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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC Jan 15, 2012.
Published in final edited form as:
PMCID: PMC3100554
NIHMSID: NIHMS278748
Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks
Suling Liu,1* Christophe Ginestier,2 Sing J. Ou,1 Shawn G. Clouthier,1 Shivani H. Patel,1 Florence Monville,2 Hasan Korkaya,1 Amber Heath,1 Julie Dutcher,1 Celina G. Kleer,1 Younghun Jung,3 Gabriela Dontu,1 Russell Taichman,3 and Max S. Wicha1*
1 Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109, USA
2 Centre de Recherche en Cancerologie de Marseille, Laboratoire d’Oncologie Moleculaire, UMR891 Inserm/Institut Paoli-Calmettes, Marseille, France
3 School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109, USA
* Corresponding authors: Max S Wicha: mwicha/at/umich.edu, Address: 1500 E. Medical Center Dr., 6303 Cancer center, Ann Arbor MI 48109-0946 USA, Telephone: 1-734-936-1831, Fax: 1-734-615-3947. Suling Liu: sulingl/at/med.umich.edu, Address: 1500 E. Medical Center Dr., 7110 Cancer center, Ann Arbor MI 48109-0942 USA, Telephone: 1-734-615-4651, Fax: 1-734-647-9480
We have utilized in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSCs) and bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase (ALDH) expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice, labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerate tumor growth by increasing the breast cancer stem cell population. Utilizing immunochemistry, we identified “MSC-CSC niches” in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow derived mesenchymal stem cell may accelerate human breast tumor growth by generating cytokine networks that regulate the cancer stem cell population.
Many human cancers, including breast cancer may be driven by a population of cells which display stem cell properties. These properties include self-renewal which drives tumorigenesis and differentiation which contributes to cancer cell heterogeneity. There is increasing evidence that these “cancer stem cells” mediate tumor metastasis and by virtue of their relative resistance to chemotherapy and radiation therapy may contribute to treatment resistance and relapse following therapy (1).
Self-renewal and cell fate determination of normal stem cells are regulated by both cell intrinsic and cell extrinsic pathways. The dysregulation of these pathways resulting in stem cell expansion may be a key event initiating carcinogenesis. Developmental pathways such as Notch, Hedgehog and Wnt play an important role in normal stem cell function and are frequently deranged in cancers (25). Extrinsic signals which regulate stem cell behavior originate in the stem cell microenvironment or “niche”. This niche contains extracellular components as well as multiple cell types.
Although there is little information on the composition and function of “cancer stem cell niches” it is clear that tumor growth and metastasis is highly dependent on the tumor microenvironment. This microenvironment is comprised of tumor associated fibroblasts, endothelial cells, adipocytes and immune cells, all of which have been demonstrated to play a role in tumor growth and metastasis (6). Mesenchymal stem cells (MSCs), which can be defined as multipotent mesenchymal stromal cells, are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues, proliferate as adherent cells, have fibroblast-like morphology, form colonies in vitro and can differentiate into adipocytes, osteocytes, and chondrocytes (7). Recently, utilizing mouse breast cancer models, it has been demonstrated that bone marrow derived mesenchymal stem cells may be recruited to sites of developing tumors influencing their metastatic potential (8). It has been shown that MSCs can produce IL6 (910), and stimulate tumor growth through the paracrine production of secreted IL6 (11). Both IL6 and IL8 have been implicated in the regulation of cancer stem cells (1213).
We have previously demonstrated that both normal and malignant mammary stem cells can be isolated by virtue of their increased expression of aldehyde dehydrogenase (ALDH) as assessed by the ALDEFLUOR assay. We have utilized this methodology to isolate functional stem cells from primary breast xenografts as well as established human breast cancer cell lines and demonstrated that these cells mediate tumor invasion and metastasis (14). In the present study, we examined the interaction between bone marrow derived mesenchymal stem cells (MSCs) and cancer stem cells (CSCs) utilizing in vitro systems and mouse models. We demonstrate that mesenchymal cells (MCs), like CSCs are organized in a cellular hierarchy and that ALDEFLUOR-positive mesenchymal cells regulate CSC self-renewal. Interaction between these cell types is mediated by a cytokine network involving CXCL7 and IL6. Furthermore, we demonstrate that labeled human bone marrow mesenchymal cells traffic from the bone marrow to accelerate growth of human breast cancer xenografts at distant sites by expanding the CSC population. These studies suggest that MSCs form an important component of the “cancer stem cell niche” where they regulate the self-renewal of breast cancer stem cells.
