Hematopoietic origins of fibroblasts: I. In vivo studies of fibroblasts associated with solid tumors

Experimental Hematology 34 (2006) 208–218

Hematopoietic origins of fibroblasts: I. In vivo studies of fibroblasts associated with solid tumors Amanda C. LaRuea,b, Masahiro Masuyaa,b, Yasuhiro Ebiharaa,b, Paul A. Flemingc, Richard P. Viscontic, Hitoshi Minamiguchia,b, Makio Ogawaa,b, and Christopher J. Drakec a Department of Veterans Affairs Medical Center; bDepartment of Medicine; Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA

c

(Received 29 August 2005; revised 11 October 2005; accepted 11 October 2005)

Objective. Recent studies have reported that bone marrow cells can give rise to tissue fibroblasts. However, the bone marrow cell(s) that gives rise to fibroblasts has not yet been identified. In the present study, we tested the hypothesis that tissue fibroblasts are derived from hematopoietic stem cells (HSCs) in vivo. Methods. These studies were conducted using mice whose hematopoiesis had been reconstituted by transplantation of a clonal population of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC in conjunction with murine tumor models. Results. When tumors propagated in the transplanted mice were evaluated for the presence of EGFP+ HSC-derived cells, two prominent populations of EGFP+ cells were found. The first were determined to be fibroblasts within the tumor stromal capsule, a subset of which expressed type I collagen mRNA and a-smooth muscle actin. The second population was a perivascular cell associated with the CD31+ tumor blood vessels. Conclusion. These in vivo findings establish an HSC origin of fibroblasts. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Introduction Tissue fibroblasts are a key source of interstitial extracellular matrix (i.e., collagens) and play a major role in both normal and pathological processes. It is generally held that new tissue fibroblasts are derived from proliferation of resident tissue fibroblasts. However, accumulating evidence suggests that tissue fibroblasts may also be derived from bone marrow cells. A bone marrow source of tissue fibroblasts has been demonstrated in studies examining the pathogenesis of kidney fibrosis [1], pulmonary fibrosis [2], wounding [3,4], and tumor formation [4–8]. While these studies strongly support the bone marrow as a source of tissue fibroblasts, the cell(s) within the bone marrow that gives rise to fibroblasts remains unclear. Bone marrow is thought to contain at least two types of stem cells, hematopoietic stem cells (HSC) and stromal/ mesenchymal stem cells (SSC/MSC). HSCs are defined Offprint requests to: Prof. Christopher J. Drake, Ph.D., Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Ave., Basic Science Building, Room 1692, Charleston, SC, 29425; E-mail: [email protected]

by their ability to repopulate the bone marrow and blood lineages of lethally irradiated mice. SSCs/MSCs are a population of bone marrow cells that were defined in vitro based on their adherent properties [9] and later by their ability to give rise to a variety of mesenchymal cell types including adipocytes [10,11], chondrocytes [10,12,13], osteoblasts/osteocytes [10,14,15], endothelial cells [16], and fibroblasts [17]. Based on their adherent properties [9] and their ability to give rise to mesenchymal cells including fibroblasts [17], it is widely held that the bone marrow source of fibroblasts is the SSC/MSC. Recent studies by our group [18,19] have raised the intriguing possibility that tissue fibroblasts may also have an HSC origin. In support of this we have demonstrated that kidney mesangial cells [18] and both brain microglial and perivascular cells [19], all of which are believed to represent specialized types of fibroblasts, are derived from HSCs. Fibroblasts are the most prominent cell type in the stroma of solid tumors and play an active role in generating the structural extracellular matrix required for tumor development [20–22], stimulating tumor growth [23], supporting neovascularization [24,25], and participating in metastasis

0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2005.10.009

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[26–28]. In the present study, we examined the ability of HSCs to give rise to matrix-producing fibroblasts within the tumor stroma. For these studies, a single-cell transplantation strategy was used in conjunction with murine solid tumor models. Clonal populations of cells were expanded from a single HSC that was isolated from the bone marrow of transgenic enhanced green fluorescent protein (EGFP) mice and transplanted into non-EGFP congenic recipient mice. After ensuring multilineage engraftment, murine melanoma or Lewis lung carcinoma (LLC) cells were delivered subcutaneously. Examination of resulting solid tumors revealed numerous HSC-derived EGFPD cells within the tumor-induced stroma. Morphological and immunohistochemical analyses led to the identification of these cells as fibroblasts and fibroblast-like perivascular cells. Findings presented in this study clearly establish an HSC origin of tumor stromal fibroblasts.

