Tumor-associated fibroblasts predominantly come fromlocal and not circulating precursorsAinhoa Arinaa,b,1,2, Christian Idela,b,3, Elizabeth M. Hyjeka, Maria-Luisa Alegreb,c, Ying Wangb,c, Vytautas P. Bindokasd,Ralph R. Weichselbaume,f, and Hans Schreibera,b,g

aDepartment of Pathology, The University of Chicago, Chicago, IL 60637; bCommittee on Immunology, The University of Chicago, Chicago, IL 60637; cSectionof Rheumatology, Department of Medicine, The University of Chicago, Chicago, IL 60637; dIntegrated Microscopy Core, Department of Neurobiology,Pharmacology and Physiology, The University of Chicago, Chicago, IL 60637; eThe Ludwig Center for Metastasis Research, The University of Chicago,Chicago, IL 60637; fDepartment of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL 60637; and gCommittee on Cancer Biology, TheUniversity of Chicago, Chicago, IL 60637

Edited by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, and approved May 16, 2016 (received for review January 12, 2016)

Fibroblasts are common cell types in cancer stroma and lay downcollagen required for survival and growth of cancer cells. Althoughsome cancer therapy strategies target tumor fibroblasts, their originremains controversial. Multiple publications suggest circulatingmesenchymal precursors as a source of tumor-associated fibro-blasts. However, we show by three independent approaches thattumor fibroblasts derive primarily from local, sessile precursors. First,transplantable tumors developing in a mouse expressing greenfluorescent reporter protein (EGFP) under control of the type Icollagen (Col-I) promoter (COL-EGFP) had green stroma, whereaswe could not find COL-EGFP+ cells in tumors developing in theparabiotic partner lacking the fluorescent reporter. Lack of incorpo-ration of COL-EGFP+ cells from the circulation into tumors was con-firmed in parabiotic pairs of COL-EGFP mice and transgenic micedeveloping autochthonous intestinal adenomas. Second, transplant-able tumors developing in chimeric mice reconstituted with bone mar-row cells from COL-EGFP mice very rarely showed stromal fibroblastsexpressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed intotumors containing green stromal cells. Using multicolor in vivo con-focal microscopy, we found that Col-I–expressing fibroblasts con-stituted approximately one-third of the stromal mass and formeda continuous sheet wrapping the tumor vessels. In summary, tu-mors form their fibroblastic stroma predominantly from precursorspresent in the local tumor microenvironment, whereas the contribu-tion of bone marrow-derived circulating precursors is rare.

stroma | origin | bone marrow | collagen | mesenchymal

Tumor-associated fibroblasts (TAFs) are a prominent cellularcomponent of tumors, often assumed to be the most abun-

dant cell type in tumor stroma, and there is an interest in TAFsas a therapeutic target in cancer. However, therapeutic interven-tions are limited by the lack of truly TAF-specific targets and un-certainty about their origins. If TAFs were predominantly recruitedfrom circulating bone marrow (BM)-derived precursors, only sys-temic but not local therapies would prevent the recruitment of TAFprecursors into tumors to suppress their growth. It is generallythought that at least a fraction of the fibroblasts in tumor stroma isderived from circulating BM-derived progenitors/stem cells. Theidea that mononuclear blood cells could differentiate into fibro-blasts was suggested already in 1892 (1–3). A century later,several reports indicated that circulating CD14+ monocytes orCD45+CD34+ collagen I+ “fibrocytes” (4) could be progenitors forfibroblasts in vitro (5–7) or in vivo (8). Other recent reports alsohighlight the derivation of TAFs from circulating BM-derivedprogenitors of either hematopoietic or mesenchymal lineage (9–12). If TAFs were predominantly recruited from local sources, site-specific differences could be exploited to develop much more selec-tive therapeutics to target TAFs with fewer systemic side effects. It isincreasingly clear that fibroblasts from different tissues can consti-tute well-specialized, tissue-specific populations. Fibroblasts fromdifferent anatomic sites have positional memory, and differ in

