Cell Transplantation, Vol. 24, pp. 631–644, 2015 Printed in the USA. All rights reserved. Copyright Ó 2015 Cognizant Comm. Corp.
0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368915X687787 E-ISSN 1555-3892 www.cognizantcommunication.com
Paracrine Effects of Mesenchymal Stem Cells Induce Senescence and Differentiation of Glioblastoma Stem-Like Cells Katja Kološa,* Helena Motaln,* Christel Herold-Mende,† Marjan Koršicˇ,‡ and Tamara T. Lah*§ *Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia †Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany ‡Department of Neurosurgery, University Medical Centre of Ljubljana, Ljubljana, Slovenia §Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Glioblastoma multiforme (GBM) displays high resistance to radiation and chemotherapy, due to the presence of a fraction of GBM stem-like cells (GSLCs), which are thus representing the target for GBM elimination. Since mesenchymal stem cells (MSCs) display high tumor tropism, we examined possible antitumor effects of the secreted factors from human MSCs on four GSLC lines (NCH421k, NCH644, NIB26, and NIB50). We found that conditioned media from bone marrow and umbilical cord-derived MSCs (MSC-CM) mediated cell cycle arrest of GSLCs by downregulating cyclin D1. PCR arrays revealed significantly deregulated expression of 13 genes associated with senescence in NCH421k cells exposed to MSC-CM. Among these, ATM, CD44, COL1A1, MORC3, NOX4, CDKN1A, IGFBP5, and SERPINE1 genes were upregulated, whereas IGFBP3, CDKN2A, CITED2, FN1, and PRKCD genes were found to be downregulated. Pathway analyses in GO and KEGG revealed their association with p53 signaling, which can trigger senescence via cell cycle inhibitors p21 or p16. For both, upregulated expression was proven in all four GSLC lines exhibiting senescence after MSC-CM exposure. Moreover, MSC paracrine signals were shown to increase the sensitivity of NCH421k and NCH644 cells toward temozolomide, possibly by altering them toward more differentiated cell types, as evidenced by vimentin and GFAP upregulation, and Sox-2 and Notch-1 downregulation. Our findings support the notion that MSCs posses an intrinsic ability to inhibit cell cycle and induce senescence and differentiation of GSLCs. Key words: Cell differentiation; Glioblastoma multiforme (GBM); Stem-like cancer cells; Mesenchymal stem cells (MSCs); Senescence; Therapy response
INTRODUCTION Glioblastoma multiforme (GBM) is the most malignant type of brain tumor that is still incurable, with the overall survival of GBM patients being less than 15 months (35). This is due to highly infiltrative growth of GBM and its resistance to chemo/radiotherapies, preventing complete elimination of tumor cells, despite improvements made in surgical techniques and therapeutic protocols (7,45). The highly tumorigenic subpopulation of glioblastoma stemlike cells (GSLCs) present in the tumor and defined as a subpopulation of cells with stem-like properties, such as self-renewal, asymmetric division, and differentiation potential, essential for tumor development and propagation in vivo (8,45) are presumably causative for GBM recurrence. Their variable abundance within the tumor mass has been confirmed in several studies (6,42,52). Their stemness and increased resistance to radiation and
chemotherapy (4,16,28) are suggesting new therapeutic approaches focusing on GSLC targeting to improve the survival of GBM patients (35). Cell therapy is being revisited in view of using normal tissue stem cells, that is, human mesenchymal stem cells (MSCs) for cancer treatment. MSCs are multipotent stem cells most often isolated from birth-associated [i.e., umbilical cord (UC-MSC), cord blood (UCB-MSC)] and adult tissues, such as bone marrow (BM-MSC) and adipose tissue (AT-MSC) (31). They are supposed to have a wide therapeutic potential because of their immunomodulatory ability, wound- and neoplasma-directed homing, and tissue repair ability, relying on their differentiation potential (21,24). Already naive UCB-MSCs were shown to suppress growth of various cancers, including GBM, as a 36% size reduction of C6 xenografts in mice was observed after UCB-MSC intratumoral injection (19). Likewise, a
Received January 27, 2015; final acceptance March 9, 2015. Online prepub date: March 24, 2015. Address correspondence to Dr. Helena Motaln, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ve na pot 111, SI-1000 Ljubljana, Slovenia. Tel: +386 5923 2870; E-mail: [email protected]
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systemic AT-MSC coadministration with 8MGA glioma cells was shown to decrease de novo tumor development in mice by 63%, compared to control mice injected with 8MGA cells only (27). Injected AT-MSCs were shown to colocalize with CD133+ cells in GBM xenografts (2). Also in rat glioma models, the injection of UCB-MSCs and BM-MSCs resulted in tumor volume reduction (23), tumor growth inhibition (13), and increased survival of animals (33). Conditioned media (CM) derived from UC-MSCs and AT-MSCs were shown to inhibit the growth of U251 glioma cells (50), whereas BM-MSCs and their CM were shown to inhibit proliferation of U87MG, U251, and U373 glioma cells (31) and induce U87 cell senescence (32). In contrast, the UCB-MSCs, UC-MSCs, and AT-MSCs were shown to induce apoptosis in glioma cells (13,20,50). All of the above is implying the potential of MSCs for the development of novel anticancer cell-based therapies. As the intrinsic resistance of GSLCs to therapy presents the main obstacle in GBM treatment, the repressive potential of MSCs on GSLCs (i.e., on their stemness potential and viability) should be defined. To clarify the MSC effect on GSLCs, we utilized the established GSLC lines and primary culture of GBM cells enriched for GSLCs and exposed them to BM-MSC- and UC-MSC-derived CM. We speculated that the paracrine activity of the MSCs, namely their secreted factors, would cause the alteration of the GSLC behavior. Indeed, we observed both MSC-CMs to cause cell cycle arrest of GSLCs in the G0/G1 phase and to induce their senescence. To our knowledge, we are also first to demonstrate that the exposure of GSLCs to MSC paracrine signals changes the gene expression of GSLCs, which is driving them toward a more differentiated phenotype. The MSC-CM also increased the sensitivity of GSLCs to chemotherapeutic temozolomide (TMZ), commonly used in GBM treatment. MATERIALS AND METHODS Cell Cultures GSLC lines NCH421k (male, age 66) and NCH644 (male, age 66) were isolated from GBM resection and checked for stemness potential, as described previously (8,10). NIB26 (female, age 75) and NIB50 (male, age 81) cells were isolated from fresh GBM samples obtained from the University Medical Centre of Ljubljana (study approved by the National Ethics Committee). Upon mechanical and enzymatic dissociation, the isolated cells were grown as spheroids (with a presumed enriched fraction of GSLCs) under the same conditions as NCH cells in neurobasal (NBE) medium (Gibco, Life Tech. Corp., Paisley, UK) supplemented with 2 mM l-glutamine (PAA Lab, Pasching, Austria), 100 U penicillin, 1,000 U streptomycin (PAA Lab), 1× B-27 (Gibco), 1 U/ml heparin (Sigma-Aldrich, Steinheim, Germany), 20 ng/ml EGF in 20 ng/ml bFGF
