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Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

  • Ting-Ting Gu
    Affiliations
    Department of Reproductive Medicine, The First Affiliated Hospital of the Medical College, Xi'an Jiaotong University, Xi'an; and the Section of Cancer Stem Cell Research, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of The People's Republic of China, Xi'an, China
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  • Shu-Yan Liu
    Affiliations
    Department of Reproductive Medicine, The First Affiliated Hospital of the Medical College, Xi'an Jiaotong University, Xi'an; and the Section of Cancer Stem Cell Research, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of The People's Republic of China, Xi'an, China
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  • Peng-Sheng Zheng
    Correspondence
    Address reprint requests to Peng-Sheng Zheng, M.D., Ph.D., Department of Reproductive Medicine, The First Affiliated Hospital of the Medical College, Xi'an Jiaotong University, Xi'an 710061, China
    Affiliations
    Department of Reproductive Medicine, The First Affiliated Hospital of the Medical College, Xi'an Jiaotong University, Xi'an; and the Section of Cancer Stem Cell Research, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of The People's Republic of China, Xi'an, China
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      Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer–derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.
      NANOG is a divergent homeodomain transcription factor in embryonic stem cells that is independent of leukemia inhibitory factor. NANOG, SOX2, and OCT4 form a core of proteins that are responsible for self-renewal and differentiation in embryonic stem cells.
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      The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.
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      Nanog safeguards pluripotency and mediates germline development.
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      Core transcriptional regulatory circuitry in human embryonic stem cells.
      Human NANOG, mapping to 12p13.31 and encoding a 305–amino acid protein, has 11 pseudogenes [namely, one tandem duplicate (NANOGP1) and 10 processed pseudogenes]. NANOGP1 has been transcribed in human hematopoietic stem cells and differentiated leukemic cells,
      • Eberle I.
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      Transcriptional properties of human NANOG1 and NANOG2 in acute leukemic cells.
      but it was reported to be nonfunctional.
      • Booth H.A.
      • Holland P.W.
      Eleven daughters of NANOG.
      Nine of the processed pseudogenes were not translated into complete proteins because of insertions, deletions, reading frame shifts, premature stop codons, and other chromosomal alterations. Only NANOGP8 (NG_004093.2), whose protein product differs in three amino acids from that of NANOG (NM_024865.2) and maps to 15q14, has expressed the functional protein in prostate, breast, and colon cancer cells.
      • Zhang J.
      • Wang X.
      • Li M.
      • Han J.
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      NANOGP8 is a retrogene expressed in cancers.
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      • Tang D.G.
      Functional evidence that the self-renewal gene NANOG regulates human tumor development.
      In addition, according to GenBank, there are two NANOGP8-like genes that are translated into functional proteins. They are NANOGP8-like-1 (NANOGP8L1) (XM_002344635.2) and NANOGP8-like-2 (NANOGP8L2) (XM_002344636.1), the former differing in one amino acid from NANOG and the later having 16 amino acids less in the middle of the gene. Therefore, NANOG, NANOGP8, NANOGP8L1, and NANOGP8L2 can potentially yield functional NANOG proteins.
      Although it was originally believed that NANOG is only expressed in pluripotent cells, more recently, NANOG protein and its pseudogene forms have also been shown to play roles in a variety of cancer cell lines and tumors, including pancreatic carcinoma, breast cancer, ovarian cancer, and endometrial carcinoma.
      • Ezeh U.I.
      • Turek P.J.
      • Reijo R.A.
      • Clark A.T.
      Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma.
      • Wen J.
      • Park J.Y.
      • Park K.H.
      • Chung H.W.
      • Bang S.