transcriptional expression (13–15), responsiveness to local stim-uli [e.g., steroid hormones (16)], and their capacity to inhibit cancercell growth (17), which could well be linked to the organotropism ofmetastases (18, 19) and could represent a mechanistic basis for the“seed and soil” hypothesis (20). We therefore examined in this studythe relative contributions of circulating cells and locally derived fi-broblasts to the development of tumor stroma.Fibroblasts are the most common cell type producing collagen,

and over 90% of collagen in the body is type I (21). Genes for type Icollagen (Col-I) are only active in mesenchymal cell types suchas osteoblasts, odontoblasts, and, most relevant for this study, fi-broblasts and circulating mesenchymal progenitors (3, 22). Wetherefore chose expression of an EGFP reporter driven by thecollagen α1(I) promoters/enhancers to unambiguously mark fibro-blasts (23). α-Smooth muscle actin (αSMA) does not exclusivelymark fibroblasts, but can be up-regulated on activated fibroblasts(called myofibroblasts) in conditions involving active ECMdeposition such as fibrosis, wound healing, and cancer (24–26).Thus, we also studied the origin of αSMA+ cells using the αSMA-Discosoma sp. Red (SMA-DsRed) mouse model (23). We foundthat Col-I+ and αSMA+ TAFs derive predominantly from localsources, whereas finding TAFs derived from circulating progenitorsis a very rare event.

Significance

Fibroblasts constitute an important element of tumors and havereceived considerable attention in recent years due to their tu-mor-promoting and immunosuppressive properties. As a conse-quence, tumor-associated fibroblasts (TAFs) are considered anattractive target for cancer therapies. However, their origin re-mains controversial, with some evidence pointing at a local origin,whereas many publications suggest a significant contribution ofprogenitors from bone marrow. We found that TAFs derive al-most exclusively from local sources. Therefore, therapeutic strat-egies to target fibroblasts must exploit local recruitment and theunique transcriptional and response patterns of fibroblasts fromdifferent sites.

Author contributions: A.A., M.-L.A., and H.S. designed research; A.A., C.I., E.M.H., and Y.W.performed research; R.R.W. and H.S. supervised research; E.M.H. and V.P.B. contributed newreagents/analytic tools; A.A. and V.P.B. analyzed data; andA.A., R.R.W., andH.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Imaging data reported in this paper have been deposited in The CellImage Library www.cellimagelibrary.org/.1To whom correspondence should be addressed. Email: aarina@bsd.uchicago.edu.2Present address: The Ludwig Center for Metastasis Research and Department of Radia-tion and Cellular Oncology, The University of Chicago, Chicago, IL 60637.

3Present address: Department of Otorhinolaryngology, University Hospital of Schleswig-Holstein, 23562 Luebeck, Germany.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1600363113/-/DCSupplemental.

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ResultsTumor Growth Is Associated with the Appearance of αSMA and theContinuous Presence of Collagen α1(I)-Expressing Fibroblasts. Tostudy the distribution of TAFs during tumor development, wegenerated dual COL-EGFP/SMA-DsRed reporter mice (23) (Fig.S1) and implanted dorsal skinfold windows and cancer cells for lon-gitudinal analysis of tumors (27). As can be seen in Fig. 1A, healthyskin (day 0) in dual reporter mice shows normal dermal fibroblastsexpressing Col-I (COL-EGFP+), whereas the expression of αSMA israre. After cancer cell inoculation, SMA-DsRed+ cells appearedaround days 5–8 and became significantly more abundant arounddays 11–15 (Fig. 1B, Fig. S2, and Table S1). Expression of COL-EGFP showed an increasing trend between the same time points,although it was not statistically significant (Fig. 1B). The appear-ance of SMA-DsRed+ cells coincided with the development of tu-mor vasculature (Fig. S2). Several fibrosarcoma (MC57, Pro4L,and 8101Pro) and one carcinoma (MC38 colon cancer) celllines growing in immunodeficient or immunocompetent micerendered similar results (Table S1).

To determine whether αSMA and Col-I were expressed by thesame or different cells, colocalization studies were performed inconfocal high-magnification images (20×) from established tumors(Fig. S3). More than half of the cells that were positive for Col-Ialso expressed αSMA and vice versa at days 14–22 of tumor growth(average Mander’s coefficients varied between 0.56 ± 0.04 and0.77 ± 0.29 for COL-EGFP and 0.56 ± 0.1 and 0.83 ± 0.21 forSMA-DsRed). Therefore, a majority of cells were positive for bothmarkers, but there was always a fraction of cells that expressed onlyone or the other.

