Tumor Microenvironment

The Aged Microenvironment Influences theTumorigenic Potential of MalignantProstate Epithelial CellsDaniella Bianchi-Frias1, Mamatha Damodarasamy2, Susana A. Hernandez1,Rui M. Gil da Costa1, Funda Vakar-Lopez3, Ilsa M. Coleman1, May J. Reed2, andPeter S. Nelson1,2,3

Abstract

The incidence of prostate cancer is directly linked to age,but age-associated changes that facilitate prostate cancerdevelopment and progression are poorly understood. Thisstudy investigated age-related changes in the prostate micro-environment for their influence on prostate cancer behavior.Prostate cancer cells implanted orthotopically into the pros-tate demonstrated accelerated tumor growth in aged com-pared with young mice. Metastatic lesions following intra-venous injection were also more numerous in aged mice.Tumors from young and aged mice showed no significantdifferences concerning their proliferation index, apoptosis,or angiogenesis. However, analysis of tumor-infiltratingimmune cells by IHC and RNA sequencing (RNA-seq)revealed elevated numbers of macrophages in prostatesfrom aged mice, which are quickly polarized towards aphenotype resembling protumorigenic tumor-associatedmacrophages upon tumor cell engraftment. Older patientswith prostate cancer (>60 years old) in The Cancer Genome

Atlas Prostate Adenocarcinoma (TCGA-PRAD) datasetdisplayed higher expression of macrophage markers(CD163 and VSIG4) which associated with higher rates ofbiochemical relapse. Remodeling of the collagenous extra-cellular matrix (ECM) was associated with prostate cancergrowth and invasion in the aged microenvironment. More-over, the collagen matrix extracted from agedmice enhancedthe invasiveness and proliferation of prostate cancer cells invitro. Together, these results demonstrate that the agedprostatic microenvironment can regulate the growth andmetastasis of malignant prostate cells, highlighting the roleof resident macrophages and their polarization towards aprotumorigenic phenotype, along with remodeling of theECM.

Implications: These findings demonstrate the importanceof age-associated tumor microenvironment alterationsin regulating key aspects of prostate cancer progression.

IntroductionOrganismal aging is associated with a spectrum of molecular,

cellular, and physiologic changes that contribute to increasinghomeostatic imbalance and the development of diseases such ascancer. Notably, the greatest risk factor for the development ofprostate cancer is advanced age. The incidence of invasive prostatecancer increases significantly with each decade of life after age 40with prostate cancer identified in 0.3%, 1.9%, 5.4%, and 9.1% ofmen aged �49, 50–59, 60–69, and >70 years (1). Although amajor contributor to prostate cancer development involves eventsintrinsic to epithelial cells encompassing DNA mutations, chro-mosomal gains and losses, and gene rearrangements, it is also

recognized that features involving the tumor microenvironmentpromote neoplasia and influence tumor behavior after initiation.Experiments using rodent and rodent/human in vivo and in vitrococulture models support the idea that paracrine interactionsbetween the luminal epithelial cells and the reactive adjacentstroma cells play an important role in the development of benignprostatic hyperplasia (BPH) and prostate cancer (2, 3). In vitroexperiments have demonstrated that cellular and molecularalterations in the prostate microenvironment including thoseassociated with stromal aging and senescence can promote neo-plastic progression (4–6).

A direct role of the aged-host tissue microenvironmentin tumorigenesis was demonstrated by McCullough and col-leagues who showed that the tumorigenic potential of neo-plastic transformed rat liver epithelial cells was differentiallyregulated after their transplantation into the normal livers ofyoung and old rats (7). The rate of tumor formation increasedwith the age of the host recipients. Conversely, althoughtumors initially formed in young hosts, they subsequentlyregressed. These results strongly suggest that the tissue micro-environment is an important determinant in the age-relatedtumorigenic potential of transformed cells, supporting theconcept that age-associated increases in cancer incidence mayresult from both the accumulation of mutations (intrinsic tothe tumor cells), and age-related pro-oncogenic changes in thetissue microenvironment (8–10).

1Divisions of Human Biology and Clinical Research, Fred Hutchinson CancerResearch Center, Seattle, Washington. 2Department of Medicine, University ofWashington, Seattle, Washington. 3Department of Pathology, University ofWashington, Seattle, Washington.

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

Corresponding Author: Peter S. Nelson, Fred Hutchinson Cancer ResearchCenter, Mail Stop D4-100, 1100 Fairview Avenue N, Seattle, WA 98109. Phone:206-667-3377; Fax: 206-667-2917; E-mail: pnelson@fredhutch.org

doi: 10.1158/1541-7786.MCR-18-0522

�2018 American Association for Cancer Research.

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Although the specific alterations associated with the cancerpromoting effects of the aged-human prostate stroma have yet tobe defined, we have previously reported that the prostate micro-environment in normal aged mice exhibits distinctive and repro-ducible differences compared with strain-matched, geneticallyidentical young mice. The age-associated changes include theupregulation of a spectrum of cytokines and paracrine-actinggrowth factors, an increase in infiltrating immune cells and adisrupted collagen matrix (11). We hypothesized that thesephenotypic and molecular characteristics could plausibly con-tribute to the striking age associated pathologies affecting theprostate. In this study, we sought to evaluate the direct effects ofthe aged prostatic microenvironment on prostate tumor growthand metastatic spread.

Materials and MethodsMice

MaleC57BL/6andFVBmice, 8weeksof age,wereobtained fromThe Jackson Laboratory and Taconic, respectively. Animals weremaintained and aged in a barrier facility and cared for in accor-dance with approved IACUC protocols (file 1618 and 1671). Allanimals were maintained pathogen-free in the Fred HutchinsonCancer Research Center animal facility that is fully accredited bythe Association for Assessment and Accreditation of LaboratoryAnimal Care.

Cell line and cultureTRAMP-C2 cells were a generous gift from Dr. Norman

Greenberg (Fred Hutchinson Cancer Research Center, Seattle,WA). The TRAMP-C2 cell line no longer expresses the SV40early genes (12) and forms tumors when grafted subcutane-ously into syngeneic C57BL/6 hosts (12, 13, 14). Myc-CaP cellswere obtained from Dr. Charles Sawyers and were establishedfrom a primary prostate carcinoma dissected from a 16-month-old Hi-Myc transgenic mouse in the FVB inbred strain (15).Cells were maintained in DMEM supplemented with 10% FBS(Omega Scientific) at 37�C.

Orthotopic injectionsFor tumor implantation, TRAMP-C2 and Myc-CaP cells were

grown to 80% confluence in T150 flasks, trypsinized, washedwith PBS and resuspended in PBS at a concentration of 106

cells/20 mL. The mice were anesthetized with isofluorane andplaced in supine position. A midline incision was made in thelower abdomen. After the abdominal wall muscles were split,the bladder was exposed and then retracted anteriorly to revealthe dorsolateral prostate. 0.5 to 1 � 106 TRAMP-C2 or Myc-CaPcells suspended in 20 mL of PBS were carefully injected into theleft dorsolateral mouse prostate using a 13 mm 17-gaugeneedle and a 1 mL syringe. A slight elevation of the leftdorsolateral prostate capsule was considered indicative of cor-rect deposition of tumor cells. The abdomen was closed bysingle-stitch sutures using 4-0, Gut-Plain.