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(Gibco). Both derived cell lines were tested for the CD133 expression by flow cytometry. The U87 cell line was grown as described previously (31). Human MSCs Human BM-MSCs were purchased from Lonza Bioscience (Walkersville, MD, USA) (BM-MSC2: male, age 19, 6F4393; BM-MSC3: female, 22, 7F3677) and cultured according to the manufacturer’s recommendations. Briefly, MSCs were grown in Dulbecco’s medium (DMEM; 5921; Sigma-Aldrich) with 10% fetal bovine serum (FBS; PAA Lab), supplemented with 100 U penicillin (PAA Lab), 1,000 U streptomycin (PAA Lab), 2 mM l-glutamine (PAA Lab), Na-pyruvate (Gibco), and nonessential amino acids (Sigma-Aldrich). UC-MSCs were isolated from Wharton’s jelly according to standard protocol (47) in the study approved by the National Ethics Committee, Doc. No. 134/01/11. Umbilical cords were collected at cesarean section (37–41 weeks) upon obtained informed consent, and isolated UC-MSCs were cultured the same as BM-MSCs. UC-MSCs were characterized for CD13+, CD29+, CD44+, CD73+, CD90+, CD105+, CD14−, CD34−, CD45−, HLA-DR− surface marker expression, and osteogenic, chondrogenic, and adipogenic differentiation as recommended (15). Two UC-MSC clones (UC-MSC-10, female and UC-MSC-13, male) with the highest proliferation potential and homogenous spindlelike morphology were used in further experiments. Conditioned Media (CM) Collection Two different clones of BM-MSCs and the two clones of UC-MSCs were plated into T75 flasks (Corning, Cambridge, MA, USA) at a density of 6,000 cells/cm2, and a day before the cells would have reached 90% confluence, the standard MSC cultivation medium, described above, was changed to 12.5 ml NBE medium (used in GSLC culture). After 24 h, this CM was collected and pooled from both BM-MSC lines or from both UC-MSC cell lines, centrifuged at 300 × g for 10 min at 4°C, and stored at −80°C. Prior to the experiment, the CM were mixed in a 1:1 ratio with fresh NBE medium and designated as BM-CM and UC-CM, respectively. Similarly, the NBE CM were collected after 24 h from cultured NCH421k and NCH644 spheroids and diluted at a 1:1 ratio with fresh NBE medium and when used designated as NBE-CM. Surface Marker Detection by Flow Cytometry UC-MSCs (106) were harvested, washed with 1× phosphate-buffered saline (PBS; Gibco), and incubated with antibodies against CD13 (BD#557454), CD29 (BD# 559883), CD44 (BD#555479), CD73 (BD#550257), CD90 (BD#555596), CD14 (BD#555397), CD34 (BD#555821), CD45 (BD#555482), HLA-DR (BD#340688) (all from
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BD Biosciences, San Jose, CA, USA), CD105 (MHCD1 0505; Molecular Probes, Eugene, OR, USA), and isotype controls [FITC-IgG1 (BD#555748), FITC-IgG2a (BD#55 5573), PE-IgG1 (BD#555749), PE-IgG2b (BD#555743), APC-IgG1 (BD#555751); all from BD Biosciences] as instructed by the manufacturer. All cells were additionally stained with propidium iodide (PI; BD Biosciences) to exclude dead cells, washed, and analyzed by flow cytometry using a BD FACSCaliburTM and the CellQuest Software (both BD Biosciences). NIB26 and NIB50 cells were tested for the expression of CD133 protein using the antibodies recognizing AC133 or CD133/2 (AC141) epitope by the protocol described below. The staining results proved to overlap properly (8). NCH421k and NCH644 (106) cells were harvested upon 72-h exposure to NBE, NBE-CM, UC-CM, and BM-CM, washed with 1× PBS, and incubated with 10 µl CD133/2-PE antibody (130-090-853, Miltenyi Biotec, San Diego, CA, USA) or 20 µl isotype control antibody (PE-IgG2b, BD Biosciences) in equal concentrations. All cells were PI (BD Biosciences) stained for live cell analysis (20 min at 4°C), washed, and analyzed by flow cytometry using a BD FACSCaliburTM and the CellQuest Software (both BD Biosciences). Differentiation of UC-MSCs Adipogenic Differentiation. The adipogenic differentiation medium was composed of DMEM supplemented with 10% FBS, 1 µM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX; SigmaAldrich), 10 µg/ml insulin (Sigma-Aldrich), 100 µM indomethacin (Sigma-Aldrich). After 72 h, cells were medium changed to adipogenic maintenance medium: DMEM supplemented with 10% FBS and 10 µg/ml insulin for 24 h, and this sequence was repeated three times, followed by cultivation for 1 week in maintenance medium. After 21 days, cells were fixed in 4% paraformaldehyde for 10 min, and accumulated lipid-rich vacuoles were stained with 0.3% Oil red O (Sigma-Aldrich) for 30 min. Osteogenic Differentiation. The osteogenic differentiation medium was prepared with DMEM containing 10% FBS, 1 µM dexamethasone (Sigma-Aldrich), 50 µg/ ml l-ascorbic acid (Sigma-Aldrich), and 10 mM glycerophosphate (Sigma-Aldrich). After 21 days, cells were fixed in 4% paraformaldehyde for 10 min and stained with 1% Alizarin red S (Sigma-Aldrich) for 30 min to detect calcified extracellular matrix. Chondrogenic Differentiation. Cells were exposed to chondrogenic differentiation medium composed of DMEM, 10% FBS, 10 ng/ml TGF-b3 (Sigma-Aldrich), and 50 µg/ml l-ascorbic acid for 21 days. To detect matrix deposition of sulfated glycosaminogycans (GAGs), cells
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were fixed in 4% paraformaldehyde for 10 min and stained with 1% Alcian blue 8-GX (Sigma-Aldrich) in 3% acetic acid (Sigma-Aldrich). Cell Cycle Analysis NCH421k cells were exposed to NBE, UC-CM, and BM-CM for 72 h, trypsinized [0.25% Trypsin-EDTA (Gibco)], washed with 1× PBS, and left in 75% ethanol overnight at 4°C. Upon washing with 1× PBS, cells were incubated in 0.5 ml PI/RNase staining buffer (BD Pharmingen, San Diego, CA, USA) for 15 min at RT. Cell cycle analysis was performed using the WinMDI and Cyclored on the BD FACSCaliburTM with the CellQuest Software. Apoptosis Assay NCH421k cells were exposed to NBE, UC-CM, and BM-CM for 72 h, harvested, and early/late apoptotic cells detected by staining with Annexin-V-FITC and PI (BD Pharmingen) according to the manufacturer’s protocol. Stained cells were analyzed using BD FACSCaliburTM and the CellQuest Software. Staurosporine (STS; SigmaAldrich) treatment (2 µM, 6 h) served as a positive control for apoptosis. Mitochondrial Membrane Potential (MMP) Detection MMP change in NCH421k cells grown in NBE, UC-CM, and BM-CM for 72 h was measured with the Mitochondrial Permeability Transition Detection Kit JC-1 (ImmunoChemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. STS (2 µM, 6 h) and carbonylcyanide-chlorophenyl-hydrazone (CCCP; 25 µM, 30 min; ImmunoChemistry Technologies)treated NCH421k cells were used as a positive control. JC-1 staining was analyzed using a Synergy™ MX Microplate Reader (Bio-Tec Instruments, Winooski, VT, USA). The change of MMP was assessed by the ratio of red versus green fluorescence readings. Cell Senescence NCH421k, NCH644, NIB26, and NIB50 spheroids were grown in NBE, UC-CM, and BM-CM for 72 h; GSLC spheroids were dissociated to single cells and fixed in 0.5% glutaraldehyde solution (Sigma-Aldrich) for 20 min at RT. The proportion of cells positive for senescence indicative of b-galactosidase (SA-b-Gal) activity was evaluated as described previously (14). Quantitative Real-Time PCR (qRT-PCR) and PCR Arrays Total RNA was isolated from three biological replicates of the U87 cell line and NCH421k, NCH644, NIB26, and NIB50 cells grown in NBE, UC-CM, and BM-CM for 72 h using TrizolTM reagent (Invitrogen Limited,
