Conceived and designed the experiments: LC MS LF. Performed the experiments: LC T. Kasai YL YS GJ. Analyzed the data: LC YS AM. Contributed reagents/materials/analysis tools: MO AV AS T. Kudoh. Wrote the paper: LC. Data discussion, manuscript revision: MJCH DSS.
The authors have declared that no competing interests exist.
Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating
A number of studies have attempted to identify the mechanisms underlying malignant tumor growth and progression. Despite significant progress, most therapeutic approaches fail to eliminate all tumor cells. The remaining tumor cells often result in recurrence and metastasis. Recently, the hypothesis of cancer stem cells (CSCs) was proposed to explain the origin of cancer cells. By definition, CSCs are a small fraction of tumor cells with the capacity of both self-renewal and unlimited slow proliferation. They are often resistant to chemotherapy and radiation and thus are responsible for continuously supplying new cancer cells
It is well known that the microenvironment can exert profound genetic or epigenetic effects on stem cells through interactions between cells, or through cell-derived factors originating from the surrounding cells within the niche. These effects can be transient, as seen in the activation of signaling pathways regulating cellular proliferation and migration, or they can be associated with more stable alterations, such as cell fate determination and differentiation
In 2007, Yamanaka's group
miPS cells should be induced to some kinds of progenitor cells, such as hematopoietic cells and neural stem cells, differentiating into various phenotypes, such as macrophage, monocytes, neural cells, cardiac cells and pancreatic β- cells, when exposed to the normal niche. We hypothesized that CSCs may also be derived from miPS cells only when exposure to a malignant niche.
In this study, we designed two procedures to treat miPS cells. miPS cells were cultured without feeder cells in a mixture of miPS medium and conditioned medium obtained from the following mouse cancer cell lines: Lewis lung carcinoma (LLC), mouse embryonal carcinoma (P19), mouse melanoma (B16) and mouse mammary carcinoma (MC.E12) for 4 weeks. miPS cells cultured under these conditions were termed miPS-LLCcm, miPS-P19 cm, miPS-B16 cm, and miPS-MC.E12 cm cells respectively. miPS cells were also cocultured with each cancer cell line that had previously been treated with mitomycin C as feeder cells for 4 weeks. These miPS cells were termed miPS-LLCc, miPS-P19c, miPS-B16c and miPS-MC.E12c cells respectively. The miPS cells that had been cultured with or without feeder cells under the different conditions were then transplanted into nude mice. After 4 weeks, miPS cells formed typical teratomas that contained differentiated tissues without metastasis (
(A) Histology of miPS-LLCcm cells derived tumor. The tumor exhibited malignant phenotype with glandular epithelial hyperplasia (asterisk), high nuclear to cytoplasmic ratio, severe nuclear atypia and multiple pathological mitotic figures (arrowheads, inset) (top left); micrometastases (arrow, top right); and hypervascularization indicative of angiogenesis (bottom left) by HE staining. The positive of CD31 (Rat monoclonal antibody, brown) by IHC staining showed multiple vascular vessels in the tumor (bottom right). Scale bars: 100 µm (top left and bottom), 50 µm (top right). (B) Primary culture derived from miPS-LLCcm tumor. The primary culture exhibited stem-like cells (asterisk in top left) expressing GFP (top right) and fibroblast-like cells (arrow in top left) without GFP expression (top right). Spheroid cells grown from the primary culture in suspension (middle left) with GFP expression (middle right). The spheroid cells were placed back in adherent culture maintained stem-like cells (asterisk in bottom left) with GFP expression (bottom right) and fibroblast-like cells (arrow in bottom left) without GFP expression (bottom right). (C) Immunofluorescence staining for Nanog and Oct 3/4 in spheroid cells. Cryosections of spheroid cells were stained with primary antibodies (Rabbit anti-Nanog or Mouse anti-Oct-3/4) followed by anti-Rabbit or anti-mouse secondary antibodies labeled with Alexa fluorophores 555 (red) or 488 (Green). The cells were counterstained with DAPI (blue). Scale bars: 20 µm. (D) The expression levels of p53, MMP-2 and MMP-9 were analyzed by quantitative real-time PCR. miPS+LIF/−MEF, miPS cells cultured with LIF in the medium but without MEFfeeder cells; miPS-LLCcm spheroid, the spheroid cells derived form miPS-LLCcm cells; miPS-LLCcm LMT spheroid, the spheroid cells derived from miPS-LLCcm cells lung metastatic tumor; LLC, Lewis lung carcinoma cells.