Cell culture
Breast cancer cell lines (SUM159 and SUM149) obtained from Dr. Stephen Ethier have been extensively characterized (http://www.asterand.com/Asterand/human_tissues/hubrcelllines.htm); (15). MCF-7 cell line was purchased from ATCC. The cell lines were grown using the recommended culture conditions as described previously (16). Human bone marrow-derived mesenchymal cells (MCs) cryopreserved at passage one were purchased from ScienCell Research Laboratories (Carlsbad, CA) and grown and passaged in the recommended Medium (Carlsbad, CA). These MCs were characterized by the expression of the MC markers CD29, CD90, CD44, CD105 but not CD45, CD34 and CD11b at both Passage 2 (Figure S1A) and Passage 10 (Figure S1B). All experiments were done with subconfluent cells in the exponential phase of growth. The primary MCs from bone marrow were purchased from Texas A&M HSC COM and cultured in the recommended culture medium.
Virus infection
A highly efficient lentiviral expression system (pLentiLox 3.7; http://www.med.umich.edu/vcore/) was used to generate DsRed-, GFP- or luciferase-expressing lentiviruses in UM Vector Core Facility. The cell lines were infected with the lentiviruses as described previously (3).
ALDEFLUOR assay and Flow cytometry
The ALDEFLUOR kit (StemCell technologies Inc) was used as described previously (17). The antibodies for IL6R and gp130 were purchased from Immunotech and Pharmingen; and flow cytometry with antibody staining was performed as described previously (17). 0.5μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) was used to access cell viability.
In vitro differentiation
In vitro differentiation of hMSC-bm (unseparated population, ALDEFLUOR-positive population, ALDEFLUOR-negative population) was evaluated in triplicates and the detailed procedure for the differentiation assay is described in the supplementary material.
RNA extraction
Total RNA was isolated using RNeasy Micro Kit, according to the manufacturer’s instructions (QIAGEN).
Gene expression profiling with DNA microarrays
Affymetrix human U133 Plus 2.0 was utilized. Preparation of cRNA, hybridizations, washes and detection were done as recommended by the supplier (http://www.affymetrix.com/index.affx). Expression data were analyzed by the RMA (Robust Multichip Average) method in R using Bioconductor and associated packages (18).
Real-time quantitative RT-PCR (qRT-PCR)
1 μg of total RNA from mammospheres or differentiated cells on collagen-coated plates was utilized for real-time qRT-PCR as described previously (19).
Conditioned medium, Antibody array and Luminex bead assay
To prepare conditioned media, breast cancer cell lines (BCC: SUM159 or SUM149) alone, MSC alone or the co-culture of BCC and MSC (1:1 mixture) were plated in 100-mm tissue culture dishes in the mixture of IHM and MSCM (1:1 mixture). The detailed procedure is described in the supplementary material.
Invasion assay
Assays were done in triplicate in invasion chambers pre-coated with reduced growth factor matrix from BD Biosciences. Cells were added to the upper chamber in 200ul of serum-free medium. For the invasion assay, 20,000 MCs were seeded on the coated chamber and the lower chamber was filled with 600ul of medium (Cambrex) with or without 100ng/ml IL6, 100ug/ml IL6 blocking antibody, 20,000 pre-seeded SUM159, SUM149 or MCF-7 cells. After 27h of incubation, the cells on the underside of the upper chambers were stained with the blue stain in the Cell Invasion Assay Kit (Chemicon Cat# ECM550) and counted using light microscopy.
