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Contextual niche signals towards colorectal tumor progression by mesenchymal stem cell in the mouse xenograft model

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Abstract

Background

The role of mesenchymal stem/stromal cells (MSCs) in tumorigenesis remains controversial. This study aimed to determine whether heterotypic interactions between MSCs and colon cancer cells can supply contextual signals towards tumor progression.

Methods

Xenografts consisting of co-implanted human colorectal cancer cells with rat MSCs in immunodeficient mice were evaluated by tumor progression, angiogenic profiles, and MSC fate. Furthermore, we investigated how MSCs function as a cancer cell niche by co-culture experiments in vitro.

Results

Tumor growth progressed in two ways, either independent of or dependent on MSCs. Such cell line-specific dependency could not be explained by host immune competency. COLO 320 xenograft angiogenesis was MSC-dependent, but less dependent on vascular endothelial growth factor (VEGF), whereas HT-29 angiogenesis was not MSC-dependent, but was VEGF-dependent. MSCs and COLO 320 cells established a functional positive feedback loop that triggered formation of a cancer cell niche, leading to AKT activation. Subsequently, MSCs differentiated into pericytes that enhanced angiogenesis as a perivascular niche. In contrast, the MSC niche conferred an anti-proliferative property to HT-29 cells, through mesenchymal–epithelial transition resulting in p38 activation.

Conclusions

In conclusion, MSCs demonstrate pleiotropic capabilities as a cancer cell or perivascular niche to modulate colorectal cancer cell fate in a cell line-dependent manner in a xenogeneic context.

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Abbreviations

MSCs:

Mesenchymal stem/stromal cells

TME:

Tumor microenvironment

CAFs:

Cancer-associated fibroblastic cells

CCL5:

C–C motif chemokine ligand 5

EMT:

Epithelial–mesenchymal transition

IL-6:

Interleukin-6

VEGF:

Vascular endothelial growth factor

eGFP:

Enhanced green fluorescence protein

αMEM:

α-Modified Eagle’s medium

FBS:

Fetal bovine serum

qRT-PCR:

Quantitative real-time reverse transcription PCR

MVD:

Tumor microvessel density

Thy-1:

Thymus cell antigen-1

NG2:

Neural/glial antigen 2

αSMA:

α-Smooth muscle actin

OE:

Overexpression

CXCL12:

Chemokine C-X-C motif ligand 12

KD:

Knock down

MSC-CM:

MSC-conditioned medium

CXCR4:

C-X-C chemokine receptor type 4

MAPKs:

Mitogen-activated protein kinases

FACS:

Fluorescence-activated cell sorting

ANOVA:

Analysis of variance

PECAM-1:

Platelet endothelial cell adhesion molecule-1

Vegfr1 (Flt1):

Vascular endothelial growth factor receptor 1

PDGF-BB:

Platelet-derived growth factor BB

Pdgfr-β:

Platelet-derived growth factor receptor-β

MET:

Mesenchymal–epithelial transition

Vcam1:

Vascular cell adhesion molecule-1

CCR5:

Chemokine (C–C motif) receptor 5

VLA-4:

Very late antigen-4

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Acknowledgments

We are very grateful to Ms. K. Fujii of First Department of Internal Medicine, for technical assistance, and Dr. Y. Sasaki of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University, for critical comments. We are also thankful to Dr. M. Tsuji of Chromosome Science Labo Inc., for providing FISH probes and technical advice. This work was supported in part by Health and Labor Sciences Research Grants for research on intractable diseases from the Ministry of Health, Labour, and Welfare of Japan (K.I. and Y.A.).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Author notes

    Authors and Affiliations

    1. Department of Gastroenterology, Rheumatology, and Clinical Immunology, Sapporo Medical University, Sapporo, 060-8543, Japan

      Suguru Nakagaki, Yoshiaki Arimura, Hiroyuki Isshiki, Masanao Nasuno, Shuhei Watanabe, Kentaro Yamashita, Yasushi Adachi & Yasuhisa Shinomura

    2. Department of Anatomy, Sapporo Medical University, Sapporo, 060-8543, Japan

      Kanna Nagaishi & Mineko Fujimiya

    3. Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, 060-8543, Japan

      Masashi Idogawa

    4. Department of Educational Development, Sapporo Medical University, Sapporo, 060-8543, Japan

      Yasuyoshi Naishiro

    5. Department of Molecular Biology, Sapporo Medical University, Sapporo, 060-8543, Japan

      Hiromu Suzuki

    6. Center for Antibody and Vaccine Therapy, The University of Tokyo, Tokyo, 108-8639, Japan

      Kohzoh Imai

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    1. Suguru Nakagaki
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    Correspondence to Yoshiaki Arimura.