Cell names |
Cell number | Tumor formation | Histologic examination |
miPS (with feeder cells) | 4×106 | 3/3 | Teratoma |
miPS (without feeder cells) | 4×106 | 3/3 | Teratoma |
miPS-LLCcm cells | 4×106 | 8/8 | Malignant tumor, angiogenesis |
miPS-LLCc cells | – |
||
miPS-P19 cm cells | 4×106 | 5/5 | Malignant tumor |
miPS-P19c cells | 4×106 | 0/3 | |
miPS-B16 cm cells | 4×106 | 3/5 | Malignant tumor |
miPS-B16c cells | 4×106 | 0/3 | |
miPS-MC.E12 cm cells | 4×106 | 3/3 | Malignant tumor |
miPS-MC.E12c cells | 4×106 | 4/5 | Malignant tumor |
: miPS cells were named with each name of cancer derived cells and “c” or “cm”. “c” stands for the miPS cells were cocultured with mouse cancer derived cells treated with mitomycin C and “cm” for the miPS cells cultured in the conditioned medium of cancer derived cells.
: Cells could not survive after several passages in subculture.
Thirty to fifty percent of the cells were GFP positive in the tumors derived from miPS-LLCcm cells while less than five percent were positive in the differentiated teratoma (
(A) Histology of tumor derived from spheroid cells. The tumor showed some glandular structure (asterisks) with multiple pathologic mitotic figures (arrowheads, inset) (left), high mitotic rates (arrowheads in middle), and hypervascularization (right) by HE staining. Scale bars: 100 µm. (B) Lung metastasis after tail vein injection of spheroid cells. Lungs were occupied by metastatic tumor nodules. (C) The metastases showed some glandular structure (asterisks) with multiple pathologic mitotic figures (arrowheads in top left); hypervascularization (top right); invasion into lung parenchymal tissue (bottom left) by HE staining. The expression of GFP (Rabbit polyclonal antibody, brown) was found in these metastatic nodules by IHC staining (bottom right). T, tumor; L, lung tissue. Scale bars: 100 µm. (D) Immunohistochemistry of CK and GFP localization in tumors derived from miPS, miPS-LLCcm and spheroid cells. Serial sections were stained with CK (mouse monoclonal antibody, brown) and GFP (Rabbit polyclonal antibody, brown), and counterstained with hematoxylin. Glandular region were CK positive but GFP negative in the tumors. Scale bars: 100 µm.
Cell number | Tumor formation | Histologic examination |
1×10 | 0/4 | NA |
1×102 | 0/4 | NA |
1×103 | 0/4 | NA |
1×104 | 0/4 | NA |
1×105 | 2/4 | Malignant tumor, angiogenesis |
8×105 | 4/4 | Malignant tumor, angiogenesis |
2×106 | 4/4 | Malignant tumor, angiogenesis |
4×106 | 4/4 | Malignant tumor, angiogenesis |
NA: not applicable.
We then investigated the type of the malignant tumor by IHC. Pan-Cytokeratin (CK, an epithelial tumor cells marker), vimentin (a marker of mesenchymal tumor), α-actin (a marker of myogenic tumor), CD31 (a marker for vasculogenesis), NF-M and GFAP (markers of neurogenic tumor) were used to stain the tumors (data not shown). CK was found to be strongly stained in the tumors. The expression of CK and GFP was then assessed in multiple serial sections. Glandular regions were CK positive but these cells were GFP negative in the tumors (
Embryonic stem cell markers and the four transcription factors that were transduced were then checked by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (RT-qPCR). miPS-LLCcm cells and spheroid cells showed expression of the embryonic stem cell markers (
(A) RT-PCR analysis of embryonic stem cell marker gene expression. (B) RT-PCR analysis of the four miPS transcription factors. The PCR products were the coding regions (Total), endogenous transcripts only (Endo.), and transgene transcripts only (tg). (C) Expression levels of embryonic stem cell marker gene were analyzed by quantitative real-time PCR. (D) Expression levels of the four miPS cell transcription factors were analyzed by quantitative real-time PCR.
