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Research Article

The Parity-Associated Microenvironmental Niche in theOmental Fat Band Is Refractory to Ovarian Cancer Metastasis

Courtney A. Cohen1, Amanda A. Shea2, C. Lynn Heffron1, Eva M. Schmelz2, and Paul C. Roberts1

AbstractOvarian cancer is an insidious and aggressive disease of older women, typically undiscovered before

peritonealmetastasis due to its asymptomatic nature and lack of early detection tools. Epidemiologic studies

suggest that child-bearing (parity) is associated with decreased ovarian cancer risk, although the molecular

mechanisms responsible for this phenomenon have not been delineated. Ovarian cancer preferentially

metastasizes to the omental fat band (OFB), a secondary lymphoid organ that aids in filtration of the

peritoneal serous fluid (PSF) and helps combat peritoneal infections. In the present study, we assessed how

parity and age impact the immune compositional profile in the OFB of mice, both in the homeostatic state

and as a consequence of peritoneal implantation of ovarian cancer. Using fluorescence-activated cell sorting

analysis and quantitative real-time PCR, we found that parity was associated with a significant reduction in

omental monocytic subsets and B1-B lymphocytes, correlating with reduced homeostatic expression levels

of key chemoattractants and polarization factors (Ccl1, Ccl2, Arg1, and Cxcl13). Of note, parous animals

exhibited significantly reduced tumor burden following intraperitoneal implantation compared with

nulliparous animals. This was associated with a reduction in tumor-associated neutrophils and macro-

phages, as well as in the expression levels of their chemoattractants (Cxcl1 and Cxcl5) in the OFB and PSF.

These findings define a preexisting "parity-associated microenvironmental niche" in the OFB that is

refractory to metastatic tumor seeding and outgrowth. Future studies designed to manipulate this niche

may provide a novel means to mitigate peritoneal dissemination of ovarian cancer. Cancer Prev Res; 6(11);

1182–93. �2013 AACR.

IntroductionOvarian cancer is responsible for 140,000 deaths a year in

womenworldwide (1)with one of the highest incidence-to-death ratios in theUnited States due to late detection, tumorheterogeneity, and a high rate of metastasis (2). However,epidemiologic studies indicate that among other factors,parity (child-bearing) may provide protection against thedevelopment of ovarian cancer (3). Nonetheless, little isknown about the persistent molecular and cellular changesthat modulate ovarian cancer development as a result ofchild-bearing.

As a largely asymptomatic disease in the early stages,ovarian cancer is rarely detected before metastasis. Duringmetastasis, ovarian cancer cells exfoliate from the primarytumor, disseminate throughout the peritoneal cavity in the

serous fluid, and preferentially seed in the omental fat band(OFB; ref. 4). As the primary metastatic site, it is typicallyremoved during surgical tumor debulking to slow diseaseprogression. The OFB is considered a secondary lymphoidorgan, contributing to immunosurveillance in the peritonealcavity and its removal results in impaired antibacterialresponses in the abdomen and increased risk of subsequentinfections (5). It is composed of fatty tissue interspersedwithimmune cell aggregates or "milky spots" (6), consisting ofleukocytes, stem and progenitor cells, fibroblasts and endo-thelial cells (7) collectively referred to as the stromal vascularfraction (SVF). Disseminated tumor cells adhere to milkyspots within hours (8), promoting a protumorigenic micro-environment that supports subsequent tumor outgrowth(6, 8, 9). However, the complex and dynamic interactionsbetween metastatic cancer cells and leukocytes within theOFB have not been well characterized.

We have shown previously that the homeostatic immunemicroenvironment of theOFB significantly differs from thatof other intraperitoneal fat depots with a distinct leukocyteprofile, and a robust cytokine signaling network (10). It isunknownhow this immunemicroenvironment is impactedby aging or parity, which may have critical implications forthe generation of a metastatic niche that determines suc-cessful tumor adherence and outgrowth. However, epide-miologic studies have reported increased ovarian cancerincidence in older women but a protective effect associated

Authors' Affiliations: Departments of 1Biomedical Sciences and Patho-biology and 2Human Nutrition, Foods and Exercise, Virginia PolytechnicInstitute and State University, Blacksburg, Virginia

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Authors: Paul C. Roberts, Virginia Polytechnic Instituteand State University, Integrated Life Sciences Building, 1981 Kraft Drive(0913), Blacksburg, VA 24061. Phone: 540-231-7948; Fax: 540-231-3426;E-mail: [email protected]; and Eva M. Schmelz, [email protected]

doi: 10.1158/1940-6207.CAPR-13-0227

�2013 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 6(11) November 20131182

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Published OnlineFirst September 10, 2013; DOI: 10.1158/1940-6207.CAPR-13-0227

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with parity (11). Here, we postulated that parity may inducechanges in the OFB that result in a preexisting immunemicroenvironment that is protective against cancer metas-tasis. As child-bearing is inherently associated with an olderdemographic, we chose three sets of female mice to distin-guishbetweenage- andparity-specific changes: 5-month-oldnulliparous youngadults, 11-month-oldnulliparousmatureadults, and 12-month-old mature parous adults. Given itsimportance as a primarymetastatic site, in the present study,we comparatively characterized the SVF and gene expressionprofiles of the OFB as a function of age and parity.

