changes in complement geneexpression before and after Cre-induced genetic events thateffectively trigger in vivo tumorigenesis. Focusing on C7 asthe prototype member of the MAC, the end-product ofcomplement activation, we show that in MKPOSE cells,C7 is present at detectable levels at baseline and is furtherupregulated (alongside other complement genes) followingexposure to AdCre (Fig. 4B–D). In addition, treatment ofcells with small molecule inhibitors that inhibit the AdCre-induced Kras and Pi3k pathways (AZD6422 or BEZ235,respectively) reverses C7 upregulation (SupplementaryFig. S2B). These drugs are in preclinical development ascancer therapeutics, AZD6244 as a highly selective inhibitorof ERK1 and ERK2 (29) and BEZ235 as a pan-PI3K-mTORinhibitor (30).

One of the consequences for complement upregulation ismediation of antibody-induced, complement-mediatedcell death. Given that humoral immunity was among the

Figure 2. Differentially expressedgenes across diseasecategories. A, heatmapsof differentially expressed (DE) genes in fivedifferent two-groupcomparisons:control versus endometriosis (E; n ¼ 39), endometriosis versus EAOC (n ¼ 73), normal versus EAOC (n ¼ 99), control versus AE (n ¼ 95), AE versus EAOC(n¼ 75). B, Venn diagram showing number differentially expressed genes that are differentially expressed in controls, E, and EAOC. Accompanying table liststhe 9 common differentially expressed genes (common intersection, arrow), fold change and P value. Complement genes are in bold. C, heatmap of7 complement genes (C4A,CFH,CFB,C3,C7,CFD,MASP1) shows a differentiating pattern among the four different disease categories (controls, E, AE andEAOC). D, Individual gene expressionprofile of five commoncomplement differentially expressedgenes (C7,CFB,CFD,CFH,MASP1) across the four cohorts(controls, E, AE, and EAOC). All genes are significantly different (FDR < 0.05 and absolute log FC greater than 1) for any of the two group comparisons (normal,endometriosis, and EAOC).






top five networks in both endometriosis and EAOC (Sup-plementary Table S3A and S3B), we explored next whethercomplement upregulation triggered by pathways down-stream of Kras and Pten/Pi3k modifies antibody-induced,complement-mediated cell death of MKPOSE cells. AdCre-treatedMKPOSE cellswere exposed toovarian cancer ascitesfluid, collected frommice challenged intraperitoneally (IP)with syngeneic, human MUC1-expressing ovarian tumors.

These mice produce anti-MUC1 antibodies at high levels inboth serum and ascites (Supplementary Fig. S3A). TheMUC1-specific antibodies in ascites have the ability to bindto MUC1 molecules present on tumor cell surface (Supple-mentary Fig. S3B). We postulated that if complementengagement would be effective, AdCre MKPOSE cellsexposed to antibody-containing ascites would undergo anti-body-induced, complement-mediated cell death. However,

0

0.5

1

1.5

2

2.5

3

3.5

Control E EAOC

Clear cellEndometrioid

Control endometrium Endometriosis Atypical endometriosisA

Endometriosis EAOC

CF

BC

FD

CF

HM

AS

P1

C

___ ___

___

___

_______

___

___

___

B

Control endometrium

C7

C7* *

Figure 3. Tissue validation ofcomplement proteins via IHC. A,tissue deposition of complementC7 protein. B, intensity staining forC7 was scored as follows: 0, nostaining; 1, weak staining; 2,moderate staining; and 3, strongstaining. y-axis: average score plusSD. Each of the three groups(controls, endometriosis, andEAOC) contained fiverepresentative samples. one-wayANOVA; P ¼ 0.001. C, tissueexpression of CFB, CFD, CFH, andMASP1 proteins in endometriosisand EAOC FFPE tissues. At least 5cases in each disease categorieswere stained for each marker.Representative images for eachmarker are shown.

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we observed either no effect or slight decrease rather thanincrease in cell death (compared to EV control cells, Fig. 4E).In contrast, control cells (consisting of similarly treatedsplenocytes exposed to same ascites, as well as mouse serumas control) are successfully lysed (Supplementary Fig. S3C).This suggests that despite upregulation in transformed OSE,complement engagement in the predicted lytic pathways israther ineffective.

Complement C7 knockdown inhibits ovarian cellproliferationComplement inhibition has been recently reported as

having a beneficial effect against tumor growth (31). Toinvestigate roles of C7 expression on proliferation of ovar-ian epithelial cells in vitro, we knocked down C7 expressionin MKPOSE AdCre-infected and control cells, using a mix-ture of siRNAs targeting four regions of mouse C7 mRNA(Fig. 5A). Mouse Gapdh was used as positive control forknockdown efficacy (Supplementary Fig. S4A), whereasnontarget siRNA, which does not affect C7 expressionwas used to control for target specificity (Supplementary

Fig. S4B). Our results show that inhibition of C7 geneexpression in MKPOSE exposed to AdCre inhibits growthcurve (Fig. 5B; P ¼ 0.03). Nontarget siRNA at increasingconcentrations does not interfere with cell growth (Supple-mentary Fig. S4C). We further tested complement inhibi-tion in combination with AZD 6422 (80 nmol/L) orBEZ235 (25 nmol/L). As expected, when cells were treatedwith C7 siRNA and either of the inhibitor drugs, growth rateof cells was significantly inhibited (P < 0.05). Combinationof complement knockdown and either drug showed addi-tional inhibitory effect (Fig. 5C; P < 0.005).