Tumor-Associated Fibroblasts Derive Predominantly from Local, andNot Circulating, Precursors. To determine the origin of TAFs, twotypes of BM chimeric mice were generated from COL-EGFP orSMA-DsRed transgenic mice, in which either only the BM or onlythe lethally irradiated host had the potential to generate fluorescentlylabeled fibroblasts. In four independent experiments using fourtumor models, TAFs were visible during the first weeks followingtumor implantation exclusively in the tumors of the chimerasexpressing the transgene in the non-BM (sessile) compartment(Fig. 2 A, D, and F and Fig. S4). After 2–4 wk of tumor growth,only a few BM-derived TAFs were observed in the chimeras thathad been reconstituted with BM from COL-EGFP transgenic mice(Fig. 2 B, C, and F and Fig. S4). No BM-derived SMA-DsRed+ cellswere found in mice reconstituted with BM from SMA-DsRedtransgenic mice in two independent experiments (Fig. S5).Because the BM chimera model could be limited by an incom-

plete deletion of mesenchymal stromal cells and engraftment of thenewly transferred mesenchymal progenitors in the BM of irradiatedmice (28), we generated parabiotic mouse pairs (Fig. 3 A and B). Insuch pairs, one mouse was COL-EGFP transgenic and the othermouse expressed DsRed ubiquitously under the chicken beta-actinpromoter. Around 2 wk after the surgery, the presence of similarpercentages of DsRed+ cells in both animals indicated that circu-lation was shared (Fig. S6). Cancer cells were injected into the distalflanks in both mice. No COL-EGFP+ cells could be found in thetumors grown in the DsRed mice parabiosed to COL-EGFP mice(Fig. 3 A and B). These data in parabiotic mice prove that thecollagen α1-expressing TAFs are derived from local sessile pre-cursors. To further investigate the local origin of TAFs, we madeuse of DsRed mice transplanted with full-thickness skin grafts fromCOL-EGFP mice (Fig. 3C). Upon s.c. injection of cancer cellsunderneath the grafts, abundant COL-EGFP+ cells were detectedin freshly explanted sections of the tumors. We performed twoexperiments, one of which is shown in Fig. 3B. In five of seven totalmice from two independent experiments, COL-EGFP+ cells couldbe detected in tumor sections cut 2.5–3 mm below the graft surfaceand, in three of seven mice, some COL-EGFP+ cells could be foundeven 4–6 mm from the surface. COL-EGFP+ cells were found inthe tumors when cancer cells had been injected either under thecenter of the grafted skin (4/5 total mice) or into its margin (2/2mice). Therefore, EGFP+ TAFs had derived from local precursorspresent in the original COL-EGFP skin grafts.

Circulating Cells Do Not Contribute Significantly to TAFs in ParabiosedMice Developing Autochthonous Intestinal Tumors. To confirm ourresults from transplantable models, we parabiosed fibroblast reportermice (COL-EGFP or SMA-DsRed) with ApcMin (Apc, adenomatosispolyposis coli; Min, multiple intestinal neoplasia) transgenic micethat develop intestinal adenomas starting at puberty (29). This tumormodel recreates more closely some aspects of the process of tu-morigenesis in humans because tumors are autochthonous. For theCOL-EGFP/ApcMin pairs, as can be seen in Fig. 4 A and C, Col-Icould readily be detected by immunofluorescence (IF) in the in-testine from the COL-EGFP mouse and the ApcMin mouse; how-ever, EGFP could exclusively be found in the COL-EGFP mousesample. Similarly, no DsRed+, but many αSMA+, cells could bedetected in the polyps from an ApcMin mouse parabiosed with