      • Park S.W.
      • Song S.Y.
      Oct4 and Nanog expression is associated with early stages of pancreatic carcinogenesis.
      • Zhang S.
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      Identification and characterization of ovarian cancer-initiating cells from primary human tumors.
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      • Liu Y.
      • Wang W.
      • Wen Y.
      • Gu J.
      Hepatitis B virus X protein blunts senescence-like growth arrest of human hepatocellular carcinoma by reducing Notch1 cleavage.
      Moreover, further research revealed that NANOG expression relates to the diagnosis, prognosis, and chemoresistance of lung, gastric, colorectal, and ovarian cancers.
      • Nirasawa S.
      • Kobayashi D.
      • Tsuji N.
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      Diagnostic relevance of overexpressed Nanog gene in early lung cancers.
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      • Zhou J.
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      Overexpression of nanog predicts tumor progression and poor prognosis in colorectal cancer.
      • Lin T.
      • Ding Y.Q.
      • Li J.M.
      Overexpression of Nanog protein is associated with poor prognosis in gastric adenocarcinoma.
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      • McArthur C.
      • Jaffe R.B.
      Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant.
      However, the role of NANOG and its pseudogenes in cervical cancer is still unclear.
      Our study demonstrates that NANOG is frequently expressed in the cytoplasm, instead of the nucleus, in cervical cancer and its stromal distribution is related to cancer progression. As a stromal component, mesenchymal stem cells (MSCs) are a population of pluripotent cells that can differentiate into osteogenic, chondrogenic, and adipogenic cells. There is strong evidence that MSCs are recruited to the tumor microenvironment to initiate the formation of the tumor. Based on this finding, numerous studies have suggested that MSCs could potentially be used as therapeutic delivery vehicles that target the tumor.
      • Liu J.
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      • Hong D.
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      • Chen H.Z.
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      Hes1/Hes5 gene inhibits differentiation via down-regulating Hash1 and promotes proliferation in cervical carcinoma cells.
      However, the function of MSCs in cancer remains controversial.
      • Barrilleaux B.
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      Transduction of human cells with polymer-complexed ecotropic lentivirus for enhanced biosafety.
      Several studies have suggested that MSCs restrict cancer growth, whereas others indicate that MSCs promote tumorigenesis.
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      FOXO1 is an essential regulator of pluripotency in human embryonic stem cells.
      • Tang S.N.
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      Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.
      • Cox J.L.
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      Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells.
      • Yang M.
      • Yan M.
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      • Li J.
      • Luo Z.
      Side population cells isolated from human osteosarcoma are enriched with tumor-initiating cells.
      • Pirozzi G.
      • Tirino V.
      • Camerlingo R.
      • Franco R.
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      • Liguori E.
      • Martucci N.
      • Paino F.
      • Normanno N.
      • Rocco G.
      Epithelial to mesenchymal transition by TGFbeta-1 induction increases stemness characteristics in primary non small cell lung cancer cell line.
      In this study, we located the expression of NANOG and NANOG-like proteins in cervical cancer cells and confirmed the relationship between NANOG-positive cells in the stroma and MSCs to assess its association with tumorigenesis and tumor progression. We show, for the first time to our knowledge, that MSCs are one type of cytoplasmic NANOG-positive cells in the cervical cancer stroma and that they promote the progression of cervical cancer in vitro and in vivo. Our findings contribute to understanding the role of the tumor stroma and MSCs in cervical cancer development.