Overall, these in vitro results link Kras and Pten/PI3Kpathways to increased complement gene expression andsuggest that complement inhibition reduces tumor cellproliferation an effect further increased, albeit moderately,by specific inhibitors of the above tumor pathways.

DiscussionSeveral inflammatory phenotypes have been associated

with endometriosis and efforts are underway to incorporate

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Figure 4. Complement biology in anEAOC preclinical mouse model. A,tissue deposition of complement inmouse ovarian tumors fromMUC1KrasPten mice. B, changesin complement gene expressionmeasured by qPCR in MKPOSEcells treated with either emptyvector (MKPOSE-EV, control) orCre-encoding adenovirus(MKPOSE-AdCre). C, MKPOSEintracellular staining for C7 byimmunocytochemistry. D,quantitation of C7 by flowcytometry. Positive cells(percentages) were gated outsideof the negative control gate. E,antibody-mediated complement-mediated cell killing assay usingMKPOSE-EV andMKPOSE -AdCrecells. Cell death was assessed viaflow cytometry, using propidiumiodide staining. Positive stainingmeasures cells death.






these into noninvasive diagnostic tests (32). However,mostof the studies to date have focused primarily on secretedchemokine profiles (20) or immune cells resident in theperitoneal cavity (macrophages, NK cells) or isolated fromcirculation (T lymphocytes, NK cells; refs. 33, 34). Weperformed here the first comprehensive immune geneexpression analysis of pelvic inflammation to date, usinga collection of 511 Nanostring probes and RNA extractedfrom affected tissues. Our results revealed immune geneprofiles that can differentiate endometriosis from bothhealthy and cancer cases. Surprisingly, however, unsuper-vised clustering shows that several of the patients withendometriosis may have an inflammation profile similarto those with EAOC (Fig. 1). This intriguing finding furtherconfirms heterogeneity of inflammatory milieu in endome-triosis and suggests that cancer-like immune signaturesmaydevelop earlier, in patients with lesions that are classified asclinically benign. Given the sensitivity and specificity ofimmune effectors that can sense early molecular changes ofcells undergoing transformation, it is conceivable that pro-filing the immune response in tissuemayprovidemolecularclues for patients with EAOC risk. This hypothesis stimu-lates further studies, with significantly larger sample sizesand additional controls, to validate and test the efficacy of

cancer-like immune signatures as potential predictors ofprogression to EAOC. We also acknowledge that all thecases used here as controls consisted of a separate cohort ofendometrial tissue, due to challenges in obtainingmatchingeutopic endometrium/ectopic glands from same patients.Inclusion of healthy ovaries, as additional reference, isequally problematic due to the fact that ovaries typicallydisplay only a delicate epithelial component (the OSEmonolayer, which is often easily detached during surgicalremoval) and that little/no immune stroma is typicallypresent in disease-free ovaries.

Analyses of differentially expressed gene sets revealed thatcomplement is the top pathway in endometriosis andEAOC cases. Although this is the first tissue profiling studyon complement, changes in peritoneal fluid levels of com-plement have been recently reported in endometriosis.Using qPCR gene profiling of 84 genes in a cohort of 20endometriosis cases, Aslan and colleagues recently reportedthat most significant upregulation was in C5 gene expres-sion, although no other complement genes were detected,possibly due to the limited breadth of the profiler employed(35). We utilized here a comprehensive immune gene setand focused not only on endometriosis but also on EAOC.Our finding that 5 of 9 genes (56%) differentially expressed

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Figure 5. Complement inhibitionand tumor cell growth. A, changesin C7 gene expression in C7 siRNA(25 nmol/L) treated cells ascompared with untreated cells. B,C7 siRNA–treated cells showdecreased cell growth ascompared with untreated cells(�, P < 0.01). C, survival curve ofMKPOSE AdCre cells treated withKras and Pten pathway inhibitordrugs AZD6244 (80 nmol/L) orBEZ235 (25 nmol/L), respectively,along with C7 siRNA. Assays wererun in triplicate. Average values andSEs are shown. Results fromoneofthe two experiments shown.Student t test. �, P < 0.05;��, P < 0.005.