Materials and methods Mice C57BL/6-Ly5.1 breeders were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Transgenic EGFP breeding pairs (C57BL/6-Ly5.2 background) were provided by Dr. M. Okabe (Osaka University, Japan) [29]. Mice were bred and maintained at the Animal Research Facility of the Veterans Affairs Medical Center (Charleston, SC, USA). Research was conducted in accordance with guidelines set by the Institutional Animal Care and Use Committee, Department of Veterans Affairs Medical Center. Antibodies, hybridomas, and cytokines Phycoerythrin (PE)-conjugated D7 (anti-Ly-6A/E [anti-Sca-1]; rat immunoglobulin G2a [IgG2a]), allophycocyanin (APC)-conjugated 2B8 (anti-c-kit; rat IgG2b), biotinylated and fluorescein isothiocyanate (FITC)-conjugated RAM34 (anti-CD34; rat IgG2a), PE-conjugated, biotinylated, or purified RB6-8C5 (anti-Ly-6G [anti-Gr-1]; rat IgG2b), PE-conjugated or biotinylated RA3-6B2 (anti-CD45R/ B220; rat IgG2a), PE-conjugated 30-H12 (anti-Thy-1.2; rat IgG2b), biotinylated or purified TER-119 (anti-erythrocytes; rat IgG2b), PE-conjugated A20 (anti-CD45.1; mouse IgG2a), biotinylated CD3e (armenian hamster IgG1k), streptavidin-conjugated PE-Cy5, streptavidin-conjugated APC-Cy7, and purified anti-CD31 were purchased from BD Pharmingen (San Diego, CA, USA). PE-conjugated M1/70.15 (anti-Mac-1; rat IgG2b) was purchased from Caltag Laboratories (Burlingame, CA, USA). Anti-GFP rabbit IgG was purchased from Molecular Probes (Eugene, OR, USA). Cy3-conjugated anti-a-smooth muscle actin was purchased from Sigma (St. Louis, MO, USA). Secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA, USA). Hybridoma 14.8 (anti-B220; rat IgG2b), GK1.5 (anti-CD4; rat IgG2b), and 53.6.72 (anti-CD8a; rat IgG2b) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Recombinant mouse stem cell factor (SCF) and human granulocyte colony-stimulating factor (G-CSF) were purchased from R&D Systems (Minneapolis, MN, USA) and Amgen (Thousand Oaks, CA, USA), respectively. Rabbit anti-NG2 chondroitin sulfate proteoglycan (AB5320) was purchased from Chemicon International (Temecula, CA, USA).