At different time points after injections (3-, 7-, 14-, and 30–45days postinjection), mice were sacrificed by deep anesthesiafollow by cervical dislocations. Prostate tumors were resected,weighed, and processed for further analyses. After careful inspec-tion for macroscopically detectable metastases, enlarged lymphnodes, lungs, livers, kidneys, mesentery, and other organs werecollected.

HistologyAll tumors were removed and either embedded in Tissue-Tek

(Sakura Finetechnical Co., Ltd.) and stored at �70�C until anal-ysis of frozen sections, or formalin-fixed and embedded in par-affin (FFPE). FFPE tissue sections, 4-mmthick, were deparaffinizedand processed for H&E staining, Masson's trichrome staining (forcollagen detection) or immunostaining. For IHC, tissue sectionswere deparaffinized, and endogenous peroxidase activity wasblocked with 3% H2O2 for 8 minutes. Antigen was retrieved bysteam heating with 10 mmol/L citrate buffer (pH 6.0) for 20minutes. Primary antibodies and working dilutions were asfollows: CD3 (1:500; Serotech MCA1477); F4/80 (1:50; SerotechMCA497GA); KI67 (TEC-3 clone; DakoM7249). CASP3 (BiocareCP229C); LY6G (1:500; BioLegend 127601); FOXP3 (1:25;eBioscience 14-5773-82), B220 (1:1,000; Chemicon CBL1342),and ARG1 (Millipore ABS535).

For Masson's trichome scores, the staining intensity of collagenfiberswas assessed at 100�. Staining intensitywas assessed as null(0); light (1: focal staining, with small occasional positive colla-gen fibers); moderate (2: multifocal to diffuse staining, withnumerous positive collagen fibers); and strong (3: diffuse stain-ing, with bundles of positive collagen fibers).

For blood vessel detection, frozen sections were air-driedand fixed in acetone, pretreated with 3% H2O2, blocked over-night in PBS with 2% normal goat serum and incubated withprimary antibody (MECA32; Developmental Studies Hybrid-oma Bank, University of Iowa, Iowa City, IA) at 2 mg/mL. For allimmunostained sections, after incubation of primary antibodyfor 1 to 2 hours, slides were washed and incubated for 30minutes with biotinylated species-specific secondary antibo-dies, washed, and then incubated with an avidin–biotin–per-oxidase complex (ABC; Vector Laboratories) for 30 minutes andvisualized using DAKO DAB system. Sections were counter-stained with hematoxylin, dehydrated with a graded series ofethanol, cleaned with xylene, mounted and visualized by lightmicroscopy. In all experiments, the secondary antibody aloneserved as a negative control.

ProliferationAnalysis with an anti-KI67 antibody identified proliferating

cells. Images were taken from four random fields at 400� mag-nification for each tumor and KI67-positive cells (nuclei showingnuclear immunoreactivity of any intensity) were counted from 4mice per age group at each time point (three, seven, fourteen, andthirty days postinjection), with the exception of the 14-daysyoung age group, where TRAMP-C2 cells were observed in onlyone mouse. The proliferation index was calculated as the ratio ofKI67-positive tumor cells to all counted tumor cells. Data arepresented as means and standard errors.

Cell deathApoptotic cells were identified by Cleaved caspase-3 staining.

The quantification of CASP3-positive cells was carried out bycalculating the percentage of area positive for DAB stain in one�100 field (3 and 7 days postinjection) and two to three random�100 fields for (14 and 30 days postinjection), using a thresholdin ImageJ of 90. We first selected the region of interest (ROI),excluding any host prostatic acini, and only calculating positivityin the tumor itself. The HDAB color separation from ImageJ wasused and total and positive DAB areas in ROI were calculated.Data are presented as means and SEs.

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Immune cell scoresThe presence of CD3þ, B220þ, FOXP3þ, and LY6Gþ cells was

analyzed by assigning a score based on the percentage of positivecells, counted in 3 random 100� fields. Scores were assigned asfollow: 1: scattered positive cells (<2%); 2: moderately abundantpositive cells, diffuse or arranged in foci (2%–10%), and 3:abundant positive cells, diffuse or arranged in foci (>20%).

The quantification of F4/80þ and ARG1þ cells was carried outusing ImageJ, by calculating the percentage of area positive forDAB stain in one 100�field (3 and 7days postinjection) or in twoto three random 100� fields (larger tumors at 14 and 30 dayspostinjection), using a threshold of 90. We first selected the ROI,excluding any host prostatic acini, and only calculating positivityin the tumor area. ARG1þ cells were also separately scored at thetumor periphery and adjacent to normal prostate epithelium asfollows: 1: scattered positive cells (5%–20%); 2: abundant pos-itive cells (20%–50%),multifocal or diffuse; and3: very abundantpositive cells (>50%), diffuse.

Blood vessel areaFor image analysis, one random imageper samplewas captured

for each tumor using the 10� objective lens. Images were subse-quently analyzed using Adobe Photoshop (Adobe, San Jose, CA)with Fovea Pro plug-in (Reindeer Graphics). Briefly, equivalentthresholds were applied to all images to generate the ROI, whichincluded only areas of positive staining. Themeasured image areawas obtained and subsequently used to infer relative areas ofstaining as of function of themean of these image area values for agiven sample.

RNA-sequencing and gene expression analysisOrthotopic TRAMP-C2 tumors resected from young and aged

mice were homogenized in Qiagen RLT buffer, and RNA wasisolated using the RNeasy Extraction Kit (Qiagen). RNA concen-tration, purity, and integrity was assessed by NanoDrop (ThermoFisher Scientific Inc.) and Agilent Bioanalyzer. RNA-seq librarieswere constructed from 1 mg total RNA using the Illumina TruSeqStranded mRNA LT Sample Prep Kit according to the manufac-turer's protocol. Barcoded librarieswerepooled and sequencedonthe Illumina HiSeq 2500 generating 50 bp paired end reads.Sequencing reads were mapped to the mm10 mouse genomeusing TopHat v2.0.12 (16). Gene-level abundance was quanti-tated from the filtered mouse alignments in R using the GenomicAlignments Bioconductor package. Differential expression wasassessed using transcript abundances as inputs to the edgeRBioconductor package in R (17).

Survival and pathway analysisThe Cancer Genome Atlas Prostate Adenocarcinoma

(TCGA-PRAD) cohort containing 490 RRP samples with clinicaland RNA-seq expression values was utilized for analysis of prog-nostic features (18). We used the clinical and expression RSEMvalues hosted by the cBioPortal (http://www.cbioportal.org) toanalyze biochemical recurrence (BCR) in patients with lowerand higher expression values of genes associated with the agedprostate microenvironment. A cutoff of each gene's mean expres-sion was used to stratify patients into different outcome groups.Survival analyses were computed and visualized using thesurvival3 CRAN package in R and GraphPad Prism v7.03.Kaplan–Meier log-rank test P-values are shown on each plot.