    Additional information

    S. Nakagaki and Y. Arimura contributed equally to this work.

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    Supplementary material 1 (DOCX 39 kb)

    535_2015_1049_MOESM2_ESM.tif

    Supplementary Fig. 1 Xenografts analysis and co-culture experiments. To examine whether promotion of tumor growth by MSCs was dependent on the ratio of tumor cells that were co-implanted, xenografts were formed at various ratios of MSCs to COLO 320 cells (2:1 to 0:1) in SCID mice [3]. The 1:1 ratio was used for subsequent analyses because of maximal ability to promote tumor growth. M represents MSCs and C indicates COLO-320 cells. Average data from multiple experiments are shown (a total of six tumors from three mice for each cell line) (a). COLO 320 xenografts without co-implanted MSCs failed to engraft even over a longer period (day 32) observation period in SCID mice (b). Angiogenic factors in cell lines and culture supernatants were quantified by qRT-PCR (c) and quantitative real-time immuno-PCR (iPCR) (d), respectively. iPCR [4] was performed as sandwich assays using an angioplex detection kit in microtiter plates pre-coated with anti-fibroblast growth factor-2 (FGF-2), anti-hepatocyte growth factor (HGF), or anti-VEGF antibodies (Synthera Technologies, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, the analyte (i.e. culture supernatant) as well as the standard and negative controls were diluted, and for subsequent detection of immunocomplexes (capture antibody–analyte antigen-detection antibody), MUSTag (multiple simultaneous tag) mix was added, followed by incubation for 1 h at room temperature (RT). After EcoRI digestion and the retrieved reaction mixture was added, real-time PCR was conducted in a PRISM 7500 Sequence Detection System (Applied Biosystems) for 50 cycles of a two-step PCR amplification protocol (95 °C for 15 s and 60 °C for 1 min). Oligo-tags of FGF-2, HGF, and VEGF antibodies were labeled by FAM, HEX, and Cy5 fluorochromes, respectively. For flow cytometry, a single cell suspension of colon cancer cells co-cultured with MSCs was fixed in 90 % cold ethanol, treated with RNase A, and stained with propidium iodide. (d) Histograms of cell cycle analysis of co-cultured colon cancer cells using FACS after eliminating MSCs by gating on GFP-negative cells. (e) The percentages of each population gated in panel d are shown. The G0/G1 peak was decreased and the G2/M was increased in co-cultured COLO 320 cells. In contrast, the G0/G1 peak was increased whereas the G2/M was decreased in co-cultured HT-29 cells compared with that in mono-cultured cells. (f) The 11 cytokines, Egf, Tgfα, Tgfβ, Vegf, Igf1, Ptgs2, Hgf, Fgf2, Tnfα, Il1β, and Il10, was analyzed by qRT-PCR in MSCs separated by FACS. Relative expression of Il1β compared with that of the steady state of a single culture of MSCs was 5.7-fold and 744-fold in MSCs co-cultured with COLO320 and HT-29 cells, respectively. Whether abundant IL-1β production induced by co-cultured MSCs (approximately 130-fold more abundant in co-cultured MSCs with HT-29 than that in COLO 320 cells) could activate p38 was not likely according to a report by Liu et al. [5]. Recently, Waterman et al. [6] classified MSCs into two distinct phenotypes: MSC1 has pro-inflammatory and anti-tumor effects, while MSC2 has an immunosuppressive role and promotes tumor growth and metastases. Although these classifications appeared to be similar to the phenotypes of MSCs co-cultured with HT-29 cells and COLO 320 cells, respectively, there were some inconsistencies concerning IL-1β and IL-10 expression in our observations (TIFF 1180 kb)