The miPS-LLCcm cells showed spheroid formation in suspension culture, a high tumorigenic potential at limited dilutions and a high metastatic potential, which were all consistent with the basic characteristics of CSCs
Self-renewal is frequently cited as a characteristic of CSCs. However, there are technical limitations to strictly evaluate self-renewal. For normal tissue stem cells, the standard test of self-renewal requires the clonal in vivo demonstration of self-renewal and multi-lineage differentiation in primary transplants of stem cells, followed by demonstration of the same properties in serial transplants of the same cells. Self-renewal in tumorigenic cancer cells has generally been evaluated by the demonstration of serial transplantability of polyclonal tumors and by the demonstration of a similar phenotypic heterogeneity in the parental and progeny tumor xenografts
Moreover,
The tumor cells developed in this study from miPS cells grew as spheroids in suspension culture, showed a high tumorigenic and metastatic potential and angiogenesis in vivo. In addition, the capacity for self-renewal and maintainance of an undifferentiated state as assessed by the expression of markers that are associated with embryonic stem cells suggest that these primary miPS-LLCcm cells and the spheroid cells which were derived from miPS-LLCcm cells contain a high proportion of CSCs. Scaffidi and Misteli have recently reported the production of CSC-like cells from fibroblast, which may be traced to somatic stem cells
The expression of specific cell surface markers has been widely used to identify CSCs. Some of these surface markers are known to be common to different CSCs population. However, these markers may still be associated with normal stem cells
Mouse induced pluripotent stem cells (miPS; cell name: iPS-MEF-Ng-20D-17; Lot No. 012) were purchased from Riken Cell Bank (Japan) and were maintained in medium (DMEM containing 15% FCS, 0.1 mM NEAA, 2 mM L-Glutamine, 0.1 mM 2-mercaptoethanol, 1000 U/ml LIF, 50 U/ml penicillin and 50 U/ml streptomycin) on feeder layers of mitomycin-C-treated mouse embryonic fibroblast (MEF) cells (Reprocell, Japan). Mouse Lewis lung cancer (LLC) cells were purchased from ATCC (USA) and were maintained in DMEM containing 10% FCS; mouse embryonal carcinoma cells (P19) were purchased from Riken Cell Bank (Japan) and were maintained in αMEM containing 10% FCS; mouse melanoma cells (B16/BL6) (ATCC, USA) and mouse mammary tumor cells (BALB-MC.E12) (Riken Cell Bank, Japan) were maintained in MEM containing 10% FCS.
For preparing conditioned medium (CM) from the different mouse cancer cell lines, medium was collected from confluent dishes and filtered using 0.45 µm filter (Millpore, Ireland). Then 3 ml CM were added into 3.5 cm dish overnight to confirm there were no surviving cancer cells in CM. For the conditioned medium experiments, miPS cells (without MEF feeder cells) were maintained in medium described above without LIF. Half of the medium was changed every day with CM for 4 weeks. miPS cells without treatment with CM were used as control. For the coculture experiments, the mouse tumor cell lines were treated with 0.4 µg/ml mitomycin C (Sigma, USA) and were then used as feeder cells and cocultured with miPS cells (without MEF feeder cells) for 4 weeks. miPS cells were passaged every 3 days and cell morphology was photographed using a Olympus IX81 microscope equipped with a light fluorescence device (Olympus, Japan).
For primary culture, mouse allografts were cut into small pieces (approximately 1 mm3) in HBSS. After washing three times, the tissues were transferred into a 15 ml tube with 0.25% trypsin of 5–6 fold volume at 37°C for 40 min. Five microliter DMEM containing 10% FCS was then added to terminate digestion. The cellular suspension was then placed into a new tube and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended in 5 ml HBSS, and centrifuged at 1000 rpm for 5 min. The cell pellet was then placed into an appropriate volume of miPS medium without LIF and the cells were seeded into a dish at a density of 5×105/ml. Cells were passaged every 3 days and cells morphology was observed and photographed using Olympus IX81 microscope equipped with a light fluorescence device (Olympus, Japan).