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Paisley, UK) following the manufacturer’s instructions. Total RNA (1 µg) was used for cDNA generation using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Gene expression of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16), cyclin-dependent kinase inhibitor 1A (CDKN1A; p21), cyclin D1 (CCND1), BCL-2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL-2), prominin 1 (PROM1; CD133), SRY (sex-determining region Y)-box 2 (Sox-2), nestin (NES), Notch-1, tubulin b 3 class III (TUBB3), vimentin (VIM), and glial fibrillary acidic protein (GFAP) was quantified by real-time quantitative PCR on ABI 7900 HT Sequence Detection System (Applied Biosystems). Real-time PCR reactions comprised of cDNA added to TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (all Applied Biosystems): CDKN2A, Hs00923894_m1; CDKN1A, Hs 00355782_m1; CCND1, Hs00765553_m1; BAX, Hs999 99001_m1; BCL-2, Hs0060823_m1; PROM1, Hs010092 50; Sox-2, Hs01053049_s1; NES, Hs00707120_s1; Notch-1, Hs01062014_m1; TUBB3, Hs00801390_s1; VIM, Hs001 85584_m1; GFAP, Hs00157674_m1; p53, Hs01034249; and glyceraldehyde 3-phosphate dehydrogenase (GADPH) TaqMan probe (Assay No. 4310884E) as an internal control. The analyses were performed with SDS v2.2 software (Applied Biosystems) and comparative Ct method (DDCt algorithm). By using generated cDNA from NCH421k exposed to control and both types of MSC-CM, the expression of 84 senescence-related genes was determined also following the Cellular Senescence RT-Prolifer PCR Array (Qiagen-SABiosciences, Hilden, Germany) protocol. On the Cellular Senescence RT-Prolifer PCR Array, the average of three different housekeeping genes, ACTB (actin b), GAPDH, and ribosomal protein L13a (RLP13A), was used in calculations as an internal control. Fold increase in mRNA levels was calculated with the DDCt method by the provided online program available at http://pcr dataanalysis.sabiosciences.com/pcr/arrayanalysis.php. Results are expressed as means of three independent experiments done in duplicate. Cytotoxicity Testing After Exposure to Temozolomide and 5-Fluoro-Uracil by MTS Cell Viability Assay NCH421k and NCH644 cells were plated into 96-well plates (Corning) at a density of 104 cells/well and left to grow for 24 h before adding NBE, UC-CM, BM-CM, and TMZ (2.5–1000 µM) or 5-fluoro-uracil (5-FU) (0.01–150 µM) (both Sigma-Aldrich). After 48 h and 72 h of TMZ and 5-FU treatment, the CellTiter 96® AQueous MTS reagent [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium; Promega Corporation, Madison, WI, USA; Sigma-Aldrich], was added to each
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well to make 333 mg/ml final concentration. Three hours later, the absorbance at 490 nm (reference 690 nm) was measured with the Synergy™ MX Microplate Reader (Bio-Tec Instruments). Statistical Analyses All the above experiments were performed in duplicate and independently repeated three times. To test the effect of MSC-CM on GSLCs compared to control medium, the ANOVA test with post hoc Dunnett’s test was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, La Jolla, CA, USA). A value of p < 0.05 was considered significant. Data are expressed as the mean ± standard deviation (SD). RESULTS Umbilical Cord-Derived MSC Lines (UC-MSCs) and Primary GSLCs Were Successfully Generated We established six MSC lines from umbilical cords (UC-MSCs) obtained from healthy donors. These UCMSCs exhibited plastic adherence in culture and typical spindle-shaped morphology. We confirmed the presence of CD13, CD29, CD44, CD73, CD90, and CD105 and the absence of CD14, CD34, CD45, and HLA-DR surface antigens in all UC-MSC clones by flow cytometry and verified their osteo-, chondro-, and adipogenic differentiation potential (data available, not shown). This characterization is in accordance with suggested criteria for MSC phenotype confirmation (15); thus, we further used two UC-MSC clones together with two BM-MSC lines for UC-CM and BM-CM production. Purified NCH421k and NCH644 GSLC lines were established previously by Campos et al. (10) and confirmed by DNA copy-number profiling and CGH array to show typical GBM chromosome aberrations (8,46) and to highly express CD133 epitope 1 antigen detected by AC133 antibody (9). Flow cytometric analysis also confirmed expression of CD133 in NIB26 (79.9% CD133+ cells) and NIB50 (71.4% CD133+ cells) that were isolated from primary GBM tissue and grown in spheroids to enrich for their GSLCs fraction (Fig. 1A). Analysis of p53 status in NCH421k, NCH644, NIB26, and NIB50 cells was determined by qRT-PCR with U87 cell line (wild-type p53) serving as reference (54). MSC-CM Caused G0 /G1 Phase Cell Cycle Arrest via Cyclin D1 Downregulation Cell cycle analysis of NCH421k cells grown with NBE or either type of CM for 72 h was performed. It revealed an increased number of NCH421k cells halted in G0/G1 phase in the presence of UC-CM, compared to NBE (Fig. 2A). Additionally, the G0/G1 cell cycle arrest of NCH421k cells, grown in UC-CM and BM-CM, was confirmed by
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Figure 1. GSLCs isolated from primary GBM tissue express high levels of CD133. NIB26 and NIB50 cells were isolated from primary GBM tissue and grown in spheroids to enrich the GSLC fraction. Flow cytometry analysis of CD133 expression showed (A) 79.9% CD133+ cells in NIB26 and (B) 71.4% CD133+ cells in NIB50 cell culture. (C) Analysis of p53 gene expression in U87, NCH421k, NCH644, NIB26, and NIB50 cells by qRT-PCR.
Figure 2. MSC-CM interferes with the cell cycle of GSLCs. (A) Cell cycle analysis of NCH421k cells performed by flow cytometry after 72-h treatment with both types of MSC-CM. (B) Cyclin D1 (CCND1) expression (qRT-PCR) was decreased in all GSLC lines after 72-h treatment with UC-CM and BM-CM. The mean ± SD of three independent experiments are provided. *p < 0.05, **p < 0.01, ***p < 0.001. Morphology of native cells: (C) Control NCH421k cells with compact spheroid morphology; (D) NCH421k spheroid morphology changed upon UC-CM treatment for 72 h; and (E) NCH421k spheroid morphology changed in the presence of BM-CM after 72 h. (F) Control NCH644 cells with compact spheroid morphology; (G) UC-CM treatment for 72 h changed NCH644 spheroid morphology to adherent type of growth; and (H) BM-CM presence after 72 h changed NCH644 spheroid morphology to adherent type of growth. Scale bar: 100 µm.
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CCND1 gene expression analysis. This showed CCND1 gene expression to decrease 4.2-fold in UC-CM and 2.9fold in BM-CM (Fig. 2B). Similarly, the CCND1 gene expression was found downregulated in NCH644, NIB26, and NIB50 cells exposed to CM. The above was associated with the indicative size reduction and loosened structure of NCH421k and NCH644 spheroids grown in both types of MSC-CM (Fig. 2C–E). The BM-CM was even shown to induce the adherent growth of NCH644 cells (Fig. 2F–H). Altogether, our data imply the capacity of MCS paracrine factors to cause the cell cycle arrest of GSLCs through decrease in the cyclin D1 expression level. MSC-CM Affected Mitochondrial Membrane Potential but Did Not Induce Apoptosis in GSLCs The induction of apoptosis in NCH421k cells grown in MSC-CM of both origins for 72 h was evaluated by MMP assay. A significant decrease in MMP was detected in NCH421k cells when exposed to UC-CM (23%) and BM-CM (31%) (Fig. 3A). Since MMP change is not solely indicative of apoptosis, BAX and BCL-2 gene expression was analyzed in the NCH421k, NCH644, NIB26, and NIB50 cells cultured in MSC-CM. The analysis of the BAX/BCL-2 gene ratio failed to demonstrate any significant change to occur in the CM-cultured GSLCs, compared to control (NBE) ones (Fig. 3B). To further exclude the onset of apoptosis, the annexin V staining was performed. This also failed to detect increased apoptosis in NCH421k cells cultured in MSC-CM versus control cells with staurosporin treatment used as positive control (Table 1). Altogether our results showed that by addition of MSC-CM containing MSC paracrine factors, the apoptosis in GSLCs cannot be induced.