Suryawanshi et al.





in both endometriosis and EAOC are complement genesstrongly supports the importance of complement cascade inthese diseases. In addition, the lower prevalence of com-plement genes in serous ovarian cancer suggests a potentialspecificity for complement in EAOC.Primarily classified as a powerful innate immune effector,

the complement system and its engagement in both acuteand chronic inflammation are well defined (36). However,emerging literature during the past decade has revealed thatcomplement activation may support tumor growth, thuscreating a paradigm shift in complement cascade in cancer(37). It is now postulated that the complement pathwaymay act via several mechanisms that coexist in the tumorenvironment: directly, by playing an active role in stimu-lating proliferation of tumor cells or indirectly, via immunesuppression (38, 39) and neovascularization (40). Never-theless, the initiating steps that link epithelial cell biologyand complement cascade are not well defined.Using our recently reported triple transgenic MUC1K-

rasPten mice with conditional mutations in Kras and Ptengenes (21), we generated a novel primary OSE-derivedmurine ovarian cancer line (MKPOSE) and a versatile invitro system that served as a surrogate for early transforma-tion in ovarian epithelial cells. By turning on Kras anddeleting Pten in MKPOSE cells, we showed for the first timethat activation through Kras and Pten/PI3K leads to com-plement upregulation, an effect reversed when small druginhibitors acting downstream of Kras and/or Pten areadded. Inhibitors of tumor-driving pathways (includingAZD6244 and BEZ325 used here) are currently tested inclinical trials for several types of tumors (41, 42). Althoughcomplement genes are likely controlled via several path-ways, molecular profiling of tumor responses in these trialsmay provide further molecular evidence on modulation ofcomplement pathways by targeted therapies.Finally, we report here that one other major biologic

pathway active in endometriosis is humoral immunity. Inaddition to changes in immune cells and cytokines, mostlydetected via peripheral blood or peritoneal fluid measure-ments, women with endometriosis often display increasedB-cell activation (43–45). Systemic antibody productionand deposition of IgG and complement in tissue has beenpreviously reported in patients with endometriosis, show-ing humoral responses to various autoantigens (43–46).Antibody-induced, complement-mediated cell death is

very effective in clearing dead cells, mostly via the classicalpathway, activated by the Fc portion of immunoglobulinsbound to various antigens on either infectious agents orapoptotic cells. The alternative pathway can be triggered viacontinuous, low level cleavage of C3, and can be activatedby bacteria, viruses, or fungi, as well as neoplastic cells. Thethird complement activation pathway, called the MBL

pathway is activated by pathogens, via pathogen-associatedmolecular patterns (36).

The expression levels for the differentially expressedcomplement genes detected here suggest that MBL pathwaywas not involved in these cases (hence ruling out potentialinfectious causes for pelvic inflammation) and that classicalpathway was likely most prominently used.

In summary, our findings reveal that chronic inflamma-tion in endometriosis is dominated by complement, whichremains active in EAOC but not tumors with serous histol-ogy, further demonstrating heterogeneity in the inflamma-tory milieu within this category commonly referred to asovarian cancer. Pharmacologic inhibition of complement iscurrently tested in clinical trials (36), and results from thesestudies will provide much needed clinical evidence tosupport (or refute) the recent paradigm shift on protumorroles of complement in cancer. Profiling studies like the onepresented here might aid in patient selection for a person-alized approach.

Disclosure of Potential Conflicts of InterestT. Lee is a consultant/advisory board member for Ethicon. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: X. Huang, E. Elishaev, R. Edwards, A.M. VladDevelopment of methodology: S.M. Suryawanshi, X. Huang, E. Elishaev,L. Zhang, S. Kim, G. Mantia-Smaldone, A.M. VladAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S.M. Suryawanshi, E. Elishaev, L. Zhang, S. Kim,N. Donellan, G. Mantia-Smaldone, S. Mansuria, R. Edwards, A.M. VladAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.M. Suryawanshi, R. Budiu, E. Elishaev,L. Zhang, S. Kim, T. Ma, G. Tseng, R. Edwards, A.M. VladWriting, review, and/or revision of the manuscript: S.M. Suryawanshi,R. Budiu, E. Elishaev, L. Zhang, S. Kim, N. Donellan, G. Mantia-Smaldone,G. Tseng, R. Edwards, A.M. VladAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.M. Suryawanshi, E. Elishaev,T. Lee, R. Edwards, A.M. VladStudy supervision: E. Elishaev, A.M. VladOther (provided the surgical specimen for the study): T. Lee

AcknowledgmentsThe authors thank Dr. Adrian Lee for access to the Nanostring platform

and Lindsay Mock and Julia Thaller for technical assistance with specimenretrieval.

Grant SupportThis study was supported by the UPMC Research Fund (to R. Edwards,

X. Huang, and A.M. Vlad), Department of Defense (DOD) Ovarian CancerAcademy Award W81XWH-10-1-0525 and NIH/NCI R01 CA163462 (toA.M. Vlad), and P50 CA159981 (to R. Edwards). X. Huang is an OvarianCancer Research Fund Liz Tilberis scholar and is supported by an AmericanCancer Society Research Scholar grant.

The costs of publication of this articlewere defrayed in part by the paymentof page charges. This article must therefore be herebymarked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 27, 2014; revised August 14, 2014; accepted September 17,2014; published OnlineFirst October 7, 2014.

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2014;20:6163-6174. Published OnlineFirst October 7, 2014.Clin Cancer Res Swati Suryawanshi, Xin Huang, Esther Elishaev, et al. Endometriosis-Associated Ovarian CancerComplement Pathway Is Frequently Altered in Endometriosis and

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