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Transplantation Donors were 10- to 12-week-old EGFP mice (C57BL/6-Ly5.2) and 10- to 14-week-old C57BL/6-Ly5.1 mice were used as irradiated recipients and for radioprotective cells. Bone marrow cells were flushed from femurs and tibiae, pooled, and washed in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). Mononuclear cells were isolated by gradient separation using Lympholyte M (Cedarlane Laboratories Limited, Ontario, Canada) and lineage-negative (Lin2) cells were prepared by negative selection using antibodies to B220, Gr-1, CD4, CD8, TER-119, and M450 sheep anti-rat IgG dynabeads (Dynal Biotech Inc, Brown Deer, WI, USA). HSCs were stained and identified using two methods: 1) in a modification of a previously described method [18,19], Lin2 cells were stained with PE-conjugated anti-Sca-1, APC-conjugated anti-c-kit, and biotinylated anti-CD34 followed by streptavidinconjugated APC-Cy7; and 2) in a modification of that described by Matsuzaki et al. for side population (SP) [30], Lin2 cells were stained (PE-conjugated anti-Sca-1, biotinylated anti-CD34, and streptavidin-conjugated PE-Cy5); resuspended (1 3 106 cells/mL) in HBSSD (Ca2D-, Mg2D-free Hanks balanced salt solution [HBSS]; Invitrogen, Gaithersburg, MD, USA) with 2% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, USA), 10 mM HEPES, 1% penicillin/streptomycin, and Hoechst 33342 (Sigma; 5 mg/mL); and incubated at 37 C for 60 minutes. For either method, propidium iodide was added (1 mg/mL) and cell sorting was performed using a FACSVantage cell sorter (BectonDickinson, San Jose, CA, USA). The gate used for SP cells corresponded to R3 and R4 subfractions described by Goodell et al. [31] and R1 and R2 described by Matsuzaki et al. [30]. Single cells were deposited into wells of Corning U-bottom 96-well culture plates (Corning, NY, USA) using the CloneCyt system (Becton-Dickinson Immunocytometry Systems). Eighteen hours after single-cell deposition, wells containing single cells were identified and the incubation continued for a total of 7 days at 5% CO2, 37 C in media containing a-modification of Eagle’s medium (ICN Biomedicals, Aurora, OH, USA), 20% FBS, 1% deionized fraction V BSA, 1 3 10-4 mol/L 2-mercaptoethanol (Sigma), 100 ng/mL SCF, and 10 ng/mL G-CSF. To study the full differentiation potentials of HSCs, it was important to generate mice exhibiting high-level, multilineage engraftment from single HSCs. Our previous studies have shown that single-cell deposition and transplantation based on Lin2Sca-1Dc-kitD CD342 phenotype alone was inefficient for producing mice with high-level engraftment. To raise the efficiency, we devised a method combining single-cell deposition with short-term cell culture. As described above, Lin2Sca-1Dc-kitD CD342 or Lin2Sca-1D CD342 SP cells were individually cultured for one week in the presence of SCF and G-CSF, a combination of cytokines that stimulates proliferation of primitive hematopoietic progenitors [32]. Because the majority of HSCs are dormant in cell cycle and begin cell division a few days after initiation of cell culture, we selected clones consisting of no more than 20 cells after incubation. This method significantly enhanced the efficiency of generating mice with high-level multilineage engraftment [18,19]. Approximately one-fourth of all mice transplanted using these methods resulted in high-level multilineage engraftment above 50%. Those mice with engraftment levels below 50% were not used for studies.

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Recipient C57BL/6-Ly5.1 mice were given a single 950-cGy dose of total-body irradiation using a 4 3 106 V linear accelerator. Contents of wells (#20 clonal cells) were injected via tail vein into lethally irradiated mice with 500 Ly5.1 bone marrow Lin2ckitDSca-1D CD34D radioprotective cells [18,19]. Osawa et al. [33] showed that these cells are effective radioprotective cells and contain almost no HSCs. For hematopoietic engraftment analysis, peripheral blood was obtained from the retroorbital plexus of anesthetized mice and red blood cells were lysed (1X PharM Lyse; BD Pharmingen). Donor-derived cells (EGFPD) in T cell, B cell, granulocyte, and monocyte/macrophage lineages were analyzed by staining with PE-conjugated anti-Thy-1.2, anti-CD45R/B220, anti-Gr-1, and anti-Mac-1, respectively. Control mice were generated to exclude the effects of pretransplantation cell culture by transplanting 100 non-cultured Lin2Sca-1Dc-kitD CD342 bone marrow cells from EGFP mice. Tumor cell lines The melanoma cell line (K1735-M2) was a gift from Dr. Mary Hendrix (Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA, USA) and is an aggressive, highly metastatic murine melanoma line. Lewis lung carcinoma (LLC; ATCC) is a highly tumorigenic, weakly metastatic murine line derived from the C57BL/6 strain of mice. Both lines tested negative for murine pathogens (IMPACT Profile I) and helicobacter (Radil Research Animal Diagnostic Laboratory, University of Missouri, Columbia, MO, USA). Cell lines were propagated and harvested using standard cell culture methods. Briefly, cells were grown to 95% confluency in 5% CO2, 37 C in media containing Dulbecco’s modified Eagle medium (Invitrogen) and 10% FBS and washed in serum-free HBSS, trypsinized (trypsin-EDTA; Invitrogen). Recovered cells were resuspended in 0.2 mL serum-free medium at a concentration of 5 3 106 cells/0.2 mL and injected subcutaneously into anesthetized engrafted mice. When tumors reached 1.0 to 1.5 cm2 in size, mice were anesthetized and perfused via intracardiac puncture with 15 mL PBS followed by 15 mL 4% paraformaldehyde. Tumors and surrounding tissues were extracted and fixed 9 to 18 hours in 4% paraformaldehyde. Immunolabeling and microscopy Tumors from engrafted mice were processed for paraffin sectioning as described [18] using protocols that allow for preservation of fluorescence intensity during tissue processing described by others [34,35]. Epifluorescence and differential interference contrast (DIC) imaging was conducted using a Leica DMR microscope with a narrow-bandpass GFP excitation cube. Laser scanning confocal microscopy (LSCM) was conducted using a Bio-RAD MRC1024 laser-scanning confocal microscope (Bio-Rad, Microscopy Division, Cambridge, MA, USA). Confocal z-series were collapsed using Image J 1.31v (National Institutes of Health, Bethesda, MD, USA) and processed using Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA, USA). In situ hybridization Complementary RNA probes were synthesized from a linearized expression plasmid containing a 321-bp fragment from the 3# untranslated region (UTR) of COL1A1 (gift from Maria Trojanowska, Medical University of South Carolina, Charleston, SC, USA). Sense and anti-sense riboprobes were labeled with digoxigenin (DIG)-11-UTP using a DIG-RNA-labeling kit (Roche Molecular