Tumorigenicity in NOD/SCID mice
Six-week old female NOD/SCID mice were purchased from Jackson Laboratories. Tumorigenicity of 1000 of ALDELFUOR-positive, -negative and unseparated SUM159-DsRed in the absence or presence of equaled number of MSCs in the mammary fatpads of NOD/SCID mice was accessed. Six mice were assayed for each group. The animals were euthanized when the tumors were about 1 – 1.5cm in the largest diameter, in compliance with regulations for use of vertebrate animal in research. A portion of each fat pad was fixed in formalin and embedded in paraffin for histological analysis. Another portion was analyzed by the ALDEFLUOR assay. In addition to the established cell lines, MC1 primary xenografts were developed and utilized as previously described (20).
Intra-tibia injection
The mouse preparation and the intramedullary injection into the tibia shaft was carried out according to previously published methods (2122). Briefly, a few hours before transplantation, mice were irradiated with 300 cGy from an x-ray irradiator (Mark I, Model 25, J.L. Shepherd). All procedures were approved by the Animal Care Committee of University of Michigan. The intramedullary injection into the tibia shaft was carried out according to previously published methods (2122). The detailed procedure is described in the supplementary material.
Bioluminescence imaging
Baseline bioluminescence was assessed before inoculation and each week thereafter. For photon flux counting, we used a charge-coupled device camera system (Xenogen) with a nose-cone isofluorane delivery system and heated stage for maintaining body temperature. Results were analyzed after 2 to 12 min of exposure using Living Image software provided with the Xenogen imaging system.
Immunostaining
For ALDH1, DsRed, GFP and DAPI quadruple fluorescent staining, paraffin-embedded sections of breast tumors from xenografts were deparaffinized in xylene and rehydrated in graded alcohol. Antigen enhancement was done by incubating the sections in citrate buffer pH6.0 (Dakocytomation, Copenhagen, Denmark) as recommended. ALDH1 antibody (BD biosciences), DsRed antibody (Santa Cruz) and GFP antibody (Neomarker) were used at a 1:25 dilution and incubated for 1 hour. PE, FITC and Cy5 labeled secondary antibodies (Jackson Labs) were used at the dilution 1:250 and incubated for 20 min. PE-conjugated secondary antibody (Red color in the staining) was used to detect the ALDH1 primary antibody, FITC-conjugated secondary antibody (Green color in the staining) was used to detect the DsRed primary antibody, and Cy5-conjugated secondary antibody (Purple color in the staining) was used to detect the GFP primary antibody. Nuclei were counterstained with DAPI/antifade (INVITROGEN) (Blue color in the sataining) and cover slipped. Sections were examined with a fluorescent microscope (Olympus FV-500 Confocal).
Statistical analysis
Results are presented as the mean ± standard deviation (STDEV) for at least 3 repeated individual experiments for each group. Mean and STDEV was determined based on analysis of at least three replicates using Microsoft Excel. Statistical differences were determined by using ANOVA and student’s t-test for independent samples. A p-value of less than 0.05 was considered statistically significant.
Bone marrow derived mesenchymal stem cells are able to expand the breast cancer stem cell population
In order to assess the ability of bone marrow mesenchymal stem cells (MSCs) to affect breast cancer stem cell functionality, we co-cultured DsRed labeled SUM159 breast cancer cells with human bone marrow derived mesenchymal cells (MCs). Following co-culture, cell populations were separated by flow cytometry and ALDH-expressing populations were assessed by the ALDEFLUOR assay. SUM159 cells cultured alone contained approximately 4% ALDEFLUOR-positive cells. Co-culture with MCs increased the proportion of ALDEFLUOR-positive cells over three fold to 14% without affecting the total cell numbers as determined by MTT assay (Figure 1A, right), suggesting that this interaction leads to an increase of cancer stem cell self-renewal (Figure 1A). To determine whether this increase required contact between cancer cells and mesenchymal cells, each of these cell types were cultured in transwells which precluded direct cell/cell contact while allowing for communication via soluble factors. As shown in Figure 1A, transwell culture recapitulated the effects of direct cell/cell contact. Furthermore, expansion of the CSC population was reproduced by addition of conditioned medium obtained from either co-culture or transwell culture of both cell compartments but not by conditioned medium obtained from culture of MCs alone (Figure 1A). Similar results were seen in two other breast cancer cell lines SUM149 and MCF-7 representing basal and luminal subtypes (Figure S2B, 2C). Co-culture of tumor cells with MCs also increased the percentage of tumor cells expressing the breast cancer stem cell (CSC) markers CD24CD44+ (23) (Figure S2). This suggests that the cancer stem cell compartment is regulated by soluble factors which are generated as a result of interaction between mesenchymal cells and cancer cells.