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MSC-CM-Activated Senescence in GSLCs As MSC-CM influenced the cell cycle of GSLCs, but did not cause apoptosis, the senescence in exposed GSLCs was investigated by using the indicative b-galactosidase staining. A significantly increased number of senescent cells was observed in NCH421k (17% and 33%), NCH644 (33% and 39%), NIB26 (20% and 25%), and NIB50 (30% and 31%) cells exposed to UC-CM and BM-CM, respectively, for 72 h, when compared to control NCH421k (Fig. 4A). In GSLCs exposed only to NBE, the percentage of senescent cells was 10.7% in NCH421k, 6.6% in NCH644, 3.3% in NIB26, and 8.2% in NIB50 cells. To get insight into the senescence signaling, NCH421k cells were subjected to both types of MSC-CM, and qRTPCR analysis of 84 genes associated with senescence was conducted by the human Senescence RT2 Profiler PCR Array (Table 2). The PCR arrays revealed changed expression of 13 genes involved in senescence (ATM, CDKN1A, CDKN2A), cell adhesion (CD44, COL1A1), oxidative stress (PRKCD, NOX4), IGF-related pathway (IGFBP3, IGFBP5), p53/pRb signaling (MORC3, CITED2), and cytoskeleton formation (FN1, SERPINE1) (Table 2). Those genes were consistently up- and downregulated in exposed NCH421k cells at least 2.1-fold. In addition, KEGG pathway analysis identified the involvement of five genes, SERPINE1, CDKN1A, IGFBP3, CDKN2A and ATM, in the p53 pathway (Table 3) and induced in senescence. The PCR Array results were validated with qRT-PCR on independent mRNA samples of GSLCs, which confirmed increased expression of the CDKN1A gene in NCH421k cells grown for 72 h in UC-CM (1.4-fold) and BM-CM (6.7-fold). Likewise, the CDKN1A expression increased
Figure 3. MSC-CM does not induce apoptosis in GSLC lines. (A) Mitochondrial membrane potential (MMP) loss was ascertained by JC-1 staining after exposure of NCH421k cells to UC-CM and BM-CM for 72 h. (B) QRT-PCR analysis of Bax and Bcl-2 gene expression in NCH421k, NCH644, NIB26, and NIB50 cells after exposure to both types of MSC-CM for 72 h. The ratio between the Bax and Bcl-2 was unaffected. Data shown are mean ± SD of three independent experiments. ***p < 0.001. CCCP, carbonylcyanidechlorophenyl-hydrazone; STS, staurosporin.
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Table 1. Annexin V/PI Staining of NCH421k Cells Exposed to Staurosporin (STS) and MSC-CM NCH421k NCH421k + STS NCH421k + UC-CM NCH421k + BM-CM
Live
Early Apoptosis
Late Apoptosis
Dead
93.34 ± 4.20 42.98 ± 12.73* 91.06 ± 2.54 88.82 ± 6.51
4.83 ± 2.63 45.26 ± 13.87* 7.01 ± 2.33 7.69 ± 2.45
1.06 ± 1.70 5.66 ± 0.56 1.24 ± 1.41 2.82 ± 4.32
0.76 ± 0.69 0.80 ± 1.02 0.56 ± 0.55 0.67 ± 0.68
The NCH421k cells were exposed to UC-CM and BM-CM, and the apoptotic cells were determined by flow cytometry as described in Materials and Methods. The exposure to STS, inducing intrinsic apoptotic pathway, was used as positive control. The data of three independent experiments are expressed as the mean ± standard deviation (SD) and *p < 0.05 was considered significant.
Figure 4. MSC-CM activates senescence in all GSLC lines. (A) Senescence-associated b-galactosidase (SA-b-Gal) staining revealed activation of senescence in NCH421k, NCH644, NIB26, and NIB50 cells after exposure to UC-CM and BM-CM upon 72-h incubation. qRT-PCR analysis of (B) p21 expression and (C) p16 expression after 72-h treatment with both types of MSC-CM in all GSLCs. Data shown are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (D) SA-b-Gal staining of dissociated and fixed NCH421k, NCH644, NIB26, and NIB50 cells after UC-CM and BM-CM presence for 72 h. Scale bar: 20 µm.
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Table 2. Differentially Expressed Genes in MSC-CM-Cultured NCH421k Cells Gene symbol ATM CD44 COL1A1 MORC3 NOX4 CDKN1A IGFBP5 SERPINE1 IGFBP3 CDKN2A CITED2 FN1 PRKCD
NCH421k + UC-CM
NCH421k + BM-CM
Fc
p Value
Fc
p Value
Pathway/Process
2.6 2.7 7.5 2.1 2.1 3.0 1.5 4.1 −2.1 1.3 −1.4 2.1 −1.4
0.032* 0.022* 0.043* 0.046* 0.045* 0.054* 0.343 0.172 0.009* 0.311 0.101 0.082 0.294
−1.2 1.4 1.5 −1.1 1.8 6.9 30.1 3.8 2.0 −2.1 −2.8 −2.4 –2.2
0.406 0.241 0.210 0.606 0.163 0.001* 0.017* 0.010* 0.048* 0.022* 0.034* 0.012* 0.016*
Senescence Cell adhesion Cell adhesion p53/pRb signaling Oxidative stress Senescence IGF related Cytoskeleton IGF related Senescence p53/pRb signaling Cytoskeleton Oxidative stress
Listed are deregulated genes in NCH421k cells exposed to either UC-CM or BM-CM relative to NBE exposed NCH421k cells. Fold increase in mRNA levels are calculated as described in Materials and Methods. The mean ± SD of three independent experiments are provided. *p < 0.05. Fc, fold change.
in NCH644 (3.2-fold) and NIB50 cells (5.0-fold) exposed to BM-CM (Fig. 4B). The expression of the CDKN2A gene also increased in NCH644 (3.4-fold), NIB26 (7.7fold), and NIB50 (1.9-fold) exposed to BM-CM (Fig. 4C), whereas it decreased 1.7-fold in NCH421k cells exposed to BM-CM (Fig. 4C). The above was consistent with the observed b-galactosidase staining (Fig. 4D), altogether implying the ability of the MSC paracrine factors to induce GSLC growth arrest and senescence. MSC-CM Affected the Expression of GSLC Markers To evaluate the impact of MSC-CM on the stemness of GSLCs, the expression of indicative GSLC marker CD133/prominin was examined by qRT-PCR (using gene expression assay recognizing all seven splice variants of the prominin transcript). An increased CD133 gene expression was observed in NCH421k cells (1.8-fold and 2.5-fold) and NIB26 cells (3.0-fold), whereas in NCH644 cells a decreased CD133 gene expression (2.3-fold and 3.9-fold) was noticed after 72 h of exposure to UC-CM and BM-CM, respectively (Fig. 5A). Flow cytometry
analysis of CD133 glycosylated epitope (CD133/2) revealed a decrease in NCH644 cells exposed to UC-CM (59.5%) and to BM-CM (61.3%) for 72 h (Fig. 5B). In NCH421k cells exposed to both types of MSC-CM, flow cytometry analysis failed to demonstrate changes in CD133 antigen expression. Also, a prolonged culture of NCH421k cells in MSC-CM for 7 days had no impact on their CD133 antigen expression (Fig. 5B). Beside CD133 expression, we also investigated the MSC-CM impact on the expression of other markers associated with stemness of GSLCs, such as Sox-2, Nestin, and Notch-1. The qRT-PCR analyses revealed decreased expression of Sox-2 in NCH421k (1.5-fold, 1.8-fold), NCH644 (2.0-fold, 1.1-fold), NIB26 (6.3-fold, 4.3-fold), and NIB50 cells (5.1-fold, 5.2-fold) exposed to UC-CM and BM-CM, respectively (Fig. 5C). Likewise, a decreased expression of Notch-1 was noted in NCH644 (1.7-fold, 1.1-fold), NIB26 (3.9-fold, 2.9-fold), and NIB50 cells (2.3-fold, 1.7-fold) when exposed to UC-CM and BM-CM, respectively, yet with no change observed NCH421k cells exposed to either type of MSC-CM (Fig. 5D). Also Nestin expression did
Table 3. Identification of Genes Involved in p53 Pathway With KEGG Pathway Analysis KEGG Term p53 signaling pathway
p Value
p FDR
Biomolecule IDs
Gene Name
11.73E-12
434.33E-12
ENSG00000106366 ENSG00000124762 ENSG00000146674 ENSG00000147889 ENSG00000149311
SERPINE1 CDKN1A IGFBP3 CDKN2A ATM
Differentially expressed genes in NCH421k cells exposed to both types MSC-CM (Table 2) were subjected to ontology enrichment analysis in KEGG to detect their involvement in cellular pathways. Biomolecular identification number (Biomolecule IDs) corresponds to ENSEMBL gene description (http://www.ensembl.org); p value (
0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368915X687787 E-ISSN 1555-3892 www.cognizantcommunication.com