Biochemicals, Indianapolis, IN, USA). In situ hybridization to 3-mm paraffin sections of excised tumors was performed following a previously published protocol [36]. Labeled RNA probes were used at a final concentration of 1 ng/mL. Hybridization was performed in a humidified chamber for 18 hours at 65 C. After posthybridization washing, DIG-labeled probes were detected using a DIG-nucleotide detection kit according to manufacturer’s protocol (Roche). Hybridized sections were counterstained with Nuclear Fast Red (VectorLabs, Burlingame, CA, USA), dehydrated through graded methanols, cleared in xylenes, and mounted. Anti-GFP immunolabeling was performed on sequential consecutive 3-mm sections using a polyclonal antibody to GFP. Bound primary antibodies were detected with a Vectastain elite ABC kit using 3,3#-diaminobenzidine (DAB) as the chromogenic substrate. Fluorescence in situ hybridization analysis of Y-chromosome Y-chromosome–specific fluorescence in situ hybridization (FISH) analysis was performed on tumor tissue from male recipients of a clonal population of cells derived from a single female EGFPD HSC as previously described [18,19]. Briefly, sections were deparaffinized, serially dehydrated, and reacted with FITC-labeled Ychromosome paint probe (Cambio Ltd., Cambridge, UK). EGFPD cells were identified by AF594-rabbit anti-GFP followed by Cy3labeled affinity-purified F(ab#)2 fragments of donkey anti-rabbit IgG (HDL). Nuclei were counterstained with bis-benzimide (Sigma-Aldrich) and sections were coverslipped and imaged.

Results Generation of mice transplanted with a clonal population derived from a single HSC To investigate the potential contribution of bone marrow– derived hematopoietic stem cells to tumor development, we first generated mice with high-level, multilineage engraftment from single HSCs. As described in the Methods, Lin2Sca-1Dc-kitD CD342 cells or Lin2Sca-1D CD342 SP cells were individually cultured for one week in the presence of SCF and G-CSF and clones consisting of 20 or fewer cells were transplanted into lethally irradiated recipients. Two to six months after cell transplantation, nucleated blood cells from these mice were analyzed for hematopoietic engraftment and mice exhibiting high-level Table 1. Lethally irradiated mice were transplanted with a clonal population of cells derived from a single EGFPD HSC. Engraftment and lineage analysis was conducted 2 months posttransplant. Lineage engraftment, % EGFPD

Mouse

Tumor cells delivered

Total engraftment, % EGFPD

Gr1DMac1D

B220D

Thy 1.2D

1 2 3 4 5

Melanoma Melanoma Melanoma LLC LLC

69.7 74.9 87.1 79.1 75.5

73.4 79.5 96.5 75 93.3

98.2 84.5 96.1 89.9 96.8

66.4 49.2 58.8 62.1 30.7

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Figure 1. Multilineage engraftment from a clonal population derived from a single EGFPD HSC. Shown is a representative flow cytometric analysis of peripheral blood from a mouse 2 months after being transplanted with a clonal population of cells derived from a single EGFPD HSC. Total engraftment by EGFPD cells was found to be 75% (C) and EGFPD cells represented 80%, 85%, and 50% of total cells in the granulocyte/macrophage (D), B cell (E), and T cell (F) lineages, respectively. (A,B): controls showing peripheral blood cells from a control Ly5.1 mouse, stained with anti-CD45.1-PE, and a control EGFP.