Materials and MethodsCell linesThe mouse ovarian surface epithelial (MOSE) cell model

used in this study was developed from C57BL/6 mice andcharacterized previously (12). TumorigenicMOSE cells werepassaged once in vivo by intraperitoneal injection intoC57BL/6 mice and recollected via peritoneal lavage follow-ing a 4- to 6-week incubation period to select for a moreaggressive phenotype. These MOSE cell variants (MOSE-LFFLv) were subsequently transduced with firefly luciferase(FFL) lentiviral particles (GeneCopoeia) as described previ-ously (13) to facilitate live in vivo imaging of cancer celloutgrowth. The characteristicsofMOSE-LFFLvwill be reportedelsewhere. MOSE-LFFLv cells were routinely maintained inhigh glucose Dulbecco’s modified Eagle medium (DMEM;Invitrogen), supplemented with 4% FBS (Hyclone), 100mg/mL penicillin and streptomycin, and 4 mg/mL puromy-cin (lentiviral particles use a puromycin resistance markerfor selection of transduced cells).

AnimalsFemale C57BL/6 mice (Harlan Laboratories) were

housed 5 per cage in a controlled environment (12 hourlight/dark cycle at 21�C) with free access to water and food(18% protein rodent chow, Teklad Diets). Young adultnulliparous mice (5 months of age, 21 g average bodyweight), mature adult nulliparous mice (11 months of age,30 g average body weight), and mature parous mice (12months of age, 31 g average body weight) were sacrificed byCO2 asphyxiation. Mouse studies were carried out in accor-dance with the guidelines approved by the Virginia TechInstitutional Animal Care and Usage Committee.

MOSE-LFFLv injectionYoung adult nulliparous andmature parousmice (n¼ 10

per group) were injected intraperitoneally with either 1 �104 MOSE-LFFLv cells in 300 mL sterile calcium- and mag-nesium-deficient PBS�/�, or mock-injected (PBS�/� alone)and sacrificed at 21 days postinjection.

Peritoneal cancer indexTo quantify relative tumor burden at the time of sacrifice,

the peritoneal cancer index (PCI) was determined asdescribed previously (14, 15), with minor modifications.The original PCI was adapted to apply to tumor size andregion inmice; "quadrant areas" weremodified to represent

distinct organs and their mesentery to evaluate preferentialseeding sites. Specific regions evaluated included peritonealcavity lining, ovaries, lesser omentum, greater omentum(OFB), diaphragm, liver, stomach, pancreas, spleen, kidney,small intestine, small intestine mesentery, large intestine,and large intestinemesentery. Tumor size was scored as (0),no visible tumor; (1), <1 mm; (2), 1–3 mm; or (3): >3 mmor solid mass. The maximum PCI score was 42, reflectingmaximal lesion size in each of the 14 designated areas.Relative PCI scores were further validated by quantitativereal-time PCR (qRT-PCR) analysis of FFL gene expressionused as a tumor cell reporter gene.

Tissue and peritoneal serous fluid harvestThe OFB was harvested from each mouse post mortem,

weighed, rinsed with PBS�/�, and processed for fluores-cence-activated cell sorting (FACS) analysis or placed intoRNAlater (Qiagen) and stored at �80�C. Resident perito-neal cavity cells were collected via peritoneal lavage with5 mL of 1 mmol/L EDTA in PBS�/�. The effluent wascentrifuged, subjected to erythrocyte lysis (155 mmol/LNH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L EDTA), andfurther processed as described below.

Tissue digestSVFs were isolated by digesting individual OFBs (n ¼ 4–

6) in GKN buffer (8.00 g/L NaCl, 0.40 g/L KCl, 3.56 g/LNa2HPO4*12H2O, 0.78 g/L NaH2PO4*2H2O, and 2 g/LD(þ)-glucose, pH 7.4) containing 1.8 mg/mL type IV colla-genase, 10% FBS, and 0.1 mg/mL DNase as previouslydescribed (10). Following digest at 37�C for 45 minutes,cells were passed through a 40-mm cell strainer, and ery-throcytes were lysed (see above).

FACS analysisSingle-cell suspensions derived fromOFB and peritoneal

serous fluid (PSF) were washed in flow buffer (2% BSA inPBS�/�), blocked with Fc block (BD Biosciences) for 10minutes at 4�C, rinsed and incubated with fluorochrome-labeled antibody combinations (available upon request)for 20minutes at 4�C. Antibodies specific for mouse CD45,CD11b, CD11c, F4/80, Ly6C, CD4, CD44, CD62L, B220,CD19, NK1.1, and Ly6G were obtained from eBioscience.CD3 and CD8 antibodies were obtained from BD Bio-sciences. Before analysis, cells were washed twice and resus-pended in PBS�/� with propidium iodide for dead cellexclusion. FACS analysis was conducted on a FACSAria(BD Biosciences) and data were analyzed using FlowJo(TreeStar) software.