Paracrine Effects of Mesenchymal Stem Cells Induce Senescence and Differentiation of Glioblastoma Stem-Like Cells Katja Kološa,* Helena Motaln,* Christel Herold-Mende,† Marjan Koršicˇ,‡ and Tamara T. Lah*§ *Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia †Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany ‡Department of Neurosurgery, University Medical Centre of Ljubljana, Ljubljana, Slovenia §Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Glioblastoma multiforme (GBM) displays high resistance to radiation and chemotherapy, due to the presence of a fraction of GBM stem-like cells (GSLCs), which are thus representing the target for GBM elimination. Since mesenchymal stem cells (MSCs) display high tumor tropism, we examined possible antitumor effects of the secreted factors from human MSCs on four GSLC lines (NCH421k, NCH644, NIB26, and NIB50). We found that conditioned media from bone marrow and umbilical cord-derived MSCs (MSC-CM) mediated cell cycle arrest of GSLCs by downregulating cyclin D1. PCR arrays revealed significantly deregulated expression of 13 genes associated with senescence in NCH421k cells exposed to MSC-CM. Among these, ATM, CD44, COL1A1, MORC3, NOX4, CDKN1A, IGFBP5, and SERPINE1 genes were upregulated, whereas IGFBP3, CDKN2A, CITED2, FN1, and PRKCD genes were found to be downregulated. Pathway analyses in GO and KEGG revealed their association with p53 signaling, which can trigger senescence via cell cycle inhibitors p21 or p16. For both, upregulated expression was proven in all four GSLC lines exhibiting senescence after MSC-CM exposure. Moreover, MSC paracrine signals were shown to increase the sensitivity of NCH421k and NCH644 cells toward temozolomide, possibly by altering them toward more differentiated cell types, as evidenced by vimentin and GFAP upregulation, and Sox-2 and Notch-1 downregulation. Our findings support the notion that MSCs posses an intrinsic ability to inhibit cell cycle and induce senescence and differentiation of GSLCs. Key words: Cell differentiation; Glioblastoma multiforme (GBM); Stem-like cancer cells; Mesenchymal stem cells (MSCs); Senescence; Therapy response
INTRODUCTION Glioblastoma multiforme (GBM) is the most malignant type of brain tumor that is still incurable, with the overall survival of GBM patients being less than 15 months (35). This is due to highly infiltrative growth of GBM and its resistance to chemo/radiotherapies, preventing complete elimination of tumor cells, despite improvements made in surgical techniques and therapeutic protocols (7,45). The highly tumorigenic subpopulation of glioblastoma stemlike cells (GSLCs) present in the tumor and defined as a subpopulation of cells with stem-like properties, such as self-renewal, asymmetric division, and differentiation potential, essential for tumor development and propagation in vivo (8,45) are presumably causative for GBM recurrence. Their variable abundance within the tumor mass has been confirmed in several studies (6,42,52). Their stemness and increased resistance to radiation and
chemotherapy (4,16,28) are suggesting new therapeutic approaches focusing on GSLC targeting to improve the survival of GBM patients (35). Cell therapy is being revisited in view of using normal tissue stem cells, that is, human mesenchymal stem cells (MSCs) for cancer treatment. MSCs are multipotent stem cells most often isolated from birth-associated [i.e., umbilical cord (UC-MSC), cord blood (UCB-MSC)] and adult tissues, such as bone marrow (BM-MSC) and adipose tissue (AT-MSC) (31). They are supposed to have a wide therapeutic potential because of their immunomodulatory ability, wound- and neoplasma-directed homing, and tissue repair ability, relying on their differentiation potential (21,24). Already naive UCB-MSCs were shown to suppress growth of various cancers, including GBM, as a 36% size reduction of C6 xenografts in mice was observed after UCB-MSC intratumoral injection (19). Likewise, a
Received January 27, 2015; final acceptance March 9, 2015. Online prepub date: March 24, 2015. Address correspondence to Dr. Helena Motaln, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ve na pot 111, SI-1000 Ljubljana, Slovenia. Tel: +386 5923 2870; E-mail: [email protected]
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systemic AT-MSC coadministration with 8MGA glioma cells was shown to decrease de novo tumor development in mice by 63%, compared to control mice injected with 8MGA cells only (27). Injected AT-MSCs were shown to colocalize with CD133+ cells in GBM xenografts (2). Also in rat glioma models, the injection of UCB-MSCs and BM-MSCs resulted in tumor volume reduction (23), tumor growth inhibition (13), and increased survival of animals (33). Conditioned media (CM) derived from UC-MSCs and AT-MSCs were shown to inhibit the growth of U251 glioma cells (50), whereas BM-MSCs and their CM were shown to inhibit proliferation of U87MG, U251, and U373 glioma cells (31) and induce U87 cell senescence (32). In contrast, the UCB-MSCs, UC-MSCs, and AT-MSCs were shown to induce apoptosis in glioma cells (13,20,50). All of the above is implying the potential of MSCs for the development of novel anticancer cell-based therapies. As the intrinsic resistance of GSLCs to therapy presents the main obstacle in GBM treatment, the repressive potential of MSCs on GSLCs (i.e., on their stemness potential and viability) should be defined. To clarify the MSC effect on GSLCs, we utilized the established GSLC lines and primary culture of GBM cells enriched for GSLCs and exposed them to BM-MSC- and UC-MSC-derived CM. We speculated that the paracrine activity of the MSCs, namely their secreted factors, would cause the alteration of the GSLC behavior. Indeed, we observed both MSC-CMs to cause cell cycle arrest of GSLCs in the G0/G1 phase and to induce their senescence. To our knowledge, we are also first to demonstrate that the exposure of GSLCs to MSC paracrine signals changes the gene expression of GSLCs, which is driving them toward a more differentiated phenotype. The MSC-CM also increased the sensitivity of GSLCs to chemotherapeutic temozolomide (TMZ), commonly used in GBM treatment. MATERIALS AND METHODS Cell Cultures GSLC lines NCH421k (male, age 66) and NCH644 (male, age 66) were isolated from GBM resection and checked for stemness potential, as described previously (8,10). NIB26 (female, age 75) and NIB50 (male, age 81) cells were isolated from fresh GBM samples obtained from the University Medical Centre of Ljubljana (study approved by the National Ethics Committee). Upon mechanical and enzymatic dissociation, the isolated cells were grown as spheroids (with a presumed enriched fraction of GSLCs) under the same conditions as NCH cells in neurobasal (NBE) medium (Gibco, Life Tech. Corp., Paisley, UK) supplemented with 2 mM l-glutamine (PAA Lab, Pasching, Austria), 100 U penicillin, 1,000 U streptomycin (PAA Lab), 1× B-27 (Gibco), 1 U/ml heparin (Sigma-Aldrich, Steinheim, Germany), 20 ng/ml EGF in 20 ng/ml bFGF