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Fig. 1. Tumor growth is associated with the appearance of αSMA and thecontinuous presence of collagen α1(I)-expressing fibroblasts. (A) Expressionof COL-EGFP and SMA-DsRed on healthy skin (day 0) and 12 d after MC57-Cerulean tumor cells were injected in a dual reporter COL-EGFP/SMA-DsRedRag−/− mouse bearing a dorsal skinfold window chamber. Dotted lines in-dicate window and tumor boundary. (B) Quantification of the area (meanand SD) occupied by COL-EGFP+, SMA-DsRed+, and Cerulean+ (cancer) cells inimages obtained from tumors developing in dual reporter mice. Data werepooled from five independent experiments using COL-EGFP/SMA-DsRedRag−/− mice injected with MC57, Pro4L, and 8101Pro fibrosarcomas (n = 1mouse each) and COL-EGFP/SMA-DsRed Rag+/+ mice injected with MC38colon carcinoma and 8101Pro fibrosarcoma (n = 1 each); all cell linesexpressed Cerulean. Two to eight tumor regions were averaged per mouseand time point before pooling the data from individual mice. *P = 0.031.

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an SMA-DsRed mouse for 10 wk (Fig. 4 B and D). Thoroughscanning of IF sections from ApcMin mice “Swiss gut rolls,” con-taining both normal gut and adenomatous tissue, showed that fi-broblasts were not recruited from circulation into either healthy ormalignant tissue (Fig. 4 C and D). Thus, our results with autoch-thonous tumors in the parabiosis model support the conclusion thatTAFs originate mainly from local sessile precursors.

TAFs Constitute One-Third of the Tumor Stroma. To determine thecontribution of each component to the total tumor mass by livemicroscopy image quantification, mice were used in which the fol-lowing compartments were color-coded: cancer cells (Cerulean),BM-derived stroma [enhanced yellow fluorescent protein (EYFP)],and non–BM-derived stroma (DsRed) including COL-EGFP+

type I collagen-expressing fibroblasts (DsRed and EGFP) (Figs.S1 and S7 and Movies S1 and S2). Using this approach, collagen-expressing fibroblasts constituted the third most prominent cell

type in four different tumors. Cancer cells and BM-derived stromalcells constituted most of the cellular volume, their ratio beingabout 1:1 (Fig. S7 A and D). The non–BM-derived cells that werenegative for COL-EGFP included cells lining the vessels (probablyendothelial cells) and most likely some COL-EGFP−αSMA+ fi-broblasts (Fig. S7 B and C and Movies S1 and S2).

TAFs Form Perivascular Structures. In the “multicolor” tumor modeldescribed above, many DsRed+COL-EGFP+ cells wrapped thevessels once the tumor was established (Fig. 5A and Movie S1).Consistently, 66–83% of the total COL-EGFP+ cells were foundin close proximity to, or in direct contact with, an endothelial cellby two-color immunohistochemistry (Fig. S8). In dual reporterCOL-EGFP/SMA-DsRed mice, COL-EGFP+ cells often formedtubular structures around vessels in well-established tumors (Fig. 5Band Fig. S2). In the same tumors, αSMA+ cells could also be foundnext to vessels (Fig. S2, day 11), although were less abundant at late

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Fig. 3. TAFs derive predominantly from local sessile precursors. (A) Parabioticpairs of mice were created where only one mouse expressed COL-EGFP and theother mouse expressed DsRed ubiquitously. Two pairs were created: pair 1 con-tained immunocompetent mice with MC38-Cerulean tumors; pair 2 had MC57-Cerulean tumors in Rag−/− mice. On each mouse of the parabiotic pairs, cancercells were injected into the distal flank. (A) Representative images are shownfrom the freshly explanted MC57 tumors from pair 2 at week 3 of tumor growth(n = 6–9 areas per tumor; mean and SD). (B) Quantification of the EGFP+ areafraction (mean and SD) in the tumors from parabiosed mice. **P = 0.0022,***P = 0.001. (C) Full-thickness skin grafts from COL-EGFP mice were trans-planted into four DsRed Rag−/− mice. One month later, MC38-Cerulean cancercells were injected s.c. into the center of the green skin grafts with the help of aUV lamp. At day 12 (mouse 1) or 25 (mice 2–4) of tumor growth, mice were killedand 1- to 2-mm-thick slices were cut from freshly explanted tumors at a distanceof 2.5–3mm from the surface. The area fraction occupied by COL-EGFP+ cells wasquantified in 4–17 optical regions per mouse (mean and SD). A second experi-ment in which cancer cells were injected 2 mo after skin graft transplantationgave similar results (n = 3 mice). N.D., nondetectable. (Scale bars, 50 μm.)