multilineage engraftment by donor EGFPD cells were selected for study (Table 1). Figure 1 is an example of flow cytometric analysis of the nucleated blood cells from a representative recipient mouse exhibiting high-level

multilineage engraftment. The majority of the granulocytes, macrophages, and B cells in this mouse were EGFPD, demonstrating an almost complete hematopoietic reconstitution by the clonal population transplanted. The

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slow turnover of T cells (i.e., memory T cells) results in the presence of recipient EGFP2 T cells. HSC-derived EGFPD cells incorporate into the tumor-induced stroma Mice demonstrating high multilineage engraftment from a clonal population of EGFPD HSCs were injected subcutaneously with murine melanoma cells (n 5 3). Analysis of thin (3-mm) paraffin sections from tumors grown in transplanted mice revealed numerous EGFPD cells in the tumor stroma (Fig. 2A and B), both within the fibrous capsule surrounding the neoplastic cells (arrows) and among the tumor cells (asterisks). These findings demonstrate that HSCs of the bone marrow can contribute cells to melanoma-induced stroma. HSCs give rise to fibroblasts within the tumor-induced stromal capsule To examine the morphology of individual HSC-derived EGFPD cells within the melanoma stroma, high-magnification epifluorescence and DIC microscopy were used. EGFPD cells (Fig. 2C, arrows) observed within the tumor capsule were found to have a morphology consistent with

that described for fibroblasts, including an elongated shape and spindle-like processes (Fig. 2D, arrows). To evaluate if the EGFPD cells within the stromal capsule also possessed a synthetic profile characteristic of fibroblasts, in addition to exhibiting phenotypic properties of fibroblasts, the pattern of mRNA and protein expression of these EGFPD cells was examined. In situ hybridization studies using a riboprobe for the a1 subunit of type I procollagen and immunolabeling studies using antibodies to GFP were conducted on sequential consecutive paraffin tissue sections (Fig. 3). Analysis showed a subpopulation of EGFPD cells (brown) within the tumor stroma that were positive for type I procollagen mRNA expression (blue, arrows). In addition, the EGFPD cells that expressed type I procollagen mRNA exhibited a fibroblastic morphology. When images from sequential tissue sections were superimposed and fibroblast populations were counted, the percentages of HSC-derived fibroblasts (EGFPD/procollagen Ia1D) ranged from 0 to 28.6% (mean 8.3 6 7.1; n 5 29). Also evident in Figure 3 are non–EGFP-derived fibroblasts (procollagen Ia1D/EGFP2 cells; arrowheads) that are morphologically indistinguishable from the EGFPD fibroblasts (arrows). The finding that HSC-derived EGFPD cells both exhibit

Figure 2. HSC-derived EGFPD cells contribute to the melanoma-induced stromal capsule. Analysis of sectioned melanoma tissue from mice transplanted with a clonal population of cells derived from a single EGFPD HSC show numerous EGFPD cells within the tumor stroma (A,B), both in the tumor capsule (arrows) and among the tumor cells (asterisks). Panels A and B depict EGFP fluorescence and the tumor-capsule boundary is indicated by dashed line. Higher-magnification examination of tumor sections using epifluorescence and DIC shows EGFPD cells within the tumor capsule with morphological properties of fibroblasts (C–E; arrows). Panel C shows an epifluorescence image, Panel D shows the corresponding DIC image, and the superimposition of the epifluorescence and DIC images is depicted in Panel E. Bar equals 25 mm (A,B); 12.5 mm (C–E).

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Figure 3. EGFPD cells express procollagen Ia1 mRNA. In situ analysis of sectioned melanoma tissue from clonally engrafted mice shows that a subset of EGFPD cells (A,C; arrows) with a fibroblastic morphology within the stromal capsule express type I collagen message (B,D; arrows). EGFP2 fibroblasts (EGFP2/procollagen Ia1D cells; arrowheads) and nonfibroblastic HSC-derived cells (EGFPD/procollagen Ia12 cells; open arrowheads) were also observed. Expression of procollagen Ia1 was detected with a RNA probe to the 3# UTR of the mouse COL1A1 gene (blue); anti-GFP expression was detected in the adjacent section (brown). Bar equals 25 mm.