      Materials and Methods

      Cell Lines and Cell Culture

      Human cervical carcinoma cell lines (HeLa, SiHa, and C-33 A) and the human embryonic carcinoma cell line (Tera-1) were obtained from ATCC (Manassas, VA). HeLa, SiHa, and C-33 A cells were cultured in Dulbecco's modified Eagle's medium (DMEM-HIGH Glucose; Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. Tera-1 cells, a human embryonic carcinoma cell line serving as a positive control, was maintained in McCoy's 5A Medium (Sigma-Aldrich) containing 10% FBS.

      Immunohistological Staining, Immunocytochemistry, and Immunofluorescence

      A total of 95 samples [27 normal cervical epithelia (NC), 21 cancer in situ (CIS), and 47 squamous cervical cancer (SCC)] without chemotherapy, immunotherapy, or radiotherapy were obtained by surgery at the First Affiliated Hospital of Xi'an Jiaotong University Medical College (Xi'an, China). The procedures followed medical ethics approval practices, and the patients provided their informed consent before specimen collection. For immunohistochemistry (IHC), procedures were performed as previously described.
      • Zhang Y.
      • Li B.
      • Ji Z.Z.
      • Zheng P.S.
      Notch1 regulates the growth of human colon cancers.
      In brief, 10% formalin-fixed, paraffin-embedded tissue sections were deparaffinized and hydrated. An endogenous antigen retrieval procedure was performed with citric acid buffer (10 mmol/L citrate buffer, pH 6.0). Slides were incubated with primary antibodies overnight at 4°C and secondary antibodies for 30 minutes at room temperature, followed by diaminobenzidine development.
      The staining intensity was scored on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; or 3, strong. The fraction of positive cells, defined as the relative positive-staining area, was scored on a scale of 0 to 4: 0, 0% to 5%; 1, 6% to 25%; 2, 26% to 50%; 3, 51% to 75%; and 4, 76% to 100%. The immunoreactive score (IRS) was calculated by multiplying the staining intensity score by the fraction of positive cells.
      • Remmele W.
      • Stegner H.E.
      Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue [in German].
      IRS values are defined as follows: 0, NANOG negative, and between 1 and 12 (1 ≤ IRS ≤ 12), NANOG positive. Specimens were scored by two investigators (T.-T.G. and S.-Y.L.) and evaluated by statistical analysis.
      For immunocytochemistry and immunofluorescence, cells were plated on coverslips and fixed with 4% paraformaldehyde for 30 minutes at room temperature, followed by 0.25% Triton X-100 permeabilization. Immunostained cells were analyzed for NANOG, colocalization of CD105 and NANOG, and CD44 and NANOG using an Olympus-CX31 microscope (Olympus, Tokyo, Japan) or a Leica TCS SP5 confocal microscope (Leica, Solms, Germany). Images were captured with an Olympus-CX31 microscope digital camera and a Leica DFC 500 digital camera and processed with LAS AF software version 2.0 (Leica). Antibodies against human NANOG (sc-30331), CD34 (sc-19621), matrix metalloproteinase (MMP) 2 (sc-10736), MMP9 (sc-21733), SNAI 1 (sc-28199), TWIST (sc-15393), and plakoglobin (sc-7900) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The other two anti-NANOG antibodies were obtained from Epitomics (3369-1; Burlingame, CA) and Cell Signaling (4903; Danvers, MA). Anti-mouse Alexa Fluor 488 and anti-goat Alexa Fluor 555 were obtained from Invitrogen, and DAPI was obtained from Sigma-Aldrich (St. Louis, MO).

      Cytoplasmic Protein and Nuclear Protein Extraction and Western Blot Analysis

      Cells were collected and washed twice with PBS. For the cytoplasmic fraction, cell pellets were suspended with buffer A (10 mmol/L HEPES, pH 7.9, and 0.1 mmol/L EDTA) supplemented with protease inhibitors (Complete Mini; Roche Diagnostics, Pleasanton, CA) and incubated on ice for 5 minutes. Supernatants were collected by centrifuging at 720 × g for 5 minutes. For the extraction of nuclear protein, pellets were resuspended and lysed with buffer C (20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1% Triton X-100, 0.2 mmol/L EDTA, 20% glycerol) supplemented with protease inhibitors and incubated on ice for 30 minutes. The lysates were centrifuged at 10,000 × g for 20 minutes and adjusted to a final concentration of 100 mmol/L NaCl. All proteins were equaled by bicinchoninic acid assay and separated on SDS-PAGE, followed by wet transfer. After antibody incubation and development, proteins of interest were visualized on film.
      Anti-Bmi1 (05-637) antibody was obtained from Millipore (Billerica, MA). Anti-NANOG antibodies (sc-30331) and anti-actin antibody (sc-47778) were obtained from Santa Cruz Biotechnology. Secondary antibodies were obtained from Pierce (Rockford, IL).