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(Gibco). Both derived cell lines were tested for the CD133 expression by flow cytometry. The U87 cell line was grown as described previously (31). Human MSCs Human BM-MSCs were purchased from Lonza Bioscience (Walkersville, MD, USA) (BM-MSC2: male, age 19, 6F4393; BM-MSC3: female, 22, 7F3677) and cultured according to the manufacturer’s recommendations. Briefly, MSCs were grown in Dulbecco’s medium (DMEM; 5921; Sigma-Aldrich) with 10% fetal bovine serum (FBS; PAA Lab), supplemented with 100 U penicillin (PAA Lab), 1,000 U streptomycin (PAA Lab), 2 mM l-glutamine (PAA Lab), Na-pyruvate (Gibco), and nonessential amino acids (Sigma-Aldrich). UC-MSCs were isolated from Wharton’s jelly according to standard protocol (47) in the study approved by the National Ethics Committee, Doc. No. 134/01/11. Umbilical cords were collected at cesarean section (37–41 weeks) upon obtained informed consent, and isolated UC-MSCs were cultured the same as BM-MSCs. UC-MSCs were characterized for CD13+, CD29+, CD44+, CD73+, CD90+, CD105+, CD14−, CD34−, CD45−, HLA-DR− surface marker expression, and osteogenic, chondrogenic, and adipogenic differentiation as recommended (15). Two UC-MSC clones (UC-MSC-10, female and UC-MSC-13, male) with the highest proliferation potential and homogenous spindlelike morphology were used in further experiments. Conditioned Media (CM) Collection Two different clones of BM-MSCs and the two clones of UC-MSCs were plated into T75 flasks (Corning, Cambridge, MA, USA) at a density of 6,000 cells/cm2, and a day before the cells would have reached 90% confluence, the standard MSC cultivation medium, described above, was changed to 12.5 ml NBE medium (used in GSLC culture). After 24 h, this CM was collected and pooled from both BM-MSC lines or from both UC-MSC cell lines, centrifuged at 300 × g for 10 min at 4°C, and stored at −80°C. Prior to the experiment, the CM were mixed in a 1:1 ratio with fresh NBE medium and designated as BM-CM and UC-CM, respectively. Similarly, the NBE CM were collected after 24 h from cultured NCH421k and NCH644 spheroids and diluted at a 1:1 ratio with fresh NBE medium and when used designated as NBE-CM. Surface Marker Detection by Flow Cytometry UC-MSCs (106) were harvested, washed with 1× phosphate-buffered saline (PBS; Gibco), and incubated with antibodies against CD13 (BD#557454), CD29 (BD# 559883), CD44 (BD#555479), CD73 (BD#550257), CD90 (BD#555596), CD14 (BD#555397), CD34 (BD#555821), CD45 (BD#555482), HLA-DR (BD#340688) (all from
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BD Biosciences, San Jose, CA, USA), CD105 (MHCD1 0505; Molecular Probes, Eugene, OR, USA), and isotype controls [FITC-IgG1 (BD#555748), FITC-IgG2a (BD#55 5573), PE-IgG1 (BD#555749), PE-IgG2b (BD#555743), APC-IgG1 (BD#555751); all from BD Biosciences] as instructed by the manufacturer. All cells were additionally stained with propidium iodide (PI; BD Biosciences) to exclude dead cells, washed, and analyzed by flow cytometry using a BD FACSCaliburTM and the CellQuest Software (both BD Biosciences). NIB26 and NIB50 cells were tested for the expression of CD133 protein using the antibodies recognizing AC133 or CD133/2 (AC141) epitope by the protocol described below. The staining results proved to overlap properly (8). NCH421k and NCH644 (106) cells were harvested upon 72-h exposure to NBE, NBE-CM, UC-CM, and BM-CM, washed with 1× PBS, and incubated with 10 µl CD133/2-PE antibody (130-090-853, Miltenyi Biotec, San Diego, CA, USA) or 20 µl isotype control antibody (PE-IgG2b, BD Biosciences) in equal concentrations. All cells were PI (BD Biosciences) stained for live cell analysis (20 min at 4°C), washed, and analyzed by flow cytometry using a BD FACSCaliburTM and the CellQuest Software (both BD Biosciences). Differentiation of UC-MSCs Adipogenic Differentiation. The adipogenic differentiation medium was composed of DMEM supplemented with 10% FBS, 1 µM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX; SigmaAldrich), 10 µg/ml insulin (Sigma-Aldrich), 100 µM indomethacin (Sigma-Aldrich). After 72 h, cells were medium changed to adipogenic maintenance medium: DMEM supplemented with 10% FBS and 10 µg/ml insulin for 24 h, and this sequence was repeated three times, followed by cultivation for 1 week in maintenance medium. After 21 days, cells were fixed in 4% paraformaldehyde for 10 min, and accumulated lipid-rich vacuoles were stained with 0.3% Oil red O (Sigma-Aldrich) for 30 min. Osteogenic Differentiation. The osteogenic differentiation medium was prepared with DMEM containing 10% FBS, 1 µM dexamethasone (Sigma-Aldrich), 50 µg/ ml l-ascorbic acid (Sigma-Aldrich), and 10 mM glycerophosphate (Sigma-Aldrich). After 21 days, cells were fixed in 4% paraformaldehyde for 10 min and stained with 1% Alizarin red S (Sigma-Aldrich) for 30 min to detect calcified extracellular matrix. Chondrogenic Differentiation. Cells were exposed to chondrogenic differentiation medium composed of DMEM, 10% FBS, 10 ng/ml TGF-b3 (Sigma-Aldrich), and 50 µg/ml l-ascorbic acid for 21 days. To detect matrix deposition of sulfated glycosaminogycans (GAGs), cells
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were fixed in 4% paraformaldehyde for 10 min and stained with 1% Alcian blue 8-GX (Sigma-Aldrich) in 3% acetic acid (Sigma-Aldrich). Cell Cycle Analysis NCH421k cells were exposed to NBE, UC-CM, and BM-CM for 72 h, trypsinized [0.25% Trypsin-EDTA (Gibco)], washed with 1× PBS, and left in 75% ethanol overnight at 4°C. Upon washing with 1× PBS, cells were incubated in 0.5 ml PI/RNase staining buffer (BD Pharmingen, San Diego, CA, USA) for 15 min at RT. Cell cycle analysis was performed using the WinMDI and Cyclored on the BD FACSCaliburTM with the CellQuest Software. Apoptosis Assay NCH421k cells were exposed to NBE, UC-CM, and BM-CM for 72 h, harvested, and early/late apoptotic cells detected by staining with Annexin-V-FITC and PI (BD Pharmingen) according to the manufacturer’s protocol. Stained cells were analyzed using BD FACSCaliburTM and the CellQuest Software. Staurosporine (STS; SigmaAldrich) treatment (2 µM, 6 h) served as a positive control for apoptosis. Mitochondrial Membrane Potential (MMP) Detection MMP change in NCH421k cells grown in NBE, UC-CM, and BM-CM for 72 h was measured with the Mitochondrial Permeability Transition Detection Kit JC-1 (ImmunoChemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. STS (2 µM, 6 h) and carbonylcyanide-chlorophenyl-hydrazone (CCCP; 25 µM, 30 min; ImmunoChemistry Technologies)treated NCH421k cells were used as a positive control. JC-1 staining was analyzed using a Synergy™ MX Microplate Reader (Bio-Tec Instruments, Winooski, VT, USA). The change of MMP was assessed by the ratio of red versus green fluorescence readings. Cell Senescence NCH421k, NCH644, NIB26, and NIB50 spheroids were grown in NBE, UC-CM, and BM-CM for 72 h; GSLC spheroids were dissociated to single cells and fixed in 0.5% glutaraldehyde solution (Sigma-Aldrich) for 20 min at RT. The proportion of cells positive for senescence indicative of b-galactosidase (SA-b-Gal) activity was evaluated as described previously (14). Quantitative Real-Time PCR (qRT-PCR) and PCR Arrays Total RNA was isolated from three biological replicates of the U87 cell line and NCH421k, NCH644, NIB26, and NIB50 cells grown in NBE, UC-CM, and BM-CM for 72 h using TrizolTM reagent (Invitrogen Limited,