Gene ontology (GO) enrichment was computed using GOrillawith default parameters (19). The "single ranked list of genes"option was used and genes in the RNA-seq dataset were rankedusing the increasing P-value generated by edgeR.

Collagen growth and invasion assaysCollagen preparation. To prepare collagen for subsequent 3D gels,an equivalent wet weight of tail tendon from 10 young and 10aged euthanized mice were pooled in respective "young" and"aged" groups, hydrated briefly in PBS, rinsed in acetone and 70%isopropanol, macerated, and stirred gently overnight at 4�C in0.05Nacetic acid. Subsequently, the "young" and "aged" collagenextracts were centrifuged to remove undissolved material. Totalprotein was quantified by a bicinchoninic acid assay (ThermoFisher Scientific Inc.), and specific collagen contentwas quantifiedby the Sircol assay (Accurate Chemical and Scientific Corp.;ref. 20). For all subsequent collagen gel assays, both young andaged collagens were polymerized to a final concentration of 0.6mg/mL collagen as measured by the Sircol assay.

Cell proliferation assays. Young or aged collagen gels (35 mLvolume) were polymerized in each well of a 96-well plate.Subsequently, 5,000 cells were placed on each gel, and an indexof the proliferative activity after 24 and 48 hours of culture wasmeasured with a Cultrex 3DCulture Cell Proliferation Assay Kit(Trevigen Inc.).

Cell invasion assay. Young or aged collagen gels were polymerizedon top of 8 mmol/L fluoroblock inserts (Fisher 08-772-142).TRAMP-C2 cells were trypsinized, filtered through a 0.40-mmnylon cell strainer, and 5,000 cells were placed on top of each3Dgel. Five-hundredmicroliters ofDMEMþ10%FBSwere addedto the bottom of the chambers, and incubated for 18 hours toallow for cell migration. After 18 hours, cells were labeled withcalcein AM dye (Invitrogen) following the company's protocol.Migratingfluorescently labeled cells were counted byfluorescencemicroscopy at �100 magnification random selected fields.

Statistical analysisComparisons between the measurements from young and

aged mice were performed with unpaired two-tailed t test usingGraphPad Prism version 4.02 for Windows, GraphPad Software,San Diego, CA. Significance was accepted at P < 0.05.

ResultsThe prostate microenvironment in aged mice promotes thegrowth of orthotopic tumors

To test the direct contribution of the aged prostatic microen-vironment toward the growth of prostate cancer cells, we injected106 TRAMP-C2 cells, a cell line derived from a tumor developingin a genetically engineered mouse (GEM) model expressing theSV40T antigen in mouse prostate epithelium (12), into thedorsolateral prostate of immune-competent syngeneic young(4 month-old; n ¼ 20) and aged (20–24 month-old; n ¼ 19)C57BL/6 mice. Thirty- to forty-five days following the introduc-tion of tumor cells, mice were sacrificed and evaluated for tumortake rate and growth. At necropsy, tumorswere evident in allmice,although tumor size varied substantially. In young mice, tumorsaveraged 0.5 � 0.1 g in weight whereas in aged mice, tumorsaveraged 1.2 � 0.1 g (P ¼ 0.0002; Fig. 1A–D). No histologic

Tumorigenic Potential of an Aged Microenvironment

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differences in tumors developing in the young versus aged micewere observed (Fig. 1B and C).

Tumor size differences could reflect slower growth rates inyoung versus old mice, or an initial similar rate of tumorestablishment and growth followed by subsequent tumorregression. To evaluate these possibilities, we injected 5 �105 TRAMP-C2 cells into the prostates of young and agedC57BL/6 mice and sacrificed cohorts for tumor assessments atfour different time points of 3, 7, 14, and 30 days postinjection.Between 3 and 14 days postinjection, no macroscopic tumorswere evident in any of the age groups. However, at 30 dayspostinjection, intraprostatic tumors were evident and signifi-cantly larger in the aged mice compared with young mice(tumors averaged 0.41 � 0.07 g, vs. 0.1575 � 0.04 g, respec-tively, P ¼ 0.023; Fig. 1E). These results indicate that the

differential age-associated effect on TRAMP-C2 cell growth isnot due to tumor regression in the young mice.

To determine if the age-associated effect on tumor growth isconsistent across tumors and host background, we evaluated theeffects of themicroenvironment of aged FVBmice on the growth ofsyngeneic Myc-CaP cells. This cell line was established from aprimary prostate cancer dissected from a 16-month-old Hi-Myctransgenic mouse in the FVB inbred strain (15). We injected 106

Myc-CaP tumor cells into the dorsolateral prostates of young(4-month-old; n ¼ 7) and aged (18-month-old; n ¼ 8) FVB miceand quantitated tumor sizes in both age groups thirty days post-injection. We observed a trend toward larger tumors in aged mice,although there was substantial variation and overall differenceswerenot statistically significant:mean tumorweightwas1.9� 0.3 gin young and 2.4 � 0.5 g in aged mice, respectively (P ¼ 0.39).

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Figure 1.

Influence of an aged prostatemicroenvironment on tumor growth. A–D, TRAMP-C2 tumor cells were injected into the dorsolateral prostate of young (4-month-old)and aged (20- to 24-month-old) C57BL/6 mice. At 30 to 45 days after tumor cell injection, mice were sacrificed and tumors removed for analysis. A, Representativemacroscopic images of the resected orthotopic prostate tumors from young (left) and aged (right) mice. Note larger tumor in the aged mouse. B and C,Representativemicroscopic images from H&E-stained TRAMP-C2 tumor sections from young (B) and aged (C) mice at low and high-power (inset).D, Final prostateweights of control mice and tumor-bearing prostates injected with TRAMP-C2 cells in young and aged mice. Y(wt), A(wt), Y(TC2) and A(TC2), Young (Y), and Aged (A)prostate weight from mice with normal prostates (wt) or prostates injected with TRAMP-C2 cells (TC2), respectively. E, Time course of tumor growth inyoung and aged mice. A total of 5 � 105 TRAMP-C2 tumor cells were injected into the dorsolateral prostate of young and aged C57BL/6 mice. The prostate wasresected and weighed at different time points: 3, 7, 14, and 30 days postinjection. Tumor growth is evident and significantly higher in aged mice at 30 dayspostinjection. F, Percentage of mice injected intraprostatically with 5 � 105 TRAMP-C2 cells that developed peritoneal seeding. G, Percentage of mice withcell seeding foci at 30 days post-intraperitoneal injections of 105 TRAMP-C2 tumor cells. H, Percentage of mice with tumor metastasis at 30 days after5� 105 TRAMP-C2 tumor cells were injected into the tail vein of young (4-month-old) and aged (20- to 24-month-old) C57BL/6 mice. I, Representative images oforgans with metastatic tumors following TRAMP-C2 cells tail vein injections in young and aged mice. P-values: �� , <0.001; � , <0.05. Scale bars, 100 mm.