      RT-PCR and Restriction Enzyme Digestion

      Total RNA was extracted from cells and fresh tissues using TRIzol (Invitrogen). Reverse transcription was performed as follows: 500 ng of total RNA from each sample was reverse transcribed with oligo(dT) primers from the PrimeScript RT Reagent Kit (Perfect Real Time; TaKaRa, Osaka, Japan), according to the manufacturer's recommendations. The generated cDNA (1 μL) was used as a template for PCR. Five sets of primers were designed as follows: F1/R1 (forward, 5′-AGGCAACTCACTTTATCC-3′; and reverse, 5′-CCACAAATCACAGGCATA-3′), F2/R2 (forward, 5′-TGAAATCTAAGAGGTGGCA-3′; and reverse, 5′-CTGGATGTTCTGGGTCTG-3′), F3/R3 (forward, 5′-GGACAACATTGATAGAAGC-3′; and reverse, 5′-TTACTCATCGAAACACTCG-3′), F4/R4 (forward, 5′-TATGGTTGGAGCCTAATCAGCG-3′; and reverse, 5′-ACTTATCTATAGCCAGAGACGGCAG-3′), and β-actin (forward, 5′-ATCGTCACCAACTGGGACGA-3′; and reverse, 5′-TCCATCACGATGCCAGTGGT-3′). All amplifications were performed as follows: predenaturing at 94°C for 3 minutes, denaturing at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, followed by an additional 10 minutes of extension. The products of the primer F4/R4 set were purified and digested by the restriction enzyme SmaI (TaKaRa) overnight. PCR amplification and digestion products were separated on a 2.5% agarose gel and observed with Transilluminator (JY04S; Junyi, Beijing, China). Each sample was processed twice to confirm consistency of processing.

      Isolation and Culture of Cervical Cancer–Derived MSCs

      The MSCs were isolated from stage II cervical cancer. Fresh tumor samples were obtained from the operating room and immediately taken to the laboratory for processing. Tissue was maintained in DMEM containing 10% FBS. A portion of the tumors was processed into single-cell suspensions, as previously described.
      • Yang V.S.
      • Carter S.A.
      • Hyland S.J.
      • Tachibana-Konwalski K.
      • Laskey R.A.
      • Gonzalez M.A.
      Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog.
      Briefly, tissues were washed with basal growth medium containing 10× penicillin-streptomycin (Sigma-Aldrich), followed by mincing and digestion with collagenase IV (GIBCO, Grand Island, NY) for 4 hours at 37°C before culturing in MesenPRO RS Medium (GIBCO). Once adherent, cells reached approximately 60% confluence, cells were digested with 0.25% trypsin, followed by centrifugation at 300 × g for 5 minutes, and cells were replated in a new dish for culture expansion. Cervical cancer–derived MSCs of <10 passages were used in the subsequent experiments.

      Flow Cytometric Analysis

      The following antibodies were used for flow cytometric analysis of putative MSCs: anti-CD44-fluorescein isothiocyanate (BD Pharmingen), anti-c-Met-fluorescein isothiocyanate (eBioscience, San Diego, CA), anti-hCD90-phycoerythrin (PE) (eBioscience), anti-CD105-PE (eBioscience), anti-CD117-PE (eBioscience), anti-CD31-PE (BD Pharmingen), anti-CD34-PE (BD Pharmingen), and anti-CD45-PE (BD Pharmingen). The MSCs of passages 2 to 4 were harvested with 0.25% trypsin, stained in PBS containing 2% FBS, and incubated with either isotype control or anti-human monoclonal antibodies conjugated with fluorescein isothiocyanate or PE for 30 minutes at 4°C. After washing, cells were analyzed with an FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ).

      In Vitro Differentiation

      To induce osteogenic differentiation, second- to fourth-passage cells were plated in DMEM (low-glucose) medium supplemented with 15% FBS and 1 ng/mL basic fibroblast growth factor. After confluence had reached 80%, cells were treated with osteogenic medium for 3 weeks, which consists of DMEM (low glucose) supplemented with 10−8 mol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μg/mL ascorbic acid (all from Sigma-Aldrich). Medium changes were performed twice weekly. Osteogenesis was detected by histochemical staining of alkaline phosphatase (ALP) activity using Gomori staining, as previously described.
      • Gomori G.
      The complex nature of alkaline phosphatase.
      To induce adipogenic differentiation, second- to fourth-passage cells were treated with adipogenic medium for 3 weeks, which consists of DMEM (high glucose) supplemented with 10% FBS, 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 5 μg/mL insulin, and 0.2 mmol/L indomethacin (all from Sigma-Aldrich). Medium changes were performed twice weekly. Adipogenesis was assessed after 2 weeks by oil red staining.