morphology characteristic of fibroblasts and express type I collagen mRNA indicated that these cells were fibroblasts. To determine if the HSC-derived fibroblasts possess properties of myofibroblasts, the EGFPD cells were evaluated for a-smooth muscle actin (aSMA) expression, a protein whose expression is associated with myofibroblasts [37–40]. Examination of tumor sections immunolabeled with antibodies to aSMA showed that a subset of EGFPD cells with a fibroblastic morphology were positive for aSMA expression (Fig. 4; arrows). These findings suggest that in addition to fibroblasts, the HSC may also give rise to myofibroblasts. Bone marrow HSCs give rise to perivascular cells within the tumor stroma In addition to the EGFPD cells within the tumor stromal capsule that exhibited a fibroblastic morphology, a second population of EGFPD cells with a more irregular, stellate morphology was observed within the tumor. As these cells were closely associated with what appeared to be blood

vessels, sections of melanoma tissue were immunolabeled with antibodies to CD31, a protein whose expression is indicative of endothelial cells. When sectioned tumors were analyzed using epifluorescence microscopy, it appeared as though these EGFPD cells also expressed CD31 (data not shown). To determine if these EGFPD cells were indeed endothelial cells, i.e., cells co-expressing EGFP and CD31, sectioned tumors were analyzed using multi-channel LSCM to sequentially analyze individual optical planes (1 mm thick) for EGFP (green channel) and CD31 (red channel) expression (Fig. 5A and B, arrows). High-magnification LSCM images were obtained (Fig. 5C) and subsets (w3– 4 mm total) of these images were collapsed (Fig. 5D–F). Analysis demonstrated that the EGFPD cells were tightly apposed to the vessel wall, were located on the abluminal side of the vasculature, and did not express CD31. These cells had long processes that appeared to wrap around the endothelial cells (ECs), a morphological characteristic of EC support cells or perivascular cells (Fig. 5D–F, arrows). To further characterize these cells, sections were

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Figure 4. EGFPD cells express aSMA. Analysis of sectioned melanoma tissue from clonally engrafted mice shows that some EGFPD (green; A,D; arrows) cells with a fibroblastic morphology within the stromal capsule also express aSMA (red; B,E; arrows). High-magnification images of cells denoted by asterisks in panels A and B and panels D and E are shown in panels C and F, respectively. Bar equals 25 mm (A, B, D, E); 12.5 mm (C, F).

immunolabeled with antibodies to either aSMA or NG2, a proteoglycan expressed by pericytes [41,42]. Analysis indicated that the EGFPD perivascular cells were negative for both aSMA and NG2 expression (data not shown). Together, these findings suggest that a population of HSC-derived EGFPD cells within the tumor were fibroblast-like perivascular cells, but did not express markers characteristic of pericytes.

HSCs possess the inherent capability to give rise to fibroblasts It was possible that the apparent ability of HSCs to generate tumor fibroblasts and perivascular cells was a result of the short-term culture period prior to transplantation rather than an inherent capacity to differentiate into these cell types. To exclude this possibility, two mice were transplanted with 100 noncultured Lin2Sca-1Dc-kitD CD342 cells. After

Figure 5. HSC-derived EGFPD cells within the tumor stroma are perivascular cells. Immunolabeling of sectioned melanoma tissue from clonally engrafted mice with antibodies to an endothelial cell marker, CD31/PECAM (red), shows that EGFPD cells are in association with CD31D ECs (arrows), but do not express CD31. Panels A–C show collapsed confocal images; panels D–F show a subset of confocal slices (3–4 mm) from panel C. Bar equals 25 mm.

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ensuring high levels of engraftment (40% and 93%), mice were injected subcutaneously with melanoma cells. Morphological examination of the resulting tumors using epifluorescence and DIC microscopy showed EGFPD fibroblast-like cell populations that were identical to those seen in clonally engrafted mice (data not shown). Tumor fibroblasts and perivascular cells are the result of HSC differentiation Several studies have reported spontaneous fusions between EGFPD embryonic stem cells and somatic cells in culture [43,44] and suggested that some data describing plasticity may be the result of spontaneous cell fusions. To exclude this, tumors from female-to-male transplanted mice were analyzed using FISH for Y-chromosome. No Y-chromosome-containing EGFPD cells were found within the tumor stroma (n 5 233 EGFPD cells; Fig. 6), suggesting that HSC-derived EGFPD tumor stromal cells are derived from HSC differentiation. This finding is consistent with our previous studies examining HSC potential [18] and those of others [45–48].