Figure 1
Figure 1
ALDEFLUOR-positive MCs regulate breast CSC populations and are capable of Osteogenic and Adipogenic differentiation
Mesenchymal cells (MCs) are organized in a cellular hierarchy in which ALDEFLUOR-positive MCs (MSCs) regulate CSC expansion
To determine whether mesenchymal cells are organized in a cellular hierarchy, we utilized the ALDEFLUOR assay to isolate ALDH-expressing subpopulations from human bone marrow-derived MCs, As indicated in Figure 1B and Figure S1, MCs contain approximately 5 – 6% ALDEFLUOR-positive cells. In both co-culture and transwell-culture, this ALDEFLUOR-positive subfraction is unaffected by the presence of SUM159 cells (Figure 1B). Defining characteristics of stem cells include their ability to self-renew and to undergo multi-lineage differentiation. When placed in appropriate induction medium, ALDEFLUOR-positive MCs (Mesenchymal Stem Cells or MSCs thereafter) displayed adipogenic or osteogenic differentiation, whereas, ALDEFLUOR-negative MCs did not (Figure 1C). Furthermore, we found that ALDEFLUOR-positive MCs can regenerate both ALDEFLUOR-positive and ALDEFLUOR-negative MCs, but ALDEFLUOR-negative MCs can not regenerate ALDEFLUOR-positive MCs (Figure S3A), a property that also applies to the Aldefluor-positive and negative breast cancer cells (Figure S3B). This indicates that MCs are organized in a hierarchy, in which ALDEFLUOR-positive MCs are able to undergo self-renewal and multilineage differentiation. To determine whether the differentiation of mesenchymal cells affected their ability to regulate breast cancer stem cells, we determined the effect of ALDEFLUOR-positive and ALDEFLUOR-negative mesenchymal cells on SUM159 cells in co-culture. As shown in Figure 1D, the effect of mesenchymal cells on the SUM159 cancer stem cell population was mediated by the ALDEFLUOR-positive MC population, whereas ALDEFLUOR-negative MCs had no effect. These results were confirmed utilizing freshly isolated and well-characterized MCs from Bone Marrow (Figure S4). These experiments demonstrate that interactions between MSC and CSC cell populations regulate the proportion of CSCs.