Quantitative real-time PCRIndividual OFBs were homogenized in Qiazol (Qiagen)

and RNA was purified using the RNeasy Lipid Tissue Kit(Qiagen), according to themanufacturer’s instructions. RNAconcentrationwas determined using aNanoDrop1000 spec-trophotometer. RNA (n ¼ 4–6) was subjected to the iScriptcDNA synthesis system (Bio-Rad) according to the manu-facturer’s protocol. qRT-PCR was conducted with 12.5 ng

Parity Reduces Metastasis in the Omental Fat Band

www.aacrjournals.org Cancer Prev Res; 6(11) November 2013 1183




cDNA per sample using gene-specific SYBR Green primers(primer sequences are available upon request) designedwithBeacon Design software. SensiMix SYBR and Fluoresceinmastermix (Bioline) was used in a 15 mL reaction volume.qRT-PCRwas conducted for 42 cycles at 95�C for 15 seconds,60�C for 15 seconds, and 72�C for 15 seconds, preceded by a10-minute incubation at 95�C on the iQ5 (Bio-Rad). Meltcurves were generated to ensure fidelity of the PCR product.The housekeeping gene was L19 (10) and the DDCt method(16) was used to determine fold differences.

Statistical analysisData were expressed as mean � SEM. FACS analysis and

qRT-PCR data were analyzed using a one-way ANOVAcoupled with a Tukey post hoc test in SigmaPlot (SystatSoftware). Differences were considered statistically signifi-cant at P < 0.05.

ResultsOFB size and cellularity

The size of the OFB increased significantly with agebetween 5- and 11-month-old nulliparous mice, coincidingwith overall age-associated weight gain of the animals (14.4� 1.5 g and 28.2� 2.2 g;P< 0.01). Paritywas associatedwithan additional increase in OFB weight (Fig. 1A; P < 0.01),although the average body weight of these age-matchedgroups were not significantly different (29.8 � 0.8 g vs.31.7 � 0.7 g). The OFB SVF cell count was significantlyelevated as a function of both age and parity, increasing

800% in parous mice (Fig. 1B; P < 0.01). Therefore, parityinduced a change in stromal vascular cell influx or prolifer-ation and not just an increase in overall OFB adiposity. Theproportion of CD45þ leukocytes represented approximately50% of the SVF in both sets of nulliparous mice but morethan 95% of the SVF in parous mice (Fig. 1C; P < 0.01). Tofurther define the age- and parity-associated signature, weevaluated expression of a set of immune-related genes usingqRT-PCRonwhole tissue samples. As shown in Fig. 1D, therewerebothage-andparity-associateddifferences in theexpres-sion of a number of important cytokines and chemokines inthe homeostatic state. Notably, in mature nulliparous ani-mals, the expressionof chemotacticmolecules formonocytes(Ccl1, Ccl4, and Ccl7; P < 0.05) as compared with youngnulliparous mice was increased. Expression of Tgf-b, animportant regulator of tumor cell invasion and metastasis,was significantlyhigher inoldermice, irrespectiveofparity (P< 0.01). Parity-specific changes included a significantly lowerexpression of neutrophil chemoattractants (Cxcl1 and Cxcl2;P < 0.05) and alternative activation-related genes character-istic of tumor-associated macrophages (Arg1 and M6pr; P <0.05). Thus, inherent changes that occur as a result of agingand child-bearing can affect the signaling milieu in the OFB.

OFB SVF characterizationThe OFB SVF was further characterized via FACS analysis

to identify age- and parity-specific differences in immunecell composition. Viable cells (propidium iodide exclusion)were separated into CD45þ leukocytes and CD45� stromal

Figure1. Parity-associated differences in the cellularity of theOFBsof youngadult nulliparous (yNP),mature adult nulliparous (aNP), and adult parousmice (aP)mice. A, whole tissue OFB weight. B, number of cells in the SVF isolated from digested OFB. C, CD45þ population in OFB SVF. D, gene expression profileof OFB. �, P < 0.05; ��, P < 0.01.

Cohen et al.

Cancer Prev Res; 6(11) November 2013 Cancer Prevention Research1184




constituents. CD45þ cells were subsequently separated intoR1 (lymphocyte) and R2 (monocyte/granulocyte) gatesbased on forward/side scatter (Fig. 2A), and leukocyte sub-sets were identified on the basis of well-defined surfacemarkers. The CD45þ subsets associated with the OFB innulliparousmicewere comparable, irrespective of age.How-ever, the immune cell composition of the parous OFB wasstrikingly different, supporting the hypothesis that parityleads to establishment of a unique protective microenviron-ment. In the OFB of nulliparous mice, the lymphocyte tomonocyte/granulocyte (R1:R2) ratios were approximately6:4 independent of age, whereas lymphocytes representedalmost 90% of the leukocytes isolated from the parous OFB(Fig. 2B). This was mirrored in the PSF, denoted by a 20%increase in the proportion of lymphocytes to monocytes/granulocytes in parous mice as compared with maturenulliparous mice (P < 0.05; Supplementary Table S1).The parity-associated increase in lymphocytes was

reflected across numerous subsets including CD3þ T cells

(P < 0.01), CD19þ B cells (P < 0.05), and CD11cþ dendriticcells (P < 0.01) with concomitant decreases in F480þ

macrophages (P < 0.01) and NK1.1þ natural killer (NK)cells (P < 0.01) compared with nulliparous mice (Fig. 2C).Compared with their nulliparous counterparts, the parousOFB also displayed significantly increased CD3þCD4þ Thcells, and significantly decreased CD3þCD8þ TC and CD3þ

CD4�CD8� cells (P < 0.05; Fig. 2D). Parity also induced asignificant increase in the proportion of T cells and B cells inthe PSF (P < 0.05; Supplementary Table S1), although levelsof NK cells and dendritic cells were unchanged.