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HSC-derived fibroblasts play a role in other solid tumors To determine if the ability of HSCs to contribute to tumor stromal fibroblasts is specific for melanoma or if the observed phenomenon may extend to other solid tumors, clonally engrafted mice were subcutaneously injected with murine LLC cells (n 5 2). Histological examination of LLC tumor tissue sections revealed populations of EGFPD fibroblasts similar to those observed in melanoma tumors both within the tumor stromal capsule (Fig. 7A and B, arrows) and deep within the tumor tissue (Fig. 7A and B, asterisks) as well as a population of EGFPD perivascular cells juxtaposed to CD31D endothelial cells (Fig. 7C and D, arrows). These findings suggest that HSCs may contribute fibroblastic populations in multiple solid tumors.

Discussion Based on our previous work demonstrating that HSCs give rise to specialized fibroblast-like cells [18,19] and recent studies suggesting a bone marrow origin of tissue fibroblasts [1–8], we tested the hypothesis that tissue fibroblasts

Figure 6. Cell fusion does not contribute to EGFPD tumor fibroblasts. (A–D) Fluorescence in situ hybridization for Y-chromosome was conducted on melanoma sections from clonally engrafted mice that were the result of a female-to-male transplant. Analysis of sections shows that no EGFPD cells (anti-GFP, red) contained Y-chromosomes (FISH Y-chromosome, green) within their HoechstD (blue) nucleus (arrows) and only EGFP2 recipient cells contained Ychromosomes (arrowheads). Inset in panel D shows a higher magnification of an EGFPD/Y-chromosome-cell next to an EGFP2/Y-chromosome-containing cell (asterisk). Bar equals 25 mm.

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Figure 7. HSC-derived fibroblasts contribute to Lewis lung carcinoma. Analysis of sectioned LLC tissue from clonally engrafted mice showed numerous EGFPD cells in the tumor stroma (A,B), both within the capsule (arrows) and among the tumor cells (asterisks). EGFPD cells found in association with the CD31D endothelium were identified as perivascular cells (C,D, arrows). The tumor-capsule boundary is indicated by dashed line; ‘‘a’’ indicates adipocytes in tumor section. Bar equals 25 mm (A,B); 12.5 mm (C,D).

are derived from HSCs. Using transplantation of a clonal population of cells derived from a single HSC and murine tumor models, we established that HSCs are a source of tumor stromal fibroblasts and perivascular cells. Our data showed that HSC-derived EGFPD stromal cells with morphological characteristics of fibroblasts expressed procollagen Ia1 and aSMA. Identification of HSC-derived perivascular cells was based on phenotype and their close approximation to the vascular endothelium, as these cells did not express either aSMA or NG2. Rajantie et al. [7] described a similar population of bone marrow–derived perivascular cells that do not express aSMA and whose expression of NG2 is limited to a subpopulation. Considering the above, and our previous observation of a perivascular cell population of HSC origin in the infarcted brain [19], we feel that our designation of such cells as perivascular cells is reasonable. In our evaluation of the melanoma and LLC vasculature, we noted no evidence of HSC differentiation to the endothelial lineage. This finding is in accord with our previous observations investigating the potential of HSCs [19] and that of others examining the endothelial cell potential of stem cells [6,7,49,50]. Considering our present findings of HSC-derived fibroblasts and perivascular cells in the tumor-induced stroma in conjunction with our previous works [18,19] and those of Ebihara et al. [51], which demonstrate that HSCs give rise to fibroblasts and their precursors in vitro, we propose that

the HSC may represent a systemic source of fibroblasts. These findings are significant in that they provide the first evidence of an HSC source of tissue fibroblasts. With regard to pathologies in which fibrosis plays a significant role, such as solid tumors, lung fibrosis, and wound healing, these studies provide a specific, readily identifiable target for treatment. Acknowledgments This work was supported by National Institutes of Health grants NIH-HL 07260 (ACL), RO1 HL52813 (MO), RO1 HL69123 (MO), RO1 HL57375 (CJD), and DAMD 17-00-1-0338 (CJD) and by the office of Research and Development, Medical Research Services, Department of Veterans Affairs. The authors would like to acknowledge Dr. Haiqun Zeng for FACS sorting, Ms. Joyce E. Edmonds for expertise in tissue processing, and the staff of the MUSC Radiation Oncology Department for assistance in irradiation of mice.

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