    535_2015_1049_MOESM3_ESM.tif

    Supplementary Fig. 2. Candidate panel of potential cancer niche signals and CXCL12 CpG island methylation. The candidate panel for MSC niche signaling molecules included wingless-type MMTV integration site family, member 3A (Wnt3a) and 5a (Wnt5a), secreted frizzled-related protein 1 (Sfrp1) and 4 (Sfrp4), dikkopf-1 (Dkk1), angiopoetin-1 (Angpt1), thyroid peroxidase (Tpo), N-Cadherin (Cdh2), integrin β1 (Itgb1) and α4 (Itga4), vascular cell adhesion molecule-1 (Vcam1), sec1 family domain containing 1 (Scfd1) and 2 (Scfd2), Nestin, glial fibrillary acidic protein (Gfap), chemokine (C-X-C motif) ligand 12 (Cxcl12), β-catenin (Ctnnb1), E-cadherin (Cdh1), Jagged 1 (Jag1) and 2 (Jag2), Delta-like 1 (Dll1), 2 (Dll2), and 4 (Dll4), and chemokine (C–C motif) ligand 5 (CCL5). This list was obtained from literature describing hematopoietic stem cell niche molecules [7], Notch signaling [8], and de novo secretion of chemokines from MSCs [9]. Relative expression of the indicated transcripts in co-cultured MSCs with COLO 320 was analyzed by qRT-PCR compared with that of single-cultured MSC. The Y-axis was a hemi-logarithmic scale. Six transcripts of Vcam1, Cxcl12, Cdh1, Jag1 and 2, and Ccl5 were upregulated in co-cultured MSCs compared with single-cultured MSCs (a). (b) Western blot analysis was conducted in COLO 320 cells co-cultured with MSCs, which was corresponding to Fig. 5e. Co-cultured COLO 320 cells without any treatment were used as a control. Data are representative of three independent experiments. (c) CXCL12 transcripts were analyzed by qPCR. (d) CpG island methylation at the CXCL12 promoter was quantified by bisulfite-pyrosequencing. The regions upstream from the transcription start site (CXCL12: -87 to -181 bp) were examined. DMTsKO indicates knockout of DNMT-1, -3a, and 3b in HCT116 cells. A normal colon, which represented histopathological normal specimens resected for medical reasons, was used for the negative control. Genomic DNA (1 μg) was modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen), and bisulfite pyrosequencing analysis was performed as described previously [10]. Although we hypothesized that epigenetic silencing of CXCL12 as reported by Wendt et al. [11] was exceptionally cancelled by DNA demethylation in COLO 320 cells, further analysis was necessary to test this hypothesis. (e) MicroRNAs, miR-126 (miRBase accession no. MIMAT0000445) and miR-126* (miRBase accession no. MIMAT0000444), the partner to miR-126 that is derived from the same transcript, EGF-like-domain, multiple 7 (EGFL7) were investigated. The total RNA of cells was isolated with the TaqMan MicroRNA Cells-to-CT Kit (Applied Biosystems) according to the manufacturer’s instructions. We used TaqMan MicroRNA Assays to quantify mature miRNA expression. Zhang et al. [12] recently reported that miR-126/miR-126* directly inhibits CXCL12 expression and independently suppresses the sequential recruitment of MSCs into the tumor stroma in a mouse xenograft model. However, since miR-126/miR-126* was expressed approximately 15–20-fold more in COLO 320 cells than in HT-29 cells in our analysis (e), thus miR-126/miR-126* was not likely to be a major regulatory mechanism of CXCL12 in this setting. As for CXCR4 upregulation, it is entirely an open question as to what cell types express it, and what mechanism of production occurs in xenograft tumors with or without MSCs (TIFF 635 kb)

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    Nakagaki, S., Arimura, Y., Nagaishi, K. et al. Contextual niche signals towards colorectal tumor progression by mesenchymal stem cell in the mouse xenograft model. J Gastroenterol 50, 962–974 (2015). https://doi.org/10.1007/s00535-015-1049-0

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    • DOI: https://doi.org/10.1007/s00535-015-1049-0

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