Notably, there was a significant parity-associated shift inthe subsets of B cells residing in the OFB. CD11bþB220lo/þ

B1 cells were the most prevalent subset of B cells in nul-liparousmice,whereas inparousmice theCD11b�B220lo/þ

B2-subset was dominant (Fig. 2E). B1 cells are the predom-inant B-cell type in the peritoneal cavity, and are considereda more "innate-like" population, with receptors for auto-antibodies and conserved bacterial and viral epitopes (17).

Figure 2. The OFB displaysparity-associated differencesin leukocyte populations. A,CD45þ leukocytes in the SVF ofOFBs harvested from youngadult nulliparous (yNP), matureadult nulliparous (aNP), andmatureparous mice (aP) were separatedinto regions R1 (lymphocytes) andR2 (monocytes/granulocytes) forfurther analysis. B, distribution ofR1:R2 populations in OFBs. C,overall immune cell populationsin OFBs. D, CD3þ subsets withinOFBs. E, CD19þ subsets withinOFBs. F, monocyte/granulocytesubsets within OFBs. �, P < 0.05;��, P < 0.01.






This increase in B2-cells could be indicative of a moresystemically active humoral B-cell response in parous mice.

Parity-associated changes were also evident within OFBmacrophage subsets, although these changes were not mir-rored in the parous PSF. In the PSF, macrophages are typ-ically subdivided into "large peritoneal macrophages"(LPM), and "small peritoneal macrophages" (SPM; ref. 18).LPMs can be distinguished via FACS analysis based onforward/side scatter and higher CD11b and F480 surfacestaining. SPMs increase in the PSF as a result of LPS stimu-lation and are derived from blood monocytes (18). Theapplicability of these subsets to theOFB is largely undefined.Here, the PSF contained mostly LPMs in the homeostaticstate, with SPMs representing only a minor fraction ofthe total macrophage population. This trend was not signif-icantly altered by parity (data not shown). In contrast,while the predominant macrophage population in theOFB of nulliparousmicewas the CD11bþF480lo population(SPMs), this population was significantly reduced in parousmice. Furthermore, the CD11bþF480þ population (LPMs)was virtually undetectable in parous mice, although it repre-sented approximately 8% of the R2 gate in nulliparous mice(Fig. 2F). Collectively, these data suggest that nulliparousmice, irrespective of age, had similar immunomodulatorymicroenvironments in the peritoneal cavity as denoted byindistinguishable leukocyte profiles at these time points.

Impact of parity on peritoneal tumor burdenAs parous women have a decreased risk of ovarian cancer,

we next evaluated whether the parous microenvironmentwas more refractory to tumor cell seeding and outgrowth.To investigate this, we used ahighly aggressive variant of ourMOSE-L cells (MOSE-LFFLv). Implantation of these cellsresults in rapid and widespread peritoneal outgrowth andascites in syngeneic C57BL/6 female mice. At 21 dayspostinjection of 1 � 104 MOSE-LFFLv cells, nulliparous andparous mice were euthanized and the PCI was assessed.Young nulliparous mice were chosen for comparison, asonly minor changes in gene expression and no notablechanges in the immune microenvironments were notedbetween young and mature nulliparous groups. We rea-soned that these comparisons would provide additionalinsights into mechanistic alterations in the immune micro-environment as a consequence of peritoneal tumor dissem-ination. Notably, parous mice had a significantly decreasedPCI compared with nulliparous mice (Fig. 3A; P < 0.01). Toconfirm this, we determined relative levels of the FFLreporter gene in the OFB using qRT-PCR. FFL expression,as evidenced by lower DCt values (normalized to the house-keeping gene L19), was significantly higher in the OFB ofMOSE-LFFLv–injected nulliparous mice than in parous mice(P < 0.05; Fig. 3B). TheOFB fromPBS�/� injectedmicewerenegative for FFL reporter gene expression.

Macroscopically, the OFBs in nulliparous mice weredevoid of residual adipose tissue and were fully overtakenby fibrous tumor tissue. The average OFB weight increasedsignificantly in cancer-bearing nulliparous mice (Fig. 3C)and 60% of mice developed bloody ascites. In contrast,

cancer-bearing parous mice presented with visible individ-ual tumornodules throughout theOFB,but residual adiposetissue was still highly evident, and no ascites was observed.The OFB weight did not change between MOSE-LFFLv- andvehicle-injected parous mice, confirming a significantlylower OFB tumor burden than in nulliparous mice.

The aggressive growth of MOSE-LFFLv cells in nulliparousanimals coincided with a 15-fold increase in the SVF cellcount of the OFB (Fig. 3D), although the proportion ofCD45þ leukocytes did not change (Fig. 3E). This suggeststhat the tumor microenvironment induced a proliferationor influx of leukocytes and CD45� stromal constituents.This trendwas not observed in parousmice (Fig. 3D and E),indicating that the OFB microenvironment of parous ani-mals is inherently more resistant to either tumor outgrowthor the recruitment of protumorigenic factors or cell types.