A cytokine network mediates interaction between breast cancer cells and mesenchymal cells
To determine the molecular mechanisms mediating the interaction between breast cancer cells and mesenchymal cells, we examined the effect of co-culture on the global gene expression profile of each cell population, as assessed by Affymetrix microarray. We compared gene expression patterns of SUM159 cells and MCs cultured alone or in co-culture conditions. Among the gene families induced by co-culture were cytokine genes. As shown in Table 1A, co-culture induced mRNA expression of CXCL5 (ENA78), CXCL6 (GCP2), CXCL1 (Gro-α), as well as IL6 and IL8, in both the SUM159 cells and MCs. These results were confirmed by real time RT-PCR in the SUM159 cell line (Table 1B) as well as in the primary xenograft MC1 (Figure S15). In addition, we detected a six-fold increase in CXCL7 (NAP2) expression only in MCs induced by co-culture with breast cancer cells. Both antibody arrays and the Luminex bead assay were utilized to quantitatively access the effects of co-culture on cytokine protein expression. As shown in Figure 2 and Figure S5A, the levels of CXCL1, CXCL5, CXCL6 and CXCL7 were all significantly increased by co-culture. In addition, co-culture significantly increased IL6 and IL8 production. Similar results were seen in other breast cancer cell lines SUM149 (FigureS6 and S5B) and MCF-7 (Figure S5C). To determine which of the cytokines induced by co-culture was responsible for affecting the CSC population, we assessed the effect of each of these cytokines on the proportion of the cancer stem cells, as assessed by the ALDEFLUOR assay. Dose-response curves were performed for each cytokine (Figure S7). The effect of optimized concentrations of each cytokine on the ALDEFLUOR-positive population of SUM159 cells is shown in Figure 3A. IL6 and IL8 induced significant increases in the ALDEFLUOR-positive population consistent with previous reports (14, 2425). CXCL1 had no significant effect and CXCL5 and CXCL6 generated smaller but statistically significant increases in ALDEFLUOR-positive SUM159 cells. In contrast, the addition of CXCL7 produced almost a three-fold increase in ALDEFLUOR-positive SUM159 cells, a level comparable to that produced by co-culture with MCs. Similar results were seen in other breast cancer cell lines SUM149 (Figure S8A) and MCF-7 (Figure S8C).
Figure 2
Figure 2
Induction of cytokine protein by co-culture of MCs and SUM159 cells as determined by antibody array
Figure 6
Figure 6
MSCs traffic from bone marrow to primary breast tumor sites in xenografts and are detected in primary human breast cancers
Figure 3
Figure 3
CXCL7 and IL6 play key roles in mediating the interaction of MCs and SUM159 cells
To determine the contribution of these cytokines in mediating the interaction of MSCs with CSCs, we utilized cytokine blocking antibodies. Dose-response curves for antibody blocking were performed (Figure S7). The concentration of antibody producing maximal inhibition were utilized and shown in Figure 3B. The interaction of CSCs with MSCs was partially inhibited by anti-IL6 and more completely by anti-CXCL7 suggesting an important role of these two cytokines in mediating MSC-CSC interactions. Similar results were seen in other breast cancer cell lines SUM149 (Figure S7B) and MCF-7 (Figure S8D).
In order to further define the role of CXCL7 and IL6 in mediating the interaction of MSCs with CSCs, we examined the effects of CXCL7 and IL6 on cytokine production by each cellular subcomponent. Addition of CXCL7 to SUM159 cells induced a cytokine expression pattern similar to that observed in co-culture (Figure 3C). Furthermore, the addition of CXCL7 blocking antibody to the co-culture completely blocked expression of induced cytokines CXCL1, CXCL5, CXCL6, IL6 and IL8 (Figure 3C). These experiments suggest that cytokine production by SUM159 cells in co-culture is largely due to CXCL7 produced by MSCs. Similar results were seen in another breast cancer cell line SUM149 (Figure S9).
CXCL7 production by mesenchymal stem cells is regulated by IL6
The previous experiments suggest a critical role for MSCs derived CXCL7 in mediating signaling between the epithelial and mesenchymal components of tumors. To determine how this cytokine was regulated we examined the effect of individual cytokines on CXCL7 production by MSCs. As shown in Figure 4A, addition of IL6 induced greater than a ten-fold increase in CXCL7 production by MCs. The effects of IL6 are mediated by a receptor complex composed of the IL6 receptor (IL6R) and GP130. We assessed expression of these receptors on ALDEFLUOR-positive and ALDEFLUOR-negative MCs. We separated the ALDEFLUOR-positive and ALDEFLUOR-negative MCs by flow cytometry, immunostained the separated populations with IL6R and GP130 antibodies and re-analyzed the cells by flow cytometry. As indicated in Figure 4B, both the IL6R and GP130 were primarily expressed in ALDEFLUOR-positive MCs (MSCs). Furthermore, althogh CXCL7 mRNA level was undetectable in the MC control group, addition of 100 ng/ml IL6 dramatically increases the expression of CXCL7 mRNA in ADLEFLUOR-positive MCs (MSCs) (Figure 4C).