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Aging enhances prostate cancer cell dissemination andmetastases

During the necropsy for removal and analysis of the prostateglands, we noted that aged mice had high rates of tumor spreadinto the abdomen with grossly visible peritoneal implants. In theexperiments involving TRAMP-C2 cells and the C57BL/6 strain,42% (8/19) of aged mice versus 10% (2/20) of young mice (P ¼0.05) had evidence of peritoneal seeding (Fig. 1F). This findingwas recapitulated in the FVBmice with intraprostatic injections ofMyc-CaP cells, where 67% (6/9) of aged mice and 50% (7/14) ofyoung mice had intraperitoneal tumor cell seeding, and thenumber of organs per mouse with tumor cell seeding was signif-icantly higher in the aged mice (P ¼ 0.05).

We further evaluated the contribution of an aged environmentto metastatic spread by directly injecting TRAMP-C2 cells into theperitoneum (i.p.) of C57BL/6 mice and evaluated tumor forma-tion after 30 days by necropsy. The rate of tumor seeding wassignificantly higher in the aged mice with 83% (15/18) showingvisible peritoneal tumormasses whereas 40% (4/10) of the youngmice exhibited peritoneal tumors (P¼ 0.018; Fig. 1G).Metastaseswere evident in multiple organs including the mesentery, kidney,liver, spleen, and epididymal fat pad (Table 1).

We next injected TRAMP-C2 cells into the tail veins (i.v.) ofaged or young mice and evaluated metastases by necropsy after30 days. After injection of 105 tumor cells, 100% (3/3) of agedmice and none (0/5) of the young mice had metastasis (P <0.001). After injection of 5 � 105 tumor cells 82% (9/11) agedmice and 13% (1/8) young mice had metastasis (P¼ 0.001; Fig.1H and I). Further, in the aged mice, multiple tumors wereoften present in the mesentery, liver, spleen, and occasionallythe kidney (Table 1).

The prostate microenvironment in aged mice does notsubstantially alter cancer cell proliferation, cell death, orangiogenesis

We next sought to determine how the aged prostatic microen-vironment influences aspects of tumorigenesis. We assessedtumor proliferation rates by KI67 IHC. At time-points followingTRAMP-C2 cell injection, the number of KI67 positive tumor cellswas variable and in aged mice did not differ significantly fromyoung mice: at 3 days, the number of KI67 positive cells/20�magnification field in tumors from young mice was: 18.3 � 4.70(n¼ 3) and fromagedmice: 28.13� 2.63 (n¼ 3; P¼ 0.14); and at30 days the number of KI67 positive cells in young mice was22.87 � 2.98 (n ¼ 3) and from aged mice 18.55 � 2.99 (n ¼ 4;P ¼ 0.36; Fig. 2A–C). We measured apoptosis by CASP3 stainingand observed no substantial differences between tumors in agedversus young mice (Fig. 2D–F). Although, at day 14, a highernumber of apoptotic cells was observed in the single evaluabletumor fromayoungmouse,we could onlyfindTRAMP-C2 cells inthe prostate of a single young mouse (out of four), precluding astatistical comparison (5.88 apoptotic cells/20� field; n ¼ 1

youngmouse vs. 1.37� 0.65 apoptotic cells/20� field; in tumorsfrom aged mice, n ¼ 3).

Wenext assessed blood vessel density in the tumors fromyoungand aged mice by staining for the pan-endothelial cell antigenMECA-32. Quantitative analysis of digital images demonstratedthat the percentage of the tumor area that was composed of bloodvessels was similar in tumors grown in agedmice and youngmice(Fig. 2G–I; young, n ¼ 3; aged, n ¼ 4; P ¼ 0.19).

Alterations in macrophage populations distinguish prostatecancers in aged microenvironments

We evaluated immune cell populations in the TRAMP-C2tumors and tumor microenvironments in the prostates resectedfrom young and aged mice using IHC (Fig. 3). Few CD3þ T cellswere present in either prostates from young or aged mice and thenumbers did not change significantly over time (Fig. 3A–C).Similarly, the number of B220þ B cells was comparable betweenprostates from young and aged mice (Fig. 3D–F). Regulatory Tcells, marked by FOXP3 expression, and neutrophils, marked byLY6G expression were few in number, scattered throughout thetumor microenvironment, and did not differ significantlybetween aged and young mice (Fig. 3G–I and J–L, respectively).

Notably, the macrophage population, defined by F4/80 stain-ing composed approximately 50% of the cellular composition ofTRAMP-C2 tumors and the abundance of macrophages was notsubstantially different in tumors developing in young versus agedmice (Fig. 3M–O). To rule out the possibility that TRAMP-C2 cellsexpress the F4/80 protein, we stained TRAMP-C2 cells, culturedin vitro, with the F4/80 antibody and found no immunoreactivity(see Supplementary Table S1). This result confirms that theextensive number of F4/80 positive cells present in the tumorsare macrophages. We evaluated the subpopulation of arginase 1(ARG1) expressing macrophages because these cells have beenreported to exert protumoral effects and associate with a pooroutcome in most solid tumors, including prostate cancer (21). Atday 3 posttumor cell injection, high numbers of ARG1 positivecells were identified in the tumors fromagedmice, 11.85� 3.08%compared with 1.24 � 0.69%, in tumors from young mice (P ¼0.015; Fig. 3P–R). At later time points, ARG1 positive cellsremained in the tumor periphery, frequently surrounding theremaining mouse prostatic acini, and most of the tumor-infiltrat-ing macrophages were ARG1 negative, resulting in an overalllower density of ARG1 positive cells (Fig. 3R). Nevertheless,analysis of ARG1þ cells surrounding the host acini remainedhigher in the old mice at the later time points (Fig. 3S).

We previously reported that the normal aged mouse prostatescontain a significantly higher number of resident F4/80þmacro-phages (11), but no ARG1macrophages were evident in any areasof the normal prostate (see Supplementary Fig. S1). We speculatethat the highnumber ofARG1macrophages at day 3postinjectionin the tumor regions might be a result of a rapid polarization ofresident macrophages towards a protumorigenic phenotype.

Table 1. Incidence and sites of TRAMP-C2 cell dissemination and metastases

Injection site Age group No. miceNo. micemetastases Mesentery Liver Spleen Kidney Epid. fat pad Othersa

i.p. Young 10 4 (40) 4 (40) 0 (0) 0 (0) 3 (30) 0 (0) 1i.p. Aged 18 15 (83) 14 (78) 3 (17) 3 (17) 7 (39) 3 (17) 6 (33)i.v. Young 8 1 (13) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (13)i.v. Aged 11 9 (82) 6 (55) 5 (45) 4 (36) 1 (9) 0 (0) 4 (36)

NOTE: Percentages of mice with tumor growth in distant organs are given in parentheses.aPancreas, thymus, intestine, diaphragm.