OFB SVF characterization with ovarian cancerThe OFB and PSF of ovarian cancer-bearing mice were

further characterized via FACS analysis to determine theimpact of cancer cell seeding on leukocyte populations.Tumor outgrowth caused a reduction in the proportion oflymphocytes and a concomitant increase in monocyte/granulocytes in the OFB, irrespective of parity (Fig. 4A).This pattern was also evident in the PSF, with an increase inthe proportion of monocytes/granulocytes of both nullip-arous (19% � 11.0%; P ¼ 0.21) and parous mice (15% �1.6%; P < 0.01; Supplementary Table S2).

Most notably, there was a significant increase in theCD11bþLy6Cþ Ly6Gþ population (Fig. 4B; P < 0.001) asa result of cancer cell seeding in the OFB. This populationresembles the previously described CD11bþLy6CþLy6Gþ

tumor-associated neutrophil (TAN) population (19). Inaddition, the cancer-associated redistribution of R1:R2 leu-kocytes in the OFB of nulliparous mice was mirrored acrossall subsets with decreased CD3þ T cells, CD19þ B cells, andNK1.1þ NK cells, and concomitant increases in F480þ

macrophages and CD11cþ dendritic cells. Although levelsof Th cells and TC cells declined with cancer seeding, bothdouble-negative (CD3þCD4�CD8�: DN-T cells) and NK-Tcell subsets increased although significance was not quitereached (Fig. 4C). The DN-T cells may represent either animmunosuppressive T-cell population that secretes Tgf-band Il-10 and directly kills CTLs (20) or gd T cells (20, 21)albeit neither gd TCR expression nor intracellular cytokineexpression were examined in this study.

No differences in the proportions of B1- and B2-lympho-cytes were noted in the OFB of cancer-bearing nulliparousanimals (Fig. 4D). However, in the PSF, the B1:B2 ratioincreased from 3:1 in the homeostatic state to 5:1 due toMOSE-LFFLv implantation (P < 0.05; data not shown). As B1cells act as the bridge between innate and adaptive immu-nity, producing low-affinity antibodies, this may be reflec-tive of an ineffective humoral immune response similar tohuman patients that present with antitumor antibodies thatafford no disease protection (22). It is important to notethat while the proportion of B1:B2 cells increased withcancer, the overall percentage of both B1- and B2-cells in

Cohen et al.





the PSF leukocyte population actually decreased as a resultof shifting R1:R2 ratios. Furthermore, the CD11blo/þF480�

monocyte subset levels significantly decreased as a result ofcancer (Fig. 4E). Therefore, the cancer-associated OFBimmune profile in nulliparous mice included increasedproportions of TANs and macrophages and decreased lym-phocyte and monocyte populations.To complement our FACS analysis, we used qRT-PCR to

characterize the overall signaling milieu of the OFB micro-environment after peritoneal cancer dissemination. Expres-sion patterns represent the tissue as a whole, and thus arereflective of adipocytes, the SVF, and cancer cells within thetissue. A panel of cytokines and chemokines was used toprovide an overview of genes that may contribute to aprotumorigenic microenvironment (Supplementary TablesS3 and S4). The expression of B-cell chemoattractants(Cxcl13 and Il-5) was significantly lower, whereas mono-cyte-associated cytokines (Ccl2, Ccl20, inos, Arg1, and Ym1)and neutrophil chemoattractants (Cxcl1, Cxcl3, Cxcl5, andCccl3) were significantly higher in nulliparous mice aftercancer seeding (Fig. 4F). These changes in the microenvi-

ronmental transcriptome support the cellular changesfound in the SVF, namely the decrease in B cells andapproximately 100-fold increase in TANs in the OFB ofcancer-bearing nulliparous mice.

In contrast to nulliparous animals, there was no signif-icant net gain of leukocytes in the OFB of parous animalsafter tumor outgrowth (see Fig. 3D and E). However, asimilar cancer-associated shift in the leukocyte subsets wasdetected, denoted by a decline in T and B cells and anincrease inmacrophages and TANs (Fig. 5A). Among CD3þ

leukocytes (Fig. 5B), only an increase in the CD4�, CD8�

double-negative subset was noted as a consequence oftumor outgrowth. The influx of the TANs was significantlyreduced, representing only 7% of the CD45þ population inparous mice, as opposed to the 25% in nulliparous animals(see also Fig. 4B). This suggests that cancer-mediated recruit-ment of TANs to the OFB may be compromised or damp-ened as a consequence of parity. It should also be noted thatall lymphocyte subsets weremaintained at high levels in theOFB of parous animals, irrespective of cancer, which mayhelp to maintain an activated state that is less conducive to

Figure 3. Parity mitigates tumorburden and metastasis-associated influx of stromal cellsto the OFB following MOSEFFL

intraperitoneal implantation.Direct comparison of the SVF ofOFBs obtained from young adultnulliparous (yNP) andmature adultparous (aP) mice without (PBS)or following MOSE-LFFLvimplantation. A, PCI (n ¼ 10).B, FFL gene expression in OFB oftumor-bearing mice comparedwith their PBS-injectedcounterparts. C,whole tissueOFBweight. D, total yield of SVF cellsisolated from digested OFBs. E,percentage of CD45þ populationin the OFB SVF. �, P < 0.05;��, P < 0.01.






cancer cell proliferation. This is further supported by the B-cell subset distribution shift toward the B2 phenotype inparous animals (Fig. 5C), which contrasts that observed innulliparous animals (Fig. 4D). Unlike nulliparous mice,there was no significant cancer-associated decline inCD11blo/þF480þ monocytes in parous mice (Fig. 5D).However, these levels were already significantly elevated inthe homeostatic parous OFB.