Figure 4
Figure 4
IL6 regulates CXCL7 production by MSCs and mediates MSC Chemotaxis, and interaction between mesenchymal cells and tumor cells is regulated by a positive feedback loop including CXCL7 and IL6
IL6 mediates MSCs chemotaxis
To address the functional significance of increased IL6 receptor expression in MSCs, we determined whether this cytokine mediated chemotaxis of these cells. Recombinant IL6 mediated chemotaxis of Aldefluor-positive but not Aldefluor-negative MCs, an effect inhibited by IL6 blocking antibody (Figure 4D). In addition, Aldefluor-positive but not Aldefluor-negative MCs were chemotactic toward breast tumor cells SUM159, SUM149 and MCF-7, an effect that was significantly inhibited by IL6 blocking antibody (Figure 4D). These results suggest that chemotaxis of MSCs toward breast cancer cells is primarily mediated by IL6.
These experiments suggest the existence of a cytokine network that mediates the interaction between mesenchymal cells and cancer cells in which IL6 produced by cancer cells interacts with IL6R/GP130 expressed on MSCs, which produce CXCL7 in response to this IL6 stimulation. CXCL7, in turn, induces the secretion of a number of cytokines from both SUM159 and MCs including IL6, IL8, CXCL6 and CXCL5. All of these cytokines are capable of expanding the ALDEFLUOR-positive cancer stem cell population. Furthermore, increased IL6 interacts with MSCs forming a positive feedback loop. This model of cytokine networks mediating the interaction between mesenchymal cells, breast cancer cells and breast cancer stem cells is illustrated in Figure 4E.
MSCs stimulate the growth of breast tumor xenografts in NOD/SCID mice by affecting the CSC population
We have previously shown that ALDEFLUOR-positive but not ALDEFLUOR-negative SUM159 cells are tumorigenic in NOD/SCID mice (14). In order to assess the contribution of different subpopulations of mesenchymal cells on tumor growth, ALDEFLUOR-positive, ALDEFLUOR-negative and unsorted MCs mixed in a ratio of 1 to 1 with SUM159 cells were orthotopically implanted in NOD/SCID mice. Unsorted MCs alone were implanted as control. Addition of ALDEFLUOR-positive MCs greatly accelerated tumor growth, whereas ALDEFLUOR-negative MCs had no effect on the growth of SUM159 tumors. Unsorted MCs were intermediate in their ability to stimulate tumor growth (Figure 5A and Figure S11A). MCs alone were not tumorigenic (Figure 5A). The introduction of GFP label into mesenchymal cells and DsRed label into SUM159 cells allowed for separation of these cells from established tumors and the assessment of the effects of added mesenchymal cells on the cancer stem cell population in vivo. Introduction of MSCs increased the proportion of ALDEFLUOR-positive SUM159 cells almost four-fold compared to tumors grown from SUM159 cells alone. This increase in ALDEFLUOR-positive SUM159 cells was seen in tumors grown in the presence of ALDEFLUOR-positive (ALDH-positive) but not ALDEFLUOR-negative (ALDH-negative) MCs (Figure 5B and Figure S11B). To provide functional data confirming the Aldefluor results, we determined the ability of serial dilutions of cells obtained from primary tumors to form secondary tumors in NOD/SCID mice. As shown in Figure 5D, cells obtained from primary tumors grown in the presence of MCs had significantly greater tumor generating capacity in secondary mice compared to cells obtained from primary tumors grown in the absence of MCs. These results confirm the Aldefluor data suggesting that MSCs have the capacity to increase the breast CSC population. Furthermore, the percentage of CSCs assessed by the Aldefluor assay or by expression of CD24CD44+ in the secondary tumors was similar in secondary tumors generated from MC supplemented or control primary tumors. This suggests that accelerated growth of primary tumors by MC results from an increase in CSCs rather than from an alteration of biological properties of these cells (Figure S11C). Similar in vivo results were in other breast cancer cell lines SUM149 (Figure S12) and MCF-7 (Figure S13). In order to demonstrate that the effect of MSCs on breast CSCs was not limited to established cell line generated xenografts, we utilized MC1, a breast xenograft established directly from a human breast tumor tissue. We have previously demonstrated that ALDEFLUOR-positive cells display CSC properties (16). As was the case with established cell lines, addition of MSCs accelerated tumor growth by increasing proportion of ALDEFLUOR-positive tumor cells in this model (Figure S14).