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The prostate cancer transcriptional program is altered in anaged microenvironment

To identify molecular alterations that could associate with thedifferential growth and metastatic spread of prostate cancer inyoung versus aged mice, we evaluated the transcriptional pro-grams of TRAMP-C2 tumors.We resected orthotopic tumors fromyoung (n ¼ 4) and aged (n ¼ 4) mice and quantitated genome-wide transcript abundance levels by RNA-seq. There were 199genes differentially expressed (out of 13,909 expressed) betweentumors from aged versus young mice with two-fold or greaterdifference in transcript abundance (uncorrected P-value ¼0.05; Fig. 4A). Notably, several of these genes such as Vsig4,Deptor, Reg3g, Hgfac, and Cxcl13 encode proteins with previouslyidentified roles in promoting tumor invasion and migration orinfluencing immune responses (22–24) or denote protumori-genic macrophage phenotypes such as Cd163 and Vsig4 (25, 26).

We evaluated the expression of genes associated with macro-phage phenotypes and determined that tumors from aged miceexpressed higher levels of transcripts associated with tumor pro-moting macrophages (e.g., Cd163, Arg1) and reduced expressionof genes associated with tumor repressive macrophages (Nos2,Cd68, Cd86; Fig. 4B). We next evaluated the expression of CD163and other macrophage markers in the human PC TCGA dataset(18) as a function of aging. Interestingly, we found a statisticallysignificant upregulation of CD163 and VSIG4 in patients olderthan 60 years comparedwith younger patients. In contrast,NOS2,a gene expressed in tumor suppressive macrophages, was signif-

icantly downregulated with aging (Fig. 4C). Transcript levels ofCD163 and VSIG4 in prostate cancer cell lines were undetectable(27), thus the expression of CD163 and VSIG4 is most likelyassociated with infiltrating macrophages. In addition, accordingto the Human Protein Atlas version 18, expression of CD163 andVSIG4 in human prostate cancer is restricted to infiltratingmacro-phages and is not observed in cancer cells (28), further supportingthe role of macrophages in cancer progression. Notably, highexpression of CD163 and VSIG4 in localized prostate cancersassociated with biochemical recurrence following primary ther-apy (CD163 HR¼ 1.579; 95% CI, 1.039–2.401; P¼ 0.03; VSIG4HR 1.739; 95% CI, 1.131–2.675; P ¼ 0.01; Fig. 4D and E). BothCD163 and VSIG4 remained significant when adjusted for age(CD163 adjusted HR ¼ 1.547; 95% CI, 1.017–2.353; P ¼ 0.030;VSIG4 adjusted HR ¼ 1.687; 95% CI, 1.095–2.599; P ¼ 0.0137,likelihood ratio test). Taken together, these results indicate thatmacrophage phenotypes associated with aggressive PC and pooroutcomes (21) predominate in tumors from aged mice and olderpatients.

Extracellularmatrix collagen isolated fromagedmice promotesprostate cancer cell invasion

In addition to gene expression changes indicating aging-asso-ciated alterations in macrophage phenotypes and chemokinesignaling, gene ontology (GO) analysis using GOrilla algorithm(19) also identified significant aging-associated enrichment inextracellular matrix organization pathways and collagen

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Assessment of proliferation, apoptosis, and angiogenesis in orthotopic prostate tumors from young and aged mice. A and B, Cell proliferation was quantitatedin TRAMP-C2 prostate tumors by KI67 IHC in young (A) and aged (B) mice. Magnification �400. C, KI67 proliferation index (%) assessed by counting the numberof KI67-positive cells over the total number of cells under randomly selected 400� fields, at 3, 7, 14, and 30 days after tumor cell injection. D and E, Representativeimages of apoptotic cell death detected in TRAMP-C2 prostate tumors by CASP3 IHC. Scattered apoptotic cells were detected in both young (D) and aged (E)mice. F, The percentage area stained positive for CASP3 in the tumor region was calculated from digital images (host prostate acini were excluded from the analysis)Y, Young;A, Aged3, 7, 14, and30days after tumor cell injections.G andH,Representative sections from tumors of young andagedmice demonstrating similar numbersof blood vessels, detected by MECA32 IHC. I, Angiogenesis was calculated quantifying the vessel area in digital images. Similar percentage areas of vesselimmunostaining were noted in tumors from both age groups. P values: #, not significant; i, insufficient tumors to determine significance. Scale bars, 100 mm.

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metabolic processes (Fig. 5A; Supplementary Table S2, containsthe results from GOrilla). We previously demonstrated that theextracellular collagen matrix in the normal prostate stroma fromagedmice exhibitedmarked disorganization and structural altera-tions (11). To evaluate the extracellular matrix in the context ofaging and prostate cancer, we stained TRAMP-C2 tumor sectionswith Masson's trichrome and found that the TRAMP-C2 tumorsfrom aged mice had a substantial increase in collagen fibers

compared with tumors in young mice (Fig. 5B–D). Moreover,collagen density was more prominent in the regions of the tumornear the remaining prostatic ducts of the original prostate gland.

To determine if aged collagen exerted functional effects towardPC cells, we constructing 3D collagen gels in vitro usingmouse tailtendon collagen extracted from young (4-month-old) and aged(20- to 24-month-old) C57BL/6 mice (20) and measured tumorcell invasion by trans-well cell culture assay. We seeded TRAMP-

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Immune cell infiltration in TRAMP-C2 tumors from young and aged mice. Representative images and quantification of immune infiltrates in TRAMP-C2 tumors fromyoung and aged mice immunostained with markers for T cells (CD3; A–C), B cells (B220; D–F), regulatory T cells (FOXP3, G–I), and neutrophils (LY6G, J–L),macrophages (F4/80;M–O), tumor-associated macrophages (ARG1, P–R: image analysis; S: peritumor manual scores). No significant differences were found in thenumber of CD3-positive T cells, B202-positive B cells, F4/80-positive macrophages, FOXP3-positive T-regulatory cells or LY6G-positive neutrophils betweenyoung and aged mice. There was a significant difference in the number of ARG1-positive macrophages between young and aged mice, with higher infiltration in theaged tumors at 3 days postinjection. By 30days postinjection the area percentage of ARG1-positivemacrophages in agedmice decreased significantly. However, thenumber of ARG1-positive macrophages near the host prostate remained higher in aged mice across all time points. P-values: #, not significant; i, insufficienttumors to determine significance; � , <0.05; �� , <0.001. Scale bars, 200 mm.