Suspension cultures to generate spheroids were performed as described in Dontu et al
Nude mice (Balb/c Slc
For transplantation studies, cells (shown in
For micrometastases studies, 1×105 of miPS-LLCcm spheroid cells were suspended in 100 µl DMEM containing 10% FCS and injected into nude mouse tail vein (n = 6).
Tumors were fixed for 24 hours and then processed using a routine wax-embedding procedure for histologic examination. Three micrometer thick sections were stained with hematoxylin and eosin (HE).
IHC for GFP, pan-Cytokeratin, Vimentin, α-Actin, CD31, NF-M, GFAP was performed using formalin-fixed paraffin embedded tissue sections and standard procedures. Briefly, 3 µm tissue sections were deparaffinized and antigen retrieved was performed using microwave exposure at 95°C for 5 minutes in a citrate buffer (pH 6.0) or incubation in proteinase K (40 µg/ml) at 37°C for 30 minutes. After hydrogen peroxide blocking and normal serum blocking (when using mouse monoclonal primary antibody, M.O.M Mouse Ig Blocking Reagent (Vector, USA) as a blocking buffer), the sections were then incubated for 2 h at 37°C with the following primary antibodies: rabbit polyclonal anti-GFP (1∶300, kindly provided by Ayano Satoh, Okayama University, Japan), mouse monoclonal anti-pan-Cytokeratin (AE1/AE3) (1∶200, Santa Cruz, USA), mouse monoclonal anti-Vimentin (1∶200, Santa Cruz, USA), mouse monoclonal anti-α-Actin (1∶200, Santa Cruz, USA), rat monoclonal anti-CD31 (1∶200, Santa Cruz, USA), mouse monoclonal anti-NF-M (1∶50, Santa Cruz, USA), and mouse monoclonal anti-GFAP (1∶200, Santa Cruz, USA). The sections were then incubated with biotinylated anti-rabbit, biotinylated anti-rat or biotinylated anti-mouse secondary antibody (Vector, USA), followed by incubation with the ABC reagent (Vector, USA). Detection was accomplished using 3, 30-diaminobenzidine tetrahydrochloride (DAB, Vector, USA). Incubation of sections with phosphate-buffered saline (PBS) served as negative controls. Counter staining were carried out using hematoxylin.
The spheroids were fixed in 10% neutral formalin buffer solution (Wako, Japan) for 1 hour and washed in PBS. After centrifugation at 500 rpm during 3 min, the supernatant was removed carefully with a pipette, and then the spheroids were counter-stained with hematoxylin during 30 s. After wash in PBS, spheroids were collected by centrifugation and embedded in OCT compound, and then 6 µm thich cryosections were cut. Cryosections were fixed with 10% neutral formalin buffer solution for 15 min at room temperature, and then incubated with block solution containing 5% BSA, 0.1% Triton X-100 in 0.01 M PBS at room temperature for 1 hour. Sections were then incubated with Rabbit anti-Nanog (1∶100, Abcam, Japan) or mouse anti-Oct-3/4 (1∶100, Santa Cruz, USA) in blocking solution overnight at 4°C. After three washes in PBS, sections were incubated with Goat anti-Rabbit secondary antibodies conjugated to Alexa fluorophores 555 or Goat anti-mouse secondary antibodies conjugated to Alexa fluorophores 488 (1∶400, Invitrogen, USA) for 30 min at room temperature. After three washes in PBS, sections were mounted with Vectashield (mounting medium for fluorescence with DAPI, Vector, USA). Images were acquired using an Olympus IX81 microscope equipped with a light fluorescence device (Olympus, Japan). Sections where the primary antibodies were PBS served as negative controls.
Total RNA from cell lines and tumor tissues were isolated by using RNeasy Mini Kit (QIAGEN, Germany) and TRIzol (Invitrogen, USA), respectively. One µg of total RNA was then reverse transcribed using SuperScript® II Reverse Transcriptase kit (Invitrogen, USA). RT-PCR was performed for 40 cycles for all markers, except
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