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Paisley, UK) following the manufacturer’s instructions. Total RNA (1 µg) was used for cDNA generation using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Gene expression of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16), cyclin-dependent kinase inhibitor 1A (CDKN1A; p21), cyclin D1 (CCND1), BCL-2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL-2), prominin 1 (PROM1; CD133), SRY (sex-determining region Y)-box 2 (Sox-2), nestin (NES), Notch-1, tubulin b 3 class III (TUBB3), vimentin (VIM), and glial fibrillary acidic protein (GFAP) was quantified by real-time quantitative PCR on ABI 7900 HT Sequence Detection System (Applied Biosystems). Real-time PCR reactions comprised of cDNA added to TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (all Applied Biosystems): CDKN2A, Hs00923894_m1; CDKN1A, Hs 00355782_m1; CCND1, Hs00765553_m1; BAX, Hs999 99001_m1; BCL-2, Hs0060823_m1; PROM1, Hs010092 50; Sox-2, Hs01053049_s1; NES, Hs00707120_s1; Notch-1, Hs01062014_m1; TUBB3, Hs00801390_s1; VIM, Hs001 85584_m1; GFAP, Hs00157674_m1; p53, Hs01034249; and glyceraldehyde 3-phosphate dehydrogenase (GADPH) TaqMan probe (Assay No. 4310884E) as an internal control. The analyses were performed with SDS v2.2 software (Applied Biosystems) and comparative Ct method (DDCt algorithm). By using generated cDNA from NCH421k exposed to control and both types of MSC-CM, the expression of 84 senescence-related genes was determined also following the Cellular Senescence RT-Prolifer PCR Array (Qiagen-SABiosciences, Hilden, Germany) protocol. On the Cellular Senescence RT-Prolifer PCR Array, the average of three different housekeeping genes, ACTB (actin b), GAPDH, and ribosomal protein L13a (RLP13A), was used in calculations as an internal control. Fold increase in mRNA levels was calculated with the DDCt method by the provided online program available at http://pcr dataanalysis.sabiosciences.com/pcr/arrayanalysis.php. Results are expressed as means of three independent experiments done in duplicate. Cytotoxicity Testing After Exposure to Temozolomide and 5-Fluoro-Uracil by MTS Cell Viability Assay NCH421k and NCH644 cells were plated into 96-well plates (Corning) at a density of 104 cells/well and left to grow for 24 h before adding NBE, UC-CM, BM-CM, and TMZ (2.5–1000 µM) or 5-fluoro-uracil (5-FU) (0.01–150 µM) (both Sigma-Aldrich). After 48 h and 72 h of TMZ and 5-FU treatment, the CellTiter 96® AQueous MTS reagent [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium; Promega Corporation, Madison, WI, USA; Sigma-Aldrich], was added to each
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well to make 333 mg/ml final concentration. Three hours later, the absorbance at 490 nm (reference 690 nm) was measured with the Synergy™ MX Microplate Reader (Bio-Tec Instruments). Statistical Analyses All the above experiments were performed in duplicate and independently repeated three times. To test the effect of MSC-CM on GSLCs compared to control medium, the ANOVA test with post hoc Dunnett’s test was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, La Jolla, CA, USA). A value of p < 0.05 was considered significant. Data are expressed as the mean ± standard deviation (SD). RESULTS Umbilical Cord-Derived MSC Lines (UC-MSCs) and Primary GSLCs Were Successfully Generated We established six MSC lines from umbilical cords (UC-MSCs) obtained from healthy donors. These UCMSCs exhibited plastic adherence in culture and typical spindle-shaped morphology. We confirmed the presence of CD13, CD29, CD44, CD73, CD90, and CD105 and the absence of CD14, CD34, CD45, and HLA-DR surface antigens in all UC-MSC clones by flow cytometry and verified their osteo-, chondro-, and adipogenic differentiation potential (data available, not shown). This characterization is in accordance with suggested criteria for MSC phenotype confirmation (15); thus, we further used two UC-MSC clones together with two BM-MSC lines for UC-CM and BM-CM production. Purified NCH421k and NCH644 GSLC lines were established previously by Campos et al. (10) and confirmed by DNA copy-number profiling and CGH array to show typical GBM chromosome aberrations (8,46) and to highly express CD133 epitope 1 antigen detected by AC133 antibody (9). Flow cytometric analysis also confirmed expression of CD133 in NIB26 (79.9% CD133+ cells) and NIB50 (71.4% CD133+ cells) that were isolated from primary GBM tissue and grown in spheroids to enrich for their GSLCs fraction (Fig. 1A). Analysis of p53 status in NCH421k, NCH644, NIB26, and NIB50 cells was determined by qRT-PCR with U87 cell line (wild-type p53) serving as reference (54). MSC-CM Caused G0 /G1 Phase Cell Cycle Arrest via Cyclin D1 Downregulation Cell cycle analysis of NCH421k cells grown with NBE or either type of CM for 72 h was performed. It revealed an increased number of NCH421k cells halted in G0/G1 phase in the presence of UC-CM, compared to NBE (Fig. 2A). Additionally, the G0/G1 cell cycle arrest of NCH421k cells, grown in UC-CM and BM-CM, was confirmed by
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Figure 1. GSLCs isolated from primary GBM tissue express high levels of CD133. NIB26 and NIB50 cells were isolated from primary GBM tissue and grown in spheroids to enrich the GSLC fraction. Flow cytometry analysis of CD133 expression showed (A) 79.9% CD133+ cells in NIB26 and (B) 71.4% CD133+ cells in NIB50 cell culture. (C) Analysis of p53 gene expression in U87, NCH421k, NCH644, NIB26, and NIB50 cells by qRT-PCR.
Figure 2. MSC-CM interferes with the cell cycle of GSLCs. (A) Cell cycle analysis of NCH421k cells performed by flow cytometry after 72-h treatment with both types of MSC-CM. (B) Cyclin D1 (CCND1) expression (qRT-PCR) was decreased in all GSLC lines after 72-h treatment with UC-CM and BM-CM. The mean ± SD of three independent experiments are provided. *p < 0.05, **p < 0.01, ***p < 0.001. Morphology of native cells: (C) Control NCH421k cells with compact spheroid morphology; (D) NCH421k spheroid morphology changed upon UC-CM treatment for 72 h; and (E) NCH421k spheroid morphology changed in the presence of BM-CM after 72 h. (F) Control NCH644 cells with compact spheroid morphology; (G) UC-CM treatment for 72 h changed NCH644 spheroid morphology to adherent type of growth; and (H) BM-CM presence after 72 h changed NCH644 spheroid morphology to adherent type of growth. Scale bar: 100 µm.
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CCND1 gene expression analysis. This showed CCND1 gene expression to decrease 4.2-fold in UC-CM and 2.9fold in BM-CM (Fig. 2B). Similarly, the CCND1 gene expression was found downregulated in NCH644, NIB26, and NIB50 cells exposed to CM. The above was associated with the indicative size reduction and loosened structure of NCH421k and NCH644 spheroids grown in both types of MSC-CM (Fig. 2C–E). The BM-CM was even shown to induce the adherent growth of NCH644 cells (Fig. 2F–H). Altogether, our data imply the capacity of MCS paracrine factors to cause the cell cycle arrest of GSLCs through decrease in the cyclin D1 expression level. MSC-CM Affected Mitochondrial Membrane Potential but Did Not Induce Apoptosis in GSLCs The induction of apoptosis in NCH421k cells grown in MSC-CM of both origins for 72 h was evaluated by MMP assay. A significant decrease in MMP was detected in NCH421k cells when exposed to UC-CM (23%) and BM-CM (31%) (Fig. 3A). Since MMP change is not solely indicative of apoptosis, BAX and BCL-2 gene expression was analyzed in the NCH421k, NCH644, NIB26, and NIB50 cells cultured in MSC-CM. The analysis of the BAX/BCL-2 gene ratio failed to demonstrate any significant change to occur in the CM-cultured GSLCs, compared to control (NBE) ones (Fig. 3B). To further exclude the onset of apoptosis, the annexin V staining was performed. This also failed to detect increased apoptosis in NCH421k cells cultured in MSC-CM versus control cells with staurosporin treatment used as positive control (Table 1). Altogether our results showed that by addition of MSC-CM containing MSC paracrine factors, the apoptosis in GSLCs cannot be induced.