      Colony Formation Assays and Xenograft Models in Nude Mice

      A colony formation assay was performed as previously described.
      • Digirolamo C.M.
      • Stokes D.
      • Colter D.
      • Phinney D.G.
      • Class R.
      • Prockop D.J.
      Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.
      Briefly, 200 SiHa cells and/or third- to fifth-passage cervical cancer–derived MSCs were seeded in a 100-mm dish, together or individually in the medium. After 2 weeks of culture, medium was removed and cells were fixed and stained with Giemsa (Sigma-Aldrich). The dishes were washed twice and dried, and the numbers of colonies with diameters >2 mm were scored. Each experiment was performed in triplicate. Results are presented as number of colonies and analyzed by t-test.
      For in vivo assays, nude female mice between 4 and 6 weeks old were obtained from the animal breeding facility (Jackson Laboratories, Inc., Bar Harbor, ME). Animals were maintained in accordance with institutional policies, and all studies were performed with approval of the University Committee on Use and Care of Animals of Xi'an Jiaotong University. Cervical cancer–derived MSCs were tested for the ability to support SiHa cervical cancer cells to form tumors. To generate tumors, 1.8 × 106 SiHa cells alone or mixed with 5 × 105 third- to eighth-passage cervical cancer–derived MSCs, with or without 10% Matrigel (GIBCO) in 100 μL of PBS, were s.c. injected into the dorsum of the mice. Three mice were used for each group, and the experiment was repeated with another five mice. Tumors were measured weekly using a Vernier caliper, and size was calculated with the following formula: (length × width2)/2. Xenograft tumor volumes were analyzed by analysis of variance, and tumor weight was analyzed by t-test. The tumor masses were removed and paraffin embedded after sacrifice for future immunochemistry staining.

      Statistical Analysis

      Statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL). For two groups, a Student's t-test was used to determine statistical significance. To examine the difference among three groups, analysis of variance was performed. In all of the tests, P < 0.05 was defined as statistically significant.