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stages (Fig. 5B and Fig. S2, MC38, day 25). These data suggest thatTAFs associate spatially and temporally with the development oftumor vasculature.

DiscussionIn contrast to other stromal cells [e.g., pericytes (30), macro-phages (31), and endothelial cells (32)], the origin of TAFs hasremained unresolved. Here we show that TAFs derive predominantly

from local sessile precursors and only rarely fromBM circulating cells.Recruitment of BM-derived circulating cells to the tumor wasuncompromised, as the majority of the stromal compartmentwas BM-derived.Three lines of experimentation support the main conclusion of

this study. (i) In BM chimeric mice, fibroblast reporter genes wereexpressed almost exclusively in cells derived from the non-BMcompartment. (ii) In parabiotic pairs of COL-EGFP and DsRedmice, DsRed cells were abundant in the tumor grown in the COL-EGFP transgenic partner, whereas COL-EGFP+ cells could not bedetected in the tumor grown in the DsRed mouse; similarly, noCOL-EGFP+ cells were found in autochthonous tumors fromApcMin mice parabiosed to COL-EGFP mice. (iii) COL-EGFP+

TAFs were found in tumors developing in skin grafts from COL-EGFP mice transplanted into nonreporter mice.

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Fig. 4. TAFs originating from circulating cells cannot be detected in au-tochthonous ApcMin intestinal adenomas. ApcMin mice that develop in-testinal adenomas were parabiosed with fibroblast reporter mice. Animalswere killed when the ApcMin mice became moribund, and intestinal sectionswere stained with antibodies against EGFP and Col-I (A and C) or DsRed andαSMA (B and D). Representative immunofluorescence images are shownfrom (A) a COL-EGFP/ApcMin pair parabiosed for 5 wk and (B) an SMA-DsRed/ApcMin pair parabiosed for 10 wk. Dotted lines indicate polyp boundary. Datafrom experiments shown in A and B are quantified in C and D, respectively, asthe ratio of (C) EGFP+ and collagen I+ or (D) DsRed+ and αSMA+ area to nucleararea (DAPI) in positive control sections from normal reporter mouse gut versussections of Swiss gut rolls containing both normal gut and polyps or onlyApcMin polyps from the ApcMin mouse (mean and SD). Quantitative data werederived using 5×magnification images from scanned IF slides, comprising mostof the sample (at least 8–11 5× regions per slide or its entirety). Data are fromthree ApcMin Swiss roll slides and five slides containing only polyps (C), threeApcMin Swiss roll slides, and one slide containing only polyps (D), plus onepositive control slide in each case. Two additional parabiosis experiments inthe COL-EGFP model showed consistent results. **P < 0.01, ***P < 0.001.

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Fig. 5. Col-I–expressing cells form perivascular structures. (A) COL-EGFP+ cellsare found in close association with tumor vessels. COL-EGFP+ structures wrap theDsRed+ vessels in MC57 fibrosarcoma and MC38 carcinoma growing in DsRed+

COL-EGFP+ chimeric mice with an EYFP+ BM. Representative images are shownfrom two out of three independent longitudinal experiments performed withMC57, MC38, and Pro4L-Cerulean tumors (n = 1 mouse each). COL-EGFP+ cellswrapping vessels were observed in normal (non-BM chimeric) COL-EGFP mice infive more independent experiments. (B) SMA-DsRed+ cells do not form tubularstructures around vessels in late-stage established tumors. In day 22–25 MC57andMC38 tumors grown in COL-EGFP/SMA-DsRed dual reporter mice, structuresthat are COL-EGFP+ but SMA-DsRed− wrap the vessels. DiD-labeled red bloodcells (DiD-RBC) were injected i.v. to visualize blood flow. Representative imagesare shown from two out of three independent longitudinal experiments per-formed using Cerulean-expressing MC57, Pro4L, and MC38 (n = 1 mouse each).The host was Rag−/− for the MC57 and Pro4L tumors.