TANs in the parous PSF were elevated as a result of cancer(P < 0.01; Supplementary Table S2), albeit at a lower levelthan in nulliparous mice. In addition, the PSF in parousanimals displayed a loss of LPMs and an increase in SPMsafter MOSE-LFFLv dissemination. The recruitment of SPMsis indicative of an innate-like inflammatory response, thusparous animals may be able to override, at least transiently,the inherent immunosuppressive program elicited by theMOSE-LFFLv cells.

Comparative assessment of the gene expression profile ofthe parous OFB revealed that the expression of neutrophilchemoattractants was significantly higher in cancer-bearingmice (Cxcl1,Cxcl2,Cxcl3, andCxcl5; Fig. 5E). This correlateswith the recruitment of TANs following cancer cell seeding,highlighting the importance of TANs in the propagation ofthe protumorigenic microenvironment. Together, our datasuggest that the preexisting parity-associatedmicroenviron-ment within the peritoneal cavity is inherently more resis-tant to tumor cell seeding and outgrowth by virtue of itsability tomodulate recruitment of protumorigenic immunecell types, either before tumor cell attachment (premeta-static niche) or following seeding and outgrowth.

DiscussionEpidemiologic studies indicate that parity reduces the risk

of both ovarian and breast cancer development. Although

Figure 4. Ovarian cancer outgrowthelicits macrophage and neutrophilinflux into the OFB of young adultnulliparous (yNP) mice. A,comparative distribution of R1(lymphocytes):R2(monocyte/granulocyte) CD45þ populations inyNP and mature adult parous (aP)OFB as a consequence of cancerdissemination. B–D, immune cellchanges in the OFB of yNP micefollowing MOSE-LFFLv seeding andoutgrowth were determined byFACS analysis. Overall leukocyteprofile (B) and changes withinCD3þ subsets (C) CD19þ subsets(D) and monocyte/granulocytesubsets (E) are depicted. F, geneexpression changes in the OFBs ofMOSE-LFFLv-bearing yNP micecompared with age-matched naïvemice (yNP PBS). All genesdisplayed were significantly(P < 0.05) changed from PBScontrol. Statistical significance(P values) are provided or denotedby �, P < 0.05; ��, P < 0.01.

Cohen et al.





the molecular mechanisms of decreased ovarian canceroccurrence remain unclear, breast cancer studies suggestthat protective effects relate to decreased cancer metastasis

(3). It is possible that child-bearing results in a naturallyoccurring protective state that could be harnessed and usedin the treatment of ovarian cancer, preventing fatal

Figure 5. Parity mitigates ovarian cancer outgrowth-associated influx of macrophages and neutrophils to the OFB. Immune cell changes in the OFB of matureadult (aP)mice followingMOSE-LFFLv seeding and outgrowthwere determined by FACS analysis. Overall leukocyte population changes in theOFB (A) aswellas cancer-associated changes within CD3þ subsets (B), CD19þ subsets (C) and monocyte/granulocyte subsets (D) are presented. E, gene expressionchanges in the OFBs ofMOSE-LFFLv-bearing aPmice compared with age-matched naïvemice (aP PBS). All genes displayed were significantly changed fromPBS controls; �, P < 0.05; ��, P < 0.01.






metastatic outgrowth. Our results show that parity, but notage, altered the OFB immune profile in the homeostaticstate. This parity-associated immune profile was positivelycorrelated with reduced peritoneal tumor burden. A morecomprehensive understanding of the dynamic cellularinteractions during metastatic outgrowth is critical for thedevelopment of treatment strategies to effectively target thetumor microenvironment at the time of disease discovery.These insights may ultimately lead to novel strategies torepolarize the immune microenvironment of the OFB in amanner that reflects a microenvironment refractory to can-cer growth.

Here, we compared the immune profile of the OFB as afunction of age and parity to assess whether a preexistingmicroenvironment exists that may be refractory to tumorgrowth. To this end, we characterized the leukocyte com-position and cytokine expression profiles of the OFB andPSF, both in the homeostatic state and after aggressiveovarian cancer cell implantation. In the homeostatic state,

parity (but not age) resulted in a marked decrease inmonocytic cell subsets and affiliated chemoattractants, anda shift to predominantly B2-, as opposed to B1 B lympho-cytes. Following syngeneic cancer cell implantation, parousmice exhibited a significantly reduced tumor burden ascompared with nulliparousmice. The tumormicroenviron-ment was denoted by higher TANs and TAMs, and anincrease in B1 cells in nulliparous animals, a trend thatwas dampened in parous mice in conjunction withdecreased tumor growth. Thus, we postulate that the parousOFB compositional profile is indicative of a preexistingniche that is partially refractory to metastasis.