      Results

      Stromal NANOG Expression Is Associated with the Progression of Cervical Cancer

      One of the stem cell markers, NANOG, was mainly located in the nucleus of human embryonic carcinoma cell line Tera-1 (Figure 1A). Herein, we detected the expression of NANOG in cervical cancer cell lines by immunocytochemical staining. The results showed that NANOG was expressed in the cytoplasm, rather than the nucleus, in three cervical cancer cell lines (HeLa, SiHa, and C-33 A; Figure 1A). To exclude potential nonspecificity of NANOG antibody sc-30331, we introduced two other NANOG antibodies from different companies. The three anti-NANOG antibodies demonstrated a similar staining pattern in cell lines (ie, NANOG was mainly located in the nucleus in human embryonic carcinoma cell line Tera-1, but in the cytoplasm in human cervical cancer cell lines HeLa, SiHa, and C-33 A; see Supplemental Figure S1A at http://ajp.amjpathol.org). For further confirmation, we extracted the protein from the cytoplasm and nucleus to perform Western blot analysis. All three antibodies showed NANOG existed both in the cytoplasm and the nucleus of Tera-1, but only in the cytoplasm in SiHa (see Supplemental Figure S1B at http://ajp.amjpathol.org). All these results implied that the three antibodies were effective, but also suggested that the location of NANOG was dependent on cell type. Moreover, in clinical sample experiments, 19 NC, 9 cervical CIS, and 35 SCC samples were used for staining with different anti-NANOG antibodies. The antibodies from Epitomics and Cell Signaling were biased to epithelia and tumor cell staining, whereas the antibody from Santa Cruz Biotechnology showed more staining of the stroma; all of them exhibited positive stromal cell staining (see Supplemental Figure S1C at http://ajp.amjpathol.org). In detail, for epithelia and cancer cell staining, NANOG demonstrated strong and universal staining for both Epitomics and Cell Signaling antibodies, but the cellular location of the staining was in the nucleus in normal epithelial cells and in the cytoplasm in tumor cells (see Supplemental Figure S1, C and D, at http://ajp.amjpathol.org). The average IRS values of epithelia/cancer nests were slightly higher for cancer tissues compared with the NC, but this difference was not statistically significant (Santa Cruz Biotechnology, P = 0.4376; Epitomics, P = 0.2404; see Supplemental Figure S1, E and H). However, for stroma staining, both Epitomics and Santa Cruz Biotechnology antibodies demonstrated a similar tendency (ie, the stromal cytoplasmic NANOG distribution gradually increased from NC via CIS to fully developed cancer, and this difference was statistically significant for antibody bought from Santa Cruz Biotechnology: Santa Cruz Biotechnology, P = 0.0005; Epitomics, P = 0.0682; see Supplemental Figure S1, F–I, at http://ajp.amjpathol.org). Thus, we decided to focus on the study of cervical cancer stroma.
      Figure thumbnail gr1
      Figure 1NANOG expression in cervical cancer cell lines and cervical tissue. A: Immunocytochemistry (ICC) staining shows that distinct brown colorations are observed in the cytoplasm of HeLa, SiHa, and C-33 A cells. Original magnification, ×1000. The human embryonic carcinoma cell line Tera-1 serves as a positive control. B: IHC shows the NANOG protein in NC, CIS, and SCC stromal and SCC tumor nests in cervical tissues. Original magnification, ×1000. The boxed areas demonstrate the typical cells and areas under ×5 further amplification. C: Bar chart showing percentages of stromal NANOG-positive cases. D: The average IRS shows significant differences between NC, CIS, and SCC (*P < 0.05, **P < 0.01.).
      For further confirmation, we used 95 clinical samples for NANOG stroma staining by Santa Cruz Biotechnology antibody, which included 27 NC, 21 CIS, and 47 SCC samples. The percentages of cases with positive stroma staining were 25.9% (7/27) in NC, 61.9% (13/21) in CIS, and 93.6% (44/47) in SCC (P < 0.0001; Figure 1, B and C). The average IRS values of stromal NANOG staining were as follows: 1.259 ± 0.500 in NC (n = 27), 3.333 ± 0.715 in CIS (n = 21), and 5.191 ± 0.458 in SCC (n = 47) (P < 0.0001, Figure 1D). Taken together, these results demonstrated that the cellular location of NANOG was related to cell type and tumor stage. Furthermore, stromal cytoplasmic NANOG expression was associated with the progression of cervical cancer, suggesting a powerful function of the stromal component during tumor progression.