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C2 and Myc-CaP cells on top of polymerized young and agedcollagen inserts, and measured invading cell numbers 18 hoursafter seeding by counting cells in randomly selected 100� fieldsin four independent chambers. For TRAMP-C2, 17.2 � 3.86cells invaded through collagen from aged mice whereas zerocells invaded through collagen from young mice (P ¼ 0.004)(Fig. 5E–F). For Myc-CaP, 153.5 � 35.12 and 2.833 � 1.493tumor cells invaded through collagen from aged versus youngmice, respectively (P ¼ 0.0016; data not shown). As the rigidityof the extracellular matrix can regulate tumor cell growth, bothin vitro and in vivo (29–32), we then analyzed the proliferationof prostate epithelial cells when plated on top of young andaged 3D collagen gels. TRAMP-C2 proliferation in the agedcollagen gels was significantly higher than in young-collagen at24 and 48 hours (0.07 vs 0.09 OD 490 mm, P ¼ 0.03; and 0.01vs. 0.08 OD 490 mm, P ¼ 0.005, respectively; Fig. 5G). Thesefindings suggest that alterations that occur with aging in thecollagenous material of the extracellular matrix may present aninductive and/or permissive environment for tumor cell growthand invasion.

DiscussionThe incidence of most malignancies, particularly those origi-

nating from epithelial cells, is directly associated with advancingage. While cell-autonomous events, primarily mutations or struc-tural genomic aberrations involving oncogenes and tumor sup-pressors drive neoplasia, other cell non-autonomous factorsderived from tumor microenvironment constituents influencecancer progression. The objectives of this study were to test thedirect effects of the aged prostate microenvironment on thegrowth of prostate cancer cells and metastatic spread. For thesepurposes, we evaluated an orthotopic model, where the samequantity of TRAMP-C2 PC cells were injected into young and agedimmunocompetent C57BL/6mouse prostates.We found a robustenhancement of tumor growth in the aged host. This observationclearly demonstrated the existence of age-dependent interactionsbetween the tumor cells and the prostate microenvironment,directly influencing tumor development. In contrast, a previousstudy showed that TRAMP-C2 cells implanted subcutaneously(rather than orthotopically) in young and aged C57BL/6mice did

Figure 4.

Age-associated alterations inTRAMP-C2 orthotopic tumor geneexpression. A, Heat map of the genesaltered between young (n ¼ 4) andaged (n ¼ 4) mice (P-value <0.05,fold-change �2), showingupregulated (yellow) anddownregulated (blue) genes. Eachrow shows the relative expressionlevel for a single gene, and eachcolumn shows the expression level ofa single sample. B, RNA-seq log fold-change (logFC) expression betweenyoung and aged TRAMP-C2 tumorsfor tumor-promoting-polarizationmarkers (red bars) and tumor-repressing-polarization markers(blue bars): �� , P <0.01; � , P < 0.05.C, FPKM expression of macrophageassociated markers in two differentage groups [�60-year-old (bluebars) vs. >60-year-old (red bars)] ofprostate cancer patient in the humanprostate cancer TCGA dataset,demonstrating the age-associatedupregulation of tumor-promotingmarkers in tumors from olderpatients. D and E, Kaplan–MeierBCR-free survival analysis in thehuman prostate cancer TCGAdataset, assessing correlation ofCD163 and VSIG4 with biochemicalrecurrence outcomes.

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not differ with respect to tumor take or growth rates (13). Ourpresent study highlights the influence of the local prostate micro-environment on the growth of prostate cancer cells, supportingthe view that age-associated organ-specific milieu contribute tothe processes driving tumor growth andmetastatic dissemination.

Our observations are consistent with other age-related cancermodels including breast, colon, liver (7, 33–35); however, thereare also discrepancies, most likely related to the tumor cell oforigin (36, 37). For instance, tumor progression of Lewis lungcancer cells is substantially inhibited in 24-month-old C57BL/6syngeneic mice compared with three younger age groups (36).This observation might be explained by the behavior of organ-specific tumors with aging: the highest percentage of lung cancerdeaths increases up to 31.3% in the 65 to 74 age group, but then itstarts to decline to 12% by the age of 84 or older (38). This isconsistent with those observations from the Lewis lung cancermodel, suggesting a more indolent disease in the aged popula-tion. In contrast, prostate cancer deaths increase with age with34.4% of deaths in the 75 to 84 age group, and remains high(33.5%) in patients aged greater than 84 years (38). Thus, our

findings recapitulate the age-associated clinical behavior ofhuman prostate cancer. In most cases, prostate cancer is relativelyslow-growing, which means that it typically takes years, evendecades to become metastatic. We speculate, based on our find-ings, that the aging tumor microenvironment can directly influ-ence the behavior of the tumor cells, thus creating a moreprotumorigenic/permissive environment over time.

We also observed an age-dependentmetastatic spread. Tail veininjections of TRAMP-C2 cells gave rise to metastatic lesions inaged but not in young mice. It is possible that metastases inyoung mice could develop later in time or with a higher load ofTRAMP-C2 cells (39). Although we did not analyzed spleensimmunologically in our orthotopic model, it is also plausiblethat systemic changes involving the immune system may haveplayed a role on the development of TRAMP-C2metastases in theaged mice (36). This conclusion is supported by the age-associated enhancement of the growth and spread of tumorcells directly introduced into the peritoneum.

To gain insights into the possible signaling mechanismsinduced by the aged microenvironment, we performed global

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Figure 5.

Influence of collagen matricesextracted from young and aged miceon prostate cancer invasion andproliferation. A, GO term enrichmentanalysis for genes differentiallyexpressed between young and agedtumors. Each column indicates arepresentative GO term for biologicalprocesses, molecular function, andcellular component. RNA-seq datawere simply rankedbyP-values andGOanalysis was performed using GOrilla.B–D, Representative trichrome stainsfrom tumor sections of young (B) andaged (C) mice. Masson's Trichromestain reveals collagen fibrosis in tumorsfrom aged mice. Note thepreponderance of collagen (in blue)near the host prostate gland (�). Scalebars, 100 mm. D, Collagen intensityscores as measured by Masson'sTrichrome stains. E and F, Invasionassay. E, Quantitated numbers ofinvaded TRAMP-C2 cells (number ofcells/�100 field) that invadedthrough young and aged collagen.F, Representative images of the lowersurfaces of the invasion membranechambers showing TRAMP-C2 cellsafter invading 3D collagen gelsharvested from tail tendons fromyoung (top) and aged (bottom) mice(�40 magnification). Note the highernumber of cells (arrows) that invadedthrough the aged collagen comparedwith young collagen. G, Proliferation ofTRAMP-C2 cells grown in young andaged collagen at 24 and 48 hours.� , P < 0.05.

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gene expression analysis on tumors from young and aged mice.Surprisingly, despite the robust difference in tumor growthbetween young and aged hosts, we found a low number ofdifferentially expressed genes. It is possible that the highly similarmolecular profiles between young and aged tumors could be dueto technical limitations, because the tumors used for gene expres-sion analyses for the young cohort came from those few youngmice that developed tumors, providing enough material for geneexpression analysis. Further, substantial phenotypic changes inlow abundance cell types may be masked in the analysis of bulktissue populations. Despite the low number of differentiallyexpressed genes, pathway analysis revealed an enrichment ofgenes associated with immune response (Th2 response), chemo-kine-mediated signaling pathways, extracellular matrix organiza-tion, and collagen metabolic processes in addition to pathwaysassociated with regulation of cell migration, cell death, andproliferation. These results suggest that the differences in growthbetween the age groups might be due to a yet-uncovered complexinteraction between the host immune cells, the aged-associateddisruption of the extracellular matrix and the response of thetumor cell to these factors.