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MSC-CM-Activated Senescence in GSLCs As MSC-CM influenced the cell cycle of GSLCs, but did not cause apoptosis, the senescence in exposed GSLCs was investigated by using the indicative b-galactosidase staining. A significantly increased number of senescent cells was observed in NCH421k (17% and 33%), NCH644 (33% and 39%), NIB26 (20% and 25%), and NIB50 (30% and 31%) cells exposed to UC-CM and BM-CM, respectively, for 72 h, when compared to control NCH421k (Fig. 4A). In GSLCs exposed only to NBE, the percentage of senescent cells was 10.7% in NCH421k, 6.6% in NCH644, 3.3% in NIB26, and 8.2% in NIB50 cells. To get insight into the senescence signaling, NCH421k cells were subjected to both types of MSC-CM, and qRTPCR analysis of 84 genes associated with senescence was conducted by the human Senescence RT2 Profiler PCR Array (Table 2). The PCR arrays revealed changed expression of 13 genes involved in senescence (ATM, CDKN1A, CDKN2A), cell adhesion (CD44, COL1A1), oxidative stress (PRKCD, NOX4), IGF-related pathway (IGFBP3, IGFBP5), p53/pRb signaling (MORC3, CITED2), and cytoskeleton formation (FN1, SERPINE1) (Table 2). Those genes were consistently up- and downregulated in exposed NCH421k cells at least 2.1-fold. In addition, KEGG pathway analysis identified the involvement of five genes, SERPINE1, CDKN1A, IGFBP3, CDKN2A and ATM, in the p53 pathway (Table 3) and induced in senescence. The PCR Array results were validated with qRT-PCR on independent mRNA samples of GSLCs, which confirmed increased expression of the CDKN1A gene in NCH421k cells grown for 72 h in UC-CM (1.4-fold) and BM-CM (6.7-fold). Likewise, the CDKN1A expression increased
Figure 3. MSC-CM does not induce apoptosis in GSLC lines. (A) Mitochondrial membrane potential (MMP) loss was ascertained by JC-1 staining after exposure of NCH421k cells to UC-CM and BM-CM for 72 h. (B) QRT-PCR analysis of Bax and Bcl-2 gene expression in NCH421k, NCH644, NIB26, and NIB50 cells after exposure to both types of MSC-CM for 72 h. The ratio between the Bax and Bcl-2 was unaffected. Data shown are mean ± SD of three independent experiments. ***p < 0.001. CCCP, carbonylcyanidechlorophenyl-hydrazone; STS, staurosporin.
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Table 1. Annexin V/PI Staining of NCH421k Cells Exposed to Staurosporin (STS) and MSC-CM NCH421k NCH421k + STS NCH421k + UC-CM NCH421k + BM-CM
Live
Early Apoptosis
Late Apoptosis
Dead
93.34 ± 4.20 42.98 ± 12.73* 91.06 ± 2.54 88.82 ± 6.51
4.83 ± 2.63 45.26 ± 13.87* 7.01 ± 2.33 7.69 ± 2.45
1.06 ± 1.70 5.66 ± 0.56 1.24 ± 1.41 2.82 ± 4.32
0.76 ± 0.69 0.80 ± 1.02 0.56 ± 0.55 0.67 ± 0.68
The NCH421k cells were exposed to UC-CM and BM-CM, and the apoptotic cells were determined by flow cytometry as described in Materials and Methods. The exposure to STS, inducing intrinsic apoptotic pathway, was used as positive control. The data of three independent experiments are expressed as the mean ± standard deviation (SD) and *p < 0.05 was considered significant.
Figure 4. MSC-CM activates senescence in all GSLC lines. (A) Senescence-associated b-galactosidase (SA-b-Gal) staining revealed activation of senescence in NCH421k, NCH644, NIB26, and NIB50 cells after exposure to UC-CM and BM-CM upon 72-h incubation. qRT-PCR analysis of (B) p21 expression and (C) p16 expression after 72-h treatment with both types of MSC-CM in all GSLCs. Data shown are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (D) SA-b-Gal staining of dissociated and fixed NCH421k, NCH644, NIB26, and NIB50 cells after UC-CM and BM-CM presence for 72 h. Scale bar: 20 µm.
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Table 2. Differentially Expressed Genes in MSC-CM-Cultured NCH421k Cells Gene symbol ATM CD44 COL1A1 MORC3 NOX4 CDKN1A IGFBP5 SERPINE1 IGFBP3 CDKN2A CITED2 FN1 PRKCD
NCH421k + UC-CM
NCH421k + BM-CM
Fc
p Value
Fc
p Value
Pathway/Process
2.6 2.7 7.5 2.1 2.1 3.0 1.5 4.1 −2.1 1.3 −1.4 2.1 −1.4
0.032* 0.022* 0.043* 0.046* 0.045* 0.054* 0.343 0.172 0.009* 0.311 0.101 0.082 0.294
−1.2 1.4 1.5 −1.1 1.8 6.9 30.1 3.8 2.0 −2.1 −2.8 −2.4 –2.2
0.406 0.241 0.210 0.606 0.163 0.001* 0.017* 0.010* 0.048* 0.022* 0.034* 0.012* 0.016*
Senescence Cell adhesion Cell adhesion p53/pRb signaling Oxidative stress Senescence IGF related Cytoskeleton IGF related Senescence p53/pRb signaling Cytoskeleton Oxidative stress
Listed are deregulated genes in NCH421k cells exposed to either UC-CM or BM-CM relative to NBE exposed NCH421k cells. Fold increase in mRNA levels are calculated as described in Materials and Methods. The mean ± SD of three independent experiments are provided. *p < 0.05. Fc, fold change.
in NCH644 (3.2-fold) and NIB50 cells (5.0-fold) exposed to BM-CM (Fig. 4B). The expression of the CDKN2A gene also increased in NCH644 (3.4-fold), NIB26 (7.7fold), and NIB50 (1.9-fold) exposed to BM-CM (Fig. 4C), whereas it decreased 1.7-fold in NCH421k cells exposed to BM-CM (Fig. 4C). The above was consistent with the observed b-galactosidase staining (Fig. 4D), altogether implying the ability of the MSC paracrine factors to induce GSLC growth arrest and senescence. MSC-CM Affected the Expression of GSLC Markers To evaluate the impact of MSC-CM on the stemness of GSLCs, the expression of indicative GSLC marker CD133/prominin was examined by qRT-PCR (using gene expression assay recognizing all seven splice variants of the prominin transcript). An increased CD133 gene expression was observed in NCH421k cells (1.8-fold and 2.5-fold) and NIB26 cells (3.0-fold), whereas in NCH644 cells a decreased CD133 gene expression (2.3-fold and 3.9-fold) was noticed after 72 h of exposure to UC-CM and BM-CM, respectively (Fig. 5A). Flow cytometry
analysis of CD133 glycosylated epitope (CD133/2) revealed a decrease in NCH644 cells exposed to UC-CM (59.5%) and to BM-CM (61.3%) for 72 h (Fig. 5B). In NCH421k cells exposed to both types of MSC-CM, flow cytometry analysis failed to demonstrate changes in CD133 antigen expression. Also, a prolonged culture of NCH421k cells in MSC-CM for 7 days had no impact on their CD133 antigen expression (Fig. 5B). Beside CD133 expression, we also investigated the MSC-CM impact on the expression of other markers associated with stemness of GSLCs, such as Sox-2, Nestin, and Notch-1. The qRT-PCR analyses revealed decreased expression of Sox-2 in NCH421k (1.5-fold, 1.8-fold), NCH644 (2.0-fold, 1.1-fold), NIB26 (6.3-fold, 4.3-fold), and NIB50 cells (5.1-fold, 5.2-fold) exposed to UC-CM and BM-CM, respectively (Fig. 5C). Likewise, a decreased expression of Notch-1 was noted in NCH644 (1.7-fold, 1.1-fold), NIB26 (3.9-fold, 2.9-fold), and NIB50 cells (2.3-fold, 1.7-fold) when exposed to UC-CM and BM-CM, respectively, yet with no change observed NCH421k cells exposed to either type of MSC-CM (Fig. 5D). Also Nestin expression did
Table 3. Identification of Genes Involved in p53 Pathway With KEGG Pathway Analysis KEGG Term p53 signaling pathway
p Value
p FDR
Biomolecule IDs
Gene Name
11.73E-12
434.33E-12
ENSG00000106366 ENSG00000124762 ENSG00000146674 ENSG00000147889 ENSG00000149311
SERPINE1 CDKN1A IGFBP3 CDKN2A ATM
Differentially expressed genes in NCH421k cells exposed to both types MSC-CM (Table 2) were subjected to ontology enrichment analysis in KEGG to detect their involvement in cellular pathways. Biomolecular identification number (Biomolecule IDs) corresponds to ENSEMBL gene description (http://www.ensembl.org); p value (