Figure 5
Figure 5
ALDEFLUOR-positive MCs induce cancer stem cells stimulating breast tumor growth in NOD/SCID mice
To document physical interactions between MSCs and CSCs in growing tumors, we used four-color fluorescence in which SUM159 cells were identified by DsRed expression (green), MCs by GFP expression (purple), and stem cells (MSCs and CSCs) by expression of the stem cell marker by ALDH1 expression (red). Nuclei were identified by DAPI staining (blue). We have previously reported that the ALDEFLUOR-positive SUM159 cells can be identified in situ utilizing ALDH1 monoclonal antibody (14). Four-color fluorescence revealed close apposition between ALDH1-positive MCs (MSCs) and ALDH1-positive SUM159 cells (CSCs) (Figure 5C).
MSCs traffic from bone marrow to primary breast tumor sites in xenografts and are detected in primary human breast cancers
To determine whether bone marrow mesenchymal stem cells are capable of trafficking to primary breast tumor sites, we labeled MCs with luciferase and DsRed. The growth of implanted MCs at the tibial site was demonstrated by bioluminescence (Figure 6A Left). One week after MC tibial implantation, we implanted SUM159 cells in the mammary fat pads. Tumor size was monitored weekly. As shown in Figure 6A (Right), breast tumor growth was significantly accelerated by human MCs introduced into the mouse tibia. Five weeks after tumor implantation, animals were sacrificed and the presence of MSCs and CSCs were assessed by immunochemistry and immunofluorescence. As shown in Figure 6B, DsRed immunochemistry revealed the presence of mesenchymal cells in tumors grown in animals with tibial MC inoculation but not in control tumors grown in the absence of MC introduction. To localize MSCs and cancer stem cells in situ, we utilized immunofluorescence to identify MCs (green) and the stem cells marker ALDH1 (red) to identify both MSCs and CSCs. Merged images show adjacent ALDH1-positive MCs (yellow) and ALDH1-positive SUM159 cells (red) suggesting the existence of a cancer stem cell “niche” characterized by ALDH1-positive CSCs and ALDH1-positive MCs.
To determine whether similar MSC-CSC niches are found in primary human breast cancers, we utilized immunochemistry to identify ALDH1-positive MCs and ALDH1-positive cancer cells in frozen sections of three independent ALDH1-positive primary breast cancers. We stained serial sections for CD105 and CD31 to identify MCs and distinguish them from endothelial cells and identified the stem cells in each population based on ALDH1 expression. Pan-cytokeratin (Pan-CK) was used as an epithelial cell marker. As shown in Figure 6C (Shown is one representative sample from six independent samples), ALDH1-positive mesenchymal cells (ALDH1+CD105+CD31Pan-CK) are found in apposition to ALDH1-positive cancer cells (ALDH1+CD105CD31Pan-CK+). This histology closely resembles that seen in the mouse xenografts.