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The process of recruitment and activation of fibroblasts ingrowing tumors is not well-understood. Leukocytes play an im-portant role in promoting skin wound healing by secreting IL-22,which activates extracellular matrix production and expression ofαSMA by dermal fibroblasts (33). Importantly, in wound healing,as well as in renal fibrosis, fibroblasts are also predominantlyderived from local tissues (22, 34–37). It is plausible that tumorstromal BM-derived cells could similarly secrete factors thatrecruit local fibroblast precursors and induce their activation.Consistently, we found an almost immediate (14-h) infiltration ofleukocytes into cancer cell inoculation sites (27) and a delay inthe expression of αSMA compared with the earlier observationof αSMA−COL-EGFP+ TAFs (Fig. 1 and Fig. S2). The close prox-imity of BM-derived stromal cells and fibroblasts in tumors (Fig. S7)would allow easy access of leukocyte-produced factors to neigh-boring cells. Alternatively, resident fibroblasts may convert intotumor-promoting TAFs through TGF-β– and SDF-1–mediatedautocrine signaling loops (38).The partial overlap between expression of αSMA+ and Col-

EGFP observed by us (Fig. S3) and others (23, 35), may reflectdifferent states of activation of the promoters. The expression ofCol-I seems to be more stable and probably detects most of thefibroblasts, whereas αSMA could be induced or decreased undercertain circumstances such as active tumor formation and/orneovascularization. Interestingly, many COL-EGFP+ cells in thetumor wrapped vessels, forming “sheaths.” This is somewhatsurprising, because Col-I is not generally considered a compo-nent of the vessel wall. This wrapping was not so evident withαSMA-expressing cells, maybe because of the more transientexpression of αSMA (Fig. 5B and Fig. S2). However, αSMA ex-pression began with tumor vascularization, therefore temporallycorrelating with the development of tumor vessels. These findingsare consistent with abundant in vitro experimental evidence show-ing the role of fibroblasts in endothelial cell tubulogenesis by pro-moting vessel sprouting and, especially, formation of intercellularlumens (reviewed in ref. 39). Endothelial cells also produce extra-cellular matrix metalloproteinase inducer (EMMPRIN), whichdrives induction of αSMA and activation of fibroblasts (40). Theproduction of ECM and VEGF by living fibroblasts in close asso-ciation with endothelial cells seems to be required for tubulogenesisto occur (41). Our conclusions are also consistent with the sug-gested local origin of pericytes in tumors (30). It is therefore pos-sible that some of the αSMA- and/or Col-I–expressing cells wedescribe are pericytes, although costaining with other markers (42)would be necessary to properly characterize them.Although we do not know the precise identity of the local

precursors of type I collagen- and αSMA-expressing cells, mes-enchymal progenitors (30) or mesenchymal stem cells (MSCs) arelikely candidates. MSCs are radioresistant (43, 44), reside inmultiple organs in perivascular locations (45), and have migratorycapability (45). It has even been speculated that all MSCs could bepericytes, although not all pericytes are MSCs (46). Therefore,MSCs, pericytes, and most fibroblasts in tumors may well repre-sent various states of differentiation of the same cellular lineage.Multiple factors contribute to the contradictory reports on the

origin of TAFs. First is the lack of specificity of most of themarkers used to identify TAFs (24). Second, there is someconfusion about the terminology referring to circulating mesen-chymal precursors. This terminology has sometimes been appliedto hematopoietic (nonmesenchymal) precursors that differenti-ate into cells with fibroblastoid morphology (47). It is thoughtthat the number of TAF precursors circulating in blood might beextremely low (47–49). Third, many studies highlight the BMcontribution to mesenchymal cells in tumors without a side-by-sidecomparison of the relative contributions of circulating versus localsources (10, 12, 50). In our hands, the BM contribution is minimalcompared with the contribution of the non-BM compartment. In-terestingly, we observed occasional BM-derived TAFs only at later