Tomore fully elucidate an expression signature indicativeof an inherently protective immune microenvironment inthe parous state, we evaluated a panel of immunoregulatorycytokines and chemokines. Figure 6 summarizes all statis-tically significant gene expression changes associated withage, parity, cancer metastasis, or any combination therein(P < 0.05; see Supplementary Tables S3 and S4 for full list of

CCL5

CCL21

IFN-g*

TNF-aIL-21*

CCL1

CCL4*

CCL19*

IFN-g*

CD36*

YM1*

TGF-b*

GATA3*

CCL2

CCL17*

CXCL1*

CXCL2*

CXCL13

CXCL16*

MCSF*

IL-5*

IL-6*

Arg1*

M6pr*

CCL7*

CXCL13*

IL-2*

IL-13

CCL7*

CCL17

CXCL2*

CCL19*

CCL21*

IDO*

iNOS*

IL-12

CXCL3*

CXCL12*

CXCL1*

CXCL5*

Arg1*

CCL2*

CCL3*

CCL20*

Ym1

MCSF

GATA3*

CD36*

CD1d*

IL-5*

CXCL13

iNOS

IL-10*

IL-4*

M6pr

IL-12

IDO*

Parity-related

(aNP vs. yNP)

Age-related

(aNP vs. yNP)

Cancer-related

(yNP+MOSE-LFFLv

vs. yNP)

Cancer-related

(aP+MOSE-LFFLv vs. aP)

Figure 6. Gene expression signatures in the OFB associated with age, parity, or cancer cell seeding and outgrowth. Venn diagram representing significantlyaltered genes �, P < 0.05, and genes trending toward significance P¼ 0.05–0.08. White boxes indicate increased expression, gray boxes indicate decreasedexpression.

Cohen et al.





genes evaluated). Age-related changes included a signifi-cantly increased expression of Cxcl13 and Il-2 in the OFB.Cxcl13 is a B-cell chemoattractant and Il-2 induces theproliferation and activation of T cells; both are importantsignaling molecules in secondary lymphoid organs. Parity(but not age) was associated with a significant decrease inseveral chemoattractants for neutrophils (Cxcl1 and Cxcl2;P<0.05) andmonocytes (Ccl2 andMcsf;P<0.05), aswell asmarkers associated with alternately activated, protumori-genic phenotypes (Arg1 andM6pr; P < 0.05). This correlateswith the observed reduction of these cell types in the parousOFB, and may represent a metastasis-resistant microenvi-ronmental signature. The most conspicuous cancer-relatedfinding was the significant increase in neutrophil chemoat-tractants (Ccl3,Cxcl1, -2, -3, and -5). Cancer-bearing groupsalso exhibited increased Arg1 expression, and young adultnulliparous mice had increased Ym1 expression, indicativeof the presence of TAMs.Given the protumorigenic nature of TAMs (23, 24), TANs

(25, 26), andmyeloid-derived suppressor cells (27, 28), thecancer-related increase in these cell types was expected.Therefore, the dampened influx of these subsets in parousmicemay partially define the preexisting protective niche inthe OFB. In other words, parity leads to a reduction in thelevels of cell subsets targeted for protumorigenic polariza-tion. A microenvironment that lacks these subsets, or ispartially refractory to cancer-associated recruitment mayslow the dynamics of tumor seeding and growth, resultingin delayed disease development.The parous OFB also contained a greater proportion of

both T- and B lymphocytes. Tumor-infiltrating lympho-cytes (TIL) correlate with improved prognosis and sur-vival in a variety of murine and human cancer models(29–31). Therefore, a net gain in lymphocytes may beanother attribute of the protective parity signature in theOFB. Although the effects of increased T-cell populationsin the tumor microenvironment have been well defined,less information is available describing tumor-infiltratingB cells (TIL-B) and their impact on cancer progression(32). The presence of CD20þ B cells together with CD8þ Tcells in the tumor microenvironment has been attributedto higher survival rates than the occurrence of either celltype alone, indicating a protective cellular signature (33).Interestingly, TIL-Bs produce granzyme B upon interleu-kin (IL)-21 stimulation, which would be cytotoxic totumors; upon IFN-a or Toll-like receptor (TLR) agoniststimulation, they can directly kill tumor cells via TRAILsignaling (34, 35), also supportive of their antitumori-genic potential. In contrast, killer B cells produce apo-ptotic death-inducing ligands including Fas ligand (FasL),TRAIL, and programmed death ligands 1 and 2 (PD-L1and PD-L2; ref. 36). However, their presence in the tumormicroenvironment actually inhibits protective immuneresponses, inducing apoptosis of cytolytic effector cellsinstead of malignant cells (36). Regulatory B cells, char-acterized by TGF-b and IL-10, also dampen and inhibitprotective immune responses against tumors (37) byimpairing T-cell priming. Hence, B lymphocyte subsets

are gaining widespread acceptance as key regulators andmodulators of both pro- and antitumorigenic responses.

In this study, there was a shift in the B-cell distribution ofB1 and B2 cells as a consequence of parity, with B2 cellsincreasing significantly in the parous OFB. B1 cells play animportant role in immunosurveillance in the peritonealcavity, and are in a constant flux between the PSF and theOFB. During infection, B1 cells generate large amounts oflow-affinity immunoglobulinM (IgM), immunoglobulin A(IgA), and immunoglobulin G3 (IgG3), ensuring earlyprotection (38). B1 cells have been described as the pre-cursors of tumor-promoting regulatory B cells (Breg;ref. 39). B1 cells also possess many of the above-mentionedregulatory properties, including constitutive FasL and PD-L2 expression and the ability to produce IL-10. Although theB2 subset is predominant in the parous OFB in the homeo-static state, with cancer cell seeding, the proportion of B1:B2cells increases slightly in both parous and nulliparousmice.Collectively, this information may support the theory thatB1 cells are subject to repolarization toward Bregs in thepresence of cancer. Alternatively, the parity associatedomental B2 cells may represent a subset such as the afore-mentioned killer B cells that exhibit antitumorigenic activ-ity. Future studies defining which B-cell subsets in parousanimals are actually antitumorigenic will help clarify thisquestion.