We have previously reported an age-dependent alteration ofthe structure and composition of the extracellular matrix, witha disorganized and fragmented collagen network in the pros-tate of aged mice (11). To study putative differences involvingthe extracellular matrix within the TRAMP-C2 tumors, westained tumor sections with Masson's trichrome and foundthat the TRAMP-C2 tumors from aged mice had fibrotic areaswith increased collagen fibers near the host's prostatic ducts,indicating the relevance of the immediate tissue microenviron-ment on the regulation of tumor growth. Previous studies inbreast cancer have established a causal link between highcollagen density, tumorigenesis, and invasive phenotypes(31). In this study, we show that TRAMP-C2 cells culturedin vitro in the presence of 3D collagen gels harvested from agedmice, show significantly increased proliferation and invasionwhen compared with cells cultured in collagen gels from youngmice, demonstrating the direct effect of the aged collagenousmatrix on tumor cell growth and migration. This is consistentwith other studies suggesting that the microenvironment of atumor, composed in part of extracellular matrix macromole-cules, plays a pivotal function in tumor progression and spread(29–31, 40).

An additional mechanism by which tumor growth can beenhanced and/or impaired is through the action of the immunesystem. The current understanding is that CD4þ T helper (Th)and tumor repressive macrophages (among other immunecells) can generate antitumor responses; and conversely, CD4þ

Th2 cells and tumor-promoting macrophages (among otherimmune cells) induce tumor tolerance and support tumorgrowth and progression (41). Tumor-repressive and tumor-promoting macrophages have been frequently designated M1and M2; however, recent studies show a more complex scenarioand propose a spectral polarization model (25). We havepreviously demonstrated increased numbers of lymphocytesand macrophages in the normal aged mouse prostate (11). Inthis study, we found that by day 3 after tumor cell injection,Arginase-1 positive cells were significantly and substantiallyincreased in tumors from aged mice, suggesting a rapid polar-ization of resident macrophages towards a tumor promoting

phenotype. Furthermore, gene expression analysis demonstrat-ed that markers of tumor-promoting TAMs (Cd163) and inhi-bitors of proinflammatory macrophages (Vsig4), together withan enrichment of Th2-immune response pathways, were upre-gulated in the aged tumors. In line with these findings, wedemonstrated that this age-associated increase in CD163 andVSIG4 transcript levels also occurs in human prostate cancer.This age-associated increase in tumor-promoting TAMs polar-ization is consistent with previous observations, both in miceand humans (35, 42). VSIG4 expression on macrophages alsofacilitates lung cancer development (26). Of note, Chil3, one ofthe genes significantly upregulated in TRAMP-C2 tumors fromaged mice is also expressed in macrophages and has beeninvolved in tissue remodeling, in agreement with a recent studyshowing that tumor-associated macrophages are capable ofdegrading collagen and display a matrix catabolic transcrip-tomic signature (43).

Taken together, our results support the hypothesis that thedifferences in tumor growth between young and aged mice aredue to a complex interaction between age-associated immuneresponses and the effects of the extracellular matrix on tumorcells behavior. This study adds to the evidence that microenvir-onments play crucial roles in modulating the behaviors oftumor cells and supports studies designed to modify age-associated microenvironment features to inhibit adverse tumorcell behavior.

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

Authors' ContributionsConception and design: D. Bianchi-Frias, P.S. NelsonDevelopment of methodology: D. Bianchi-Frias, M. Damodarasamy,P.S. NelsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Bianchi-Frias, M. Damodarasamy, F. Vakar-Lopez,M.J. Reed, P.S. NelsonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Bianchi-Frias, M. Damodarasamy, R.M. Gil daCosta, I.M. Coleman, M.J. Reed, P.S. NelsonWriting, review, and/or revision of the manuscript: D. Bianchi-Frias, R.M. Gilda Costa, I.M. Coleman, M.J. Reed, P.S. NelsonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.A. Hernandez, I.M. Coleman, P.S. NelsonStudy supervision: P.S. Nelson

AcknowledgmentsThe authors thank the FredHutch Scientific Imaging shared resource for

support with image acquisition and analysis. We thank Roger Coleman and theFredHutch Genomic Core Facility for support with RNA-seq data acquisitionand bioinformatics advice. We gratefully acknowledge research support fromFred Hutch/University of Washington Cancer Consortium P30 CA015704 (toP. Nelson), and grants P50 CA097186 (to D. Bianchi-Frias, S. Hernandez,R.M. Gil da Costa, F. Vakar-Lopez, I Coleman, P. Nelson) and R01 CA165573(to P. Nelson) from the NIH and W81XWH-15-1-0562 (to D. Bianchi-Frias,I. Coleman, P. Nelson) from the DOD/CDMRP.

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 May 20, 2018; revised July 27, 2018; accepted September 4, 2018;published first September 17, 2018.

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References1. American Cancer Society. Cancer facts & figures 2017 (cited 2017 Nov 16).

Available from: https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2017.

2. Thompson TC, Timme TL, Park SH, Yang G, Ren C. Mouse prostatereconstitution model system: a series of in vivo and in vitro models forbenign and malignant prostatic disease. Prostate 2000;43:248–54.

3. Hayward SW, Wang Y, Cao M, Hom YK, Zhang B, Grossfeld GD, et al.Malignant transformation in a nontumorigenic human prostatic epithelialcell line. Cancer Res 2001;61:8135–42.

4. Bavik C, Coleman I, Dean JP, Knudsen B, Plymate S, Nelson PS. The geneexpression program of prostate fibroblast senescencemodulates neoplasticepithelial cell proliferation through paracrine mechanisms. Cancer Res2006;66:794–802.

5. Begley L, Monteleon C, Shah RB, Macdonald JW, Macoska JA. CXCL12overexpression and secretion by aging fibroblasts enhance human prostateepithelial proliferation in vitro. Aging Cell 2005;4:291–8.

6. Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR.Carcinoma-associated fibroblasts direct tumor progression of initiatedhuman prostatic epithelium. Cancer Res 1999;59:5002–11.

7. McCullough KD, Coleman WB, Smith GJ, Grishan JW. Age-dependentregulation of the tumorigenic potential of neoplastically transformed ratliver epithelial cells by the liver microenvironment. Cancer Res 1994;54:3668–71.

8. Krtolica A, Campisi J. Cancer and aging: a model for the cancer promotingeffects of the aging stroma. Int J Biochem Cell Biol 2002;34:1401–14.

9. Miller RA. Gerontology as oncology. Research on aging as the key to theunderstanding of cancer. Cancer 1991;68:2496–501.

10. Anisimov VN. The relationship between aging and carcinogenesis: a criticalappraisal. Crit Rev Oncol Hematol 2003;45:277–304.