time points during tumor development and only in the BM chimerasetting, whereas we could not detect any BM-derived TAFs in theexperiments in parabiotic mice. We cannot exclude the possibilitythat under certain pathophysiological conditions, such as the injuryinduced by total-body irradiation, the contribution of circulatingprecursors to TAFs can be increased. Finally, identification of TAFsin most previous studies relied on morphology and immunofluo-rescence of tumor sections. This is reminiscent of the controversyinvolving the origin of endothelial cells in tumors, in that markercolocalization on a single cell in tumor sections can be easily mis-taken for two juxtaposing/overlapping cells, as shown previously forendothelial cells (32). The predominant origin of endothelial cellswas eventually shown to be local (32). Here, we reach the sameconclusion about TAFs through the direct observation of live orfreshly explanted tumors.We have shown previously that both BM and non-BM com-

partments of tumor stroma must be targeted for T cell-mediatedtumor eradication (51, 52). TAFs are also targets for other typesof cancer therapy in preclinical studies (53–57) and even inclinical trials (58–60) because of their role in tumor promotionand resistance to drug therapy (reviewed in ref. 26) and immu-nosuppression (61). A better understanding of the origins anddevelopment of TAFs is essential for successful targeting. Ourresults should not discourage strategies to target tumor stromasuch as genetic manipulation of mesenchymal stem cells. How-ever, this manipulation has to occur at the site of tumor growth.If mesenchymal stem cells were to be used as “Trojan horses”(62), we would need to learn to attract them efficiently from distantsources. Until this is achieved, local therapies such as radiotherapymay be more appropriate than systemic treatments to target theformation of mesenchymal stroma. Our findings should also en-courage the search for more selective targets for destroying orinhibiting TAFs, based on the unique transcriptional and responsepatterns of fibroblasts from different sites/organs of the body.

Experimental ProceduresCOL-EGFP (63) and SMA-DsRed (αSMA-RFP) mice (23) were a generousgift from D. A. Brenner (University of California, San Diego, La Jolla, CA).Tg(ACTB-DsRed*MST)1Nagy/J and 129-Tg(ACTB-EYFP)7AC5Nagy/J mice express-ing the red fluorescent protein variant DsRed.MST or EYFP gene, respectively,under the control of the chicken beta-actin promoter coupled with the cyto-megalovirus immediate-early enhancer were backcrossed to C57BL/6 mice for 20generations and then crossed to Rag-1 KOmice to obtain DsRed and EYFP Rag-1KO mice, respectively. All strains were purchased from The Jackson Laboratory.Mice were bred andmaintained in a specific pathogen-free barrier facility at TheUniversity of Chicago according to Institutional Animal Care and UseCommittee (IACUC) guidelines. All animal experiments were approvedby the IACUC of The University of Chicago. Methylcholanthrene-inducedMC57G fibrosarcoma was provided by P. Ohashi (University of Toronto,Toronto, Canada), with permission of H. Hengartner (University HospitalZurich, Zurich, Switzerland). The fibrosarcoma cell lines Pro4L and 8101Prowere induced by UV light in C3H/HeN and C57BL/6 mice, respectively (64, 65).MC38 colon adenocarcinoma was induced by s.c. injection of dimethylhy-drazine in C57BL/6 mice (66), and was generously provided by A. Schietingerand P. Greenberg (University of Washington, Seattle, WA). MC57, Pro4L,and 8101Pro fibrosarcomas and MC38 carcinoma were retrovirally infectedwith pMFG-Cerulean (27) and FACS-sorted to generate lines expressingthe fluorescent protein Cerulean. More methods are available in SIExperimental Procedures.

ACKNOWLEDGMENTS. We thank David A. Brenner for the generous dona-tion of the COL-EGFP and SMA-DsRed mice, and F. Gounari for the generousdonation of the ApcMin mice. We also thank Christine Labno, Shirley Bond,Rolando Torres, Dorothy Kane, Christian Friese, and Khashayarsha Khazaiefor expert assistance with technical protocols; Karin Schreiber for providinganimals for experiments; Sydeaka Watson from the University of ChicagoBiostatistics Core for help with statistical analysis; and Donald A. Rowley,Boris Engels, David Binder, and Douglas Fearon for productive discussions.This work was supported by National Institutes of Health Grants P01-CA97296, R01-CA22677, and R01-CA37516; the Berlin Institute of Healthand Einstein Foundation (H.S.); and the Ludwig Foundation (R.R.W.). A.A.had a fellowship from Fundación Alfonso Martín Escudero (Spain).

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