Parity was indeed associated with a significant reductionin tumor burden following syngeneic ovarian cancer cellimplantation in the peritoneal cavity. The most significantcancer-associated findingwas the increase in the proportionof TANs in the OFB. Hence, the ability to modulate earlyTAN infiltration may represent another protective elementof the parous OFB. TANs are a significant portion of theinflammatory infiltrate in a variety of tumor microenviron-ments and numerous cancer cell lines express neutrophilchemoattractants, highlighting their importance in sup-porting the tumor milieu (40–43). Although depletion ofTANs limits tumor metastasis, neutrophil accumulation intumors is correlated with poor prognosis in clinical settings(41, 44, 45). The presence of TGF-b in the tumor microen-vironment is the driving factor in the polarization of neu-trophils to a protumorigenic (or "N2") type (46).We foundthat Tgf-b expression was increased 100-fold in the OFB inboth older nulliparous andparous animals. This age-relatedincrease may play a crucial role in the increased ovariancancer incidence in postmenopausal women. The decreasedtumor burden observed in parous mice may be a result ofdecreased protumorigenic subsets and chemoattractantexpression (e.g., TANs and Cxcl1, -2) immediately availablewithin the OFB, despite increased Tgf-b expression.

Parity also resulted in an almost complete loss of CD45�

progenitor populations present within the OFB. An impor-tant component in the tumor cellular milieu is the stromalcontingent. These cells are recruited fromadipose tissue andbone marrow to provide crucial growth factors, matrices,cytokines, andnutrients to support rapid tumor growth (47,48). The loss of progenitor cells in the OFB may reflectanother aspect of the "protective signature" associated with






child-bearing, in that the scaffolding or accessory cellscrucial to rapid tumor growth are not initially available insitu. This may indicate a parity-associated differentiation oregress of CD45� progenitor populations within the OFB, inagreement with an increase in differentiation signals pre-viously reported as part of a "parity signature" inmammarytissue (3).

The OFB is not only important as a preferential site forovarian cancer, but also because of its role in peritonealimmunosurveillance. Its removal results in subsequentimpaired antibacterial responses in the abdomen, and anincreased chance of sepsis following surgery (5). Clearly,removal of this vital organ is not ideal, but it is generallyregarded as necessary to help minimize recurrent disease.Our study suggests that a more comprehensive analysis ofthe parity-induced molecular and cellular changes withinthis immunologically and metastatically relevant microen-vironment is needed in humans. Confirmation of a protec-tive "parity-associated signature" in humans could help inthe design of novel immunotherapies that induce or estab-lish this inherent protective state (3).

In conclusion, our data support epidemiologic findingssuggesting that child-bearing provides inherent partial pro-tection against ovarian cancer (11, 49, 50). It isworthnotingthat the parity-associated protective microenvironment inthe peritoneal cavity is only transient and eventually theoutgrowth of cancer cells shifts the balance back toward aprotumorigenic state. Understanding what defines the tran-sient nature of this refractory state may provide novelinsights tomodulate theOFB and prevent recurrent disease.Although our studies do not address whether parity miti-gates early stages of ovarian tumorigenesis, they do high-light a protective effect against peritoneal seeding and

outgrowth. Future studies are clearly warranted to under-stand how the OFB could be repolarized toward a parity-associated protective state in high-risk patients, or modu-lated to help delay or prevent recurrent disease in patientswith advanced-stage ovarian cancer.

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

Authors' ContributionsConception and design:C.A. Cohen, A.A. Shea, E.M. Schmelz, P.C. RobertsDevelopment of methodology: C.A. Cohen, A.A. Shea, P.C. RobertsAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C.A. Cohen, A.A. Shea, C.L. Heffron, P.C. RobertsAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C.A. Cohen, P.C. RobertsWriting, review, and/or revision of the manuscript: C.A. Cohen, A.A.Shea, E.M. Schmelz, P.C. RobertsStudy supervision: E.M. Schmelz, P.C. Roberts

AcknowledgmentsThe authors thankMelissaMakris for helpwith FACS experimental design

and analysis. This article fulfills in part the PhDdissertation requirements forC.A. Cohen in the Department of Biomedical Sciences and Pathobiology atVirginia Tech (Blacksburg, VA). C.A. Cohen was supported in part by agraduate research assistant fellowship provided by the Virginia-MarylandRegional College of Veterinary Medicine (VMRCVM) at Virginia Tech.

Grant SupportThis research was supported in part by NCI research grant CA118846 (to

E. Schmelz and P. Roberts) and funds provided by the Fralin Life SciencesInstitute at Virginia Tech (to P. Roberts and E. Schmelz).

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.

Received June 21, 2013; revised August 7, 2013; accepted August 30, 2013;published OnlineFirst September 10, 2013.

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2013;6:1182-1193. Published OnlineFirst September 10, 2013.Cancer Prev Res Courtney A. Cohen, Amanda A. Shea, C. Lynn Heffron, et al. Fat Band Is Refractory to Ovarian Cancer MetastasisThe Parity-Associated Microenvironmental Niche in the Omental

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