Stem cells are regulated by interplay between extrinsic factors and cell intrinsic regulatory pathways. During normal development and tissue homeostasis, these extrinsic factors are provided by cellular and extracellular elements which define the stem cell “niche”. There is increasing evidence that many tumors including breast cancer may be driven by a cellular subcomponent that displays stem cell properties. Although it is clear that the tumor microenvironment influences tumor growth and metastasis (28), it is unclear whether these effects are mediated by CSCs. In this study we utilized in vitro systems and mouse models to demonstrate an important role for bone marrow-derived MSCs in regulating breast CSCs. The use of mesenchymal cells and cancer cells both of human origin facilitated the study of cytokine interaction obviating known species differences in these factors. In fact, the significant facilitation of breast tumor growth by human MCs introduced into the mouse tibia may reflect these species differences. We demonstrate that the interaction between MSCs and CSCs is mediated by a positive feedback cytokine loop in which IL6 and CXCL7 play pivotal roles. This loop requires the simultaneous presence of both cell types but does not require cell-cell contact as demonstrated by transwell and conditioned medium experiments. Furthermore, we demonstrate that MCs, like cancer cells, are organized in a hierarchy in which primitive ALDH-expressing mesenchymal cells capable of self-renewal and multi-lineage differentiation interact with cancer cells to regulate CSC self-renewal. IL6 produced by cancer cells interacts with IL6 receptor and GP130 on ALDEFLUOR-positive MCs. IL6 mediated chemotaxis may facilitate the homing of MSCs to the sites of primary tumor growth, as well as inducing CXCL7 production by these cells. MSC-derived CXCL7 in turn, interacts with cancer cells through the CXCR2 receptor (29), where it induces the synthesis of a number of cytokines including IL6 and IL8. These pleiotropic effects of CXCL7 are consistent with previous reports (29). The expression of CXCL7 and its receptor CXCR2 have been shown to be increased in breast carcinomas (30). Furthermore, CXCL7 transfection increased the invasive capacity of breast cancer cells (30) consisted with the previously demonstrated increased invasive and metastatic properties of cancer stem cells (31). We have previously shown that IL8 interacts with the CXCR1 receptor on cancer stem cells triggering their self-renewal and invasive properties (14, 32). IL6 also has been reported to be capable of regulating breast stem cells (24) and colon cancer stem cells (25). In addition to regulating CSCs, IL6 produced by cancer cells interacts with MSCs, further increasing their CXCL7 production generating a positive feedback loop (Figure 5A). We confirmed the functional importance of these interactions by demonstrating that MSCs accelerate breast tumor growth in NOD/SCID mice. Furthermore as was the case in vitro, in this mouse model, this effect was mediated by ALDEFLUOR-positive MCs which were capable of increasing the CSC population in vivo. The close apposition of ALDH1+ tumor cells and MSCs was also demonstrated in frozen sections of primary human breast cancers.
It is clear that the tumor microenvironment plays an important role in tumor growth and metastasis (8). Previous elegant studies have suggested a role for mesenchymal stem cells in tumor metastasis, which is mediated by CCL5 (8). In addition, researchers reported that IL1a produced by MSC mediates similar effects (33) and that MSCs promote LTC-IC expansion (34). Our studies extend these previous findings by demonstrating that MSCs regulate cancer cell behavior through their effect on cancer stem cells. On the other hand, MSCs have been shown to actually inhibit tumor growth in some models (35). This suggests that the effects of MSCs on tumor growth are complex and may be context dependent.
The homing of bone marrow-derived mesenchymal stem cells to sites of tumor growth may rely on similar mechanisms to the homing of these cells to sites of tissue injury. Developing tumors may recruit MSCs from the bone marrow where they interact with and regenerate CSCs. If this is the case, then the development of strategies aimed at interfering with these pathways may provide a means of targeting cancer stem cells. Since these cells may mediate tumor growth and metastasis as well as contribute to treatment resistance, these strategies may lead to improved clinical outcomes for patients with advanced breast cancers.
Supplementary Material
Supllemental Datales
Acknowledgments
Thanks to Dr. Stephen Ethier for generously providing the breast cancer cell lines SUM159 and SUM149, and to Chris Neeley for technical support. This work was supported by NIH grants CA129765, CA101860 (M.W.), and 5 P 30 CA46592 (M.W.), R01CA125577 and R01 CA107469 (C.G.K). The Taubman Research Institute (M.W.). We thank the University of Michigan core facilities. MW has financial holdings in OncoMed Pharmaceuticals, which has applied for a patent on cancer stem cell technologies.
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