11. Bianchi-Frias D, Vakar-Lopez F, Coleman IM, Plymate SR, ReedMJ, NelsonPS. The effects of aging on the molecular and cellular composition of theprostate microenvironment. PLoS One 2010;5:e12501.

12. Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM. Charac-terization of prostatic epithelial cell lines derived from transgenicadenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res1997;57:3325–30.

13. Reed MJ, Karres N, Eyman D, Cruz A, Brekken RA, Plymate S. The effects ofaging on tumor growth and angiogenesis are tumor-cell dependent. Int JCancer 2007;120:753–60.

14. Said N, Frierson HF Jr, Chernauskas D, Conaway M, Motamed K,Theodorescu D. The role of SPARC in the TRAMP model of prostatecarcinogenesis and progression. Oncogene 2009;28:3487–98.

15. Watson PA, Ellwood-YenK, King JC,Wongvipat J, LebeauMM, Sawyers CL.Context-dependent hormone-refractory progression revealed throughcharacterization of a novel murine prostate cancer cell line. Cancer Res2005;65:11565–71.

16. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2:accurate alignment of transcriptomes in the presence of insertions, dele-tions and gene fusions. Genome Biol 2013;14:R36.

17. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor packagefor differential expression analysis of digital gene expression data.Bioinformatics 2010;26:139–40.

18. Cancer Genome Atlas Research Network. The molecular taxonomy ofprimary prostate cancer. Cell 2015;163:1011–25.

19. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z. GOrilla: a tool fordiscovery and visualization of enrichedGO terms in ranked gene lists. BMCBioinform 2009;10:48.

20. Damodarasamy M, Vernon RB, Karres N, Chang CH, Bianchi-Frias D,Nelson PS, et al. Collagen extracts derived from young and aged micedemonstrate different structural properties and cellular effects in three-dimensional gels. J Gerontol A Biol Sci Med Sci 2010;65:209–18.

21. Cao J, Liu J, Xu R, Zhu X, Zhao X, Qian BZ. Prognostic role of tumour-associated macrophages and macrophage scavenger receptor 1 in pros-tate cancer: a systematic review and meta-analysis. Oncotarget 2017;8:83261–9.

22. Liu X, Zhou Z, Cheng Q, Wang H, Cao H, Xu Q, et al. Acceleration ofpancreatic tumorigenesis under immunosuppressive microenvironmentinduced by Reg3g overexpression. Cell Death Dis 2017;8:e3033.

23. Zhang XH, Qian Y, Li Z, Zhang NN, Xie YJ. Let-7g-5p inhibits epithelial-mesenchymal transition consistent with reduction of glioma stem cellphenotypes by targeting VSIG4 in glioblastoma. Oncol Rep 2016;36:2967–75.

24. Byun JM, Jeong DH, Choi IH, Lee DS, Kang MS, Jung KO, et al. Thesignificance of VSIG4 expression in ovarian cancer. Int J Gynecol Cancer2017;27:872–8.

25. Aras S, ZaidiMR. TAMeless traitors:macrophages in cancer progression andmetastasis. Br J Cancer 2017;117:1583–91.

26. Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, et al. VSIG4 expression onmacrophages facilitates lung cancer development. Lab Invest 2014;94:706–15.

27. Urbanucci A, Barfeld SJ, Kytola V, ItkonenHM,Coleman IM, VodakD, et al.Androgen receptor deregulation drives bromodomain-mediated chroma-tin alterations in prostate cancer. Cell Rep 2017;19:2045–59.

28. Uhlen M, Zhang C, Lee S, Sjostedt E, Fagerberg L, Bidkhori G, et al. Apathology atlas of the human cancer transcriptome. Science 2017;357:eaan2507.

29. Reid SE, Kay EJ, Neilson LJ, Henze AT, Serneels J, McGhee EJ, et al. Tumormatrix stiffness promotes metastatic cancer cell interaction with the endo-thelium. EMBO J 2017;36:2373–89.

30. PickupMW,Mouw JK, Weaver VM. The extracellular matrix modulates thehallmarks of cancer. EMBO Rep 2014;15:1243–53.

31. Provenzano PP, InmanDR, Eliceiri KW, Knittel JG, Yan L, Rueden CT, et al.Collagen density promotes mammary tumor initiation and progression.BMC Med 2008;6:11.

32. Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, Keely PJ.Collagen reorganization at the tumor-stromal interface facilitates localinvasion. BMC Med 2006;4:38.

33. Schneider H, Lohmann B, Wirsching HG, Hasenbach K, Rushing EJ, FreiK, et al. Age-associated and therapy-induced alterations in thecellular microenvironment of experimental gliomas. Oncotarget2017;8:87124–35.

34. Grizzle WE, Xu X, Zhang S, Stockard CR, Liu C, Yu S, et al. Age-relatedincrease of tumor susceptibility is associated with myeloid-derived sup-pressor cell mediated suppression of T cell cytotoxicity in recombinantinbred BXD12 mice. Mech Ageing Dev 2007;128:672–80.

35. Ly LV, Baghat A, Versluis M, Jordanova ES, Luyten GP, van Rooijen N, et al.In aged mice, outgrowth of intraocular melanoma depends on proangio-genic M2-type macrophages. J Immunol 2010;185:3481–8.

36. Beheshti A, Wage J, McDonald JT, Lamont C, Peluso M, Hahnfeldt P,et al. Tumor-host signaling interaction reveals a systemic, age-depen-dent splenic immune influence on tumor development. Oncotarget2015;6:35419–32.

37. Parikh N, Shuck RL, Gagea M, Shen L, Donehower LA. Enhanced inflam-mation and attenuated tumor suppressor pathways are associated withoncogene-induced lung tumors in aged mice. Aging Cell 2018;17(1).

38. Howlader NNA, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, et al.Based on November 2016 SEER data submission, posted to the SEERwebsite, April 2017. SEER Cancer Statistics Review, 1975–2014, NationalCancer Institute. Bethesda, MD. [Accessed January 2018]. Available from:https://seer.cancer.gov/csr/1975_2014/.

39. Kwon ED, Foster BA, Hurwitz AA, Madias C, Allison JP, Greenberg NM,et al. Elimination of residual metastatic prostate cancer after surgery andadjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockadeimmunotherapy. Proc Natl Acad Sci U S A 1999;96:15074–9.

40. Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, et al.Tensional homeostasis and the malignant phenotype. Cancer Cell2005;8:241–54.

41. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in thetumor microenvironment. Nat Immunol 2013;14:1014–22.

42. Rauh MJ, Ho V, Pereira C, Sham A, Sly LM, Lam V, et al. SHIP represses thegeneration of alternatively activated macrophages. Immunity 2005;23:361–74.

43. Madsen DH, Jurgensen HJ, Siersbaek MS, Kuczek DE, Grey Cloud L, Liu S,et al. Tumor-associated macrophages derived from circulating inflamma-tory monocytes degrade collagen through cellular uptake. Cell Rep2017;21:3662–71.

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Tumorigenic Potential of an Aged Microenvironment

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