      NANOG Is Mainly Translated from NANOG in Cervical Cancer Cell Lines and Tissues

      As previously reported, NANOG, NANOGP8, NANOGP8L1, and NANOGP8L2 are four genes that can be translated into proteins resembling NANOG. To determine which NANOG gene is the transcript for NANOG in cervical cancer cell lines and tissues, we designed four sets of primers to distinguish NANOG transcripts by RT-PCR (Figure 2A). In detail, F1/R1 amplified a 235-bp fragment from all four transcripts, whereas F2/R2 amplified a 200-bp PCR fragment from NANOG, NANOGP8, and NANOGP8L1 and a 152-bp PCR fragment from NANOGP8L2. F3/R3 was specific to amplify a 410-bp PCR fragment from NANOGP8L1 based on its extra sequence. A 420-bp PCR fragment was amplified by F4/R4 from both NANOG and NANOGP8, which could be digested into 260- and 160-bp fragments, respectively, by SmaI in NANOG but not in NANOGP8 because of a C-to-G conversion.
      Figure thumbnail gr2
      Figure 2NANOG transcriptions in cervical cancer cell lines and cervical cancer tissues. A: Schematic of NANOG, NANOGP8, NANOGP8L1, and NANOGP8L2 gene structures. Regions of homology are indicated, as are all primer locations used for isotype-specific analysis. The SmaI restriction site (red line) is lost in NANOGP8, but not in NANOG. Sequences are not to scale. B: Expression of NANOG transcripts in cervical cancer cell lines (left section) and tissues (right section) is detected by different primer sets. β-Actin is used as loading control. NT, no template. C: Restriction enzyme SmaI digestion of F4/R4 products to identify NANOG and NANOGP8 in cervical cancer cell lines and tissues. D, digested; UD, undigested. D: The table shows types of NANOG transcripts in cervical cancer cell lines and tissues. +, presence; −, absence.
      In our experiments, the F1/R1 set amplified a 235-bp NANOG band from Tera-1 and three other cervical cancer cell lines. The F2/R2 set showed that the expression of NANOGP8L2 was absent in any cell line, whereas the F3/R3 set produced NANOGP8L1 from Tera-1 and C-33 A (Figure 2B). The F4/R4 set and SmaI digestion demonstrated that all four cell lines expressed NANOG. Altogether, the RT-PCR results revealed that NANOG protein in four cervical cell lines was mainly transcribed from the NANOG gene. Only in Tera-1 and C-33 A, a small part was transcribed from NANOGP8L1 or NANOGP8 genes (Figure 2, B and C).
      To lend further support to the presupposition that the NANOG gene was the major source of NANOG protein in cervical cancer, we performed RT-PCR on 10 cervical cancer tissues. As shown in Figure 2B, one case of cervical cancer (SCC 6) did not express any NANOG transcripts (IHC confirmed, data not shown), whereas the other nine NANOG-positive cervical cancer tissues all exhibited NANOG transcription. Among them, three cases (SCC 5, 8, and 9) expressed the NANOGP8L1 transcript and seven cases (SCC 1, 4, 5, and 7-10) expressed NANOGP8 (Figure 2, B and C), in addition to NANOG. Comparing the undigested and digested bands, NANOGP8 only absorbed a minute part, so the protein was largely derived from the NANOG gene in cervical cancer tissues. Therefore, the clear conclusion was that the NANOG protein in cervical cancer tissues and cells was predominantly derived from the NANOG transcript. The detailed expression of NANOG and NANOG-like genes is summarized in Figure 2D.

      Verification of Cytoplasmic NANOG-Positive Stromal Cells as MSCs in Cervical Cancer

      Because NANOG was usually expressed in cells with stem traits, we proposed that the cytoplasmic NANOG-positive stromal cells we detected in the cervical cancer stroma were a subpopulation of MSCs. To clarify this hypothesis, we first performed immunofluorescence in the cervical cancer tissue to determine colocalization of NANOG and MSC-specific marker. Fluorescence microscopy demonstrated that NANOG was mainly expressed in the cytoplasm of tumor stromal cells that also expressed CD44 and CD105 on the surface or cytoplasmic face of the cell membrane. This strongly implied that the stromal cytoplasmic NANOG-positive cells were MSCs in human cervical cancer (Figure 3).
      Figure thumbnail gr3
      Figure 3NANOG, CD44, and CD105 expression and colocalization in the cervical cancer stroma. Human cervical cancer tissue exhibit consistent localization of NANOG in the cytoplasm in the cervical cancer stroma, whereas CD44 and CD105 are located at the membranes and in the cytoplasm. Top row: Uniform localization of NANOG in the cytoplasm and of CD44 at the membrane. Bottom row: Cytoplasmic NANOG and membrane/cytoplasmic CD105. NANOG is stained with Alexa Fluor 555 (red), and CD44 and CD105 are stained with Alexa Fluor 488 (green). DAPI is used to show nuclei (blue). The yellow signal in the merged image represents spatial overlap of CD105 and NANOG. Scale bar = 50 μm. Original magnification, ×630 (all images). Arrows indicate typical colocalization of CD44, NANOG, and CD105 in the cervical cancer stroma. The boxed areas demonstrate the typical colocalization (arrows) cells under ×3 further amplification.
      To further confirm our hypothesis, we isolated the MSCs from fresh cervical cancer tissue. The isolated cervical cancer–derived adherent cells were spindle shaped (Figure 4A). We confirmed the mesenchymal phenotype of the cells based on both functional differentiation capacity and surface marker expression. The MSCs were capable of differentiating into multiple tissue types, including bone, cartilage, and adipose tissue.
      • Arima Y.
      • Hayashi N.
      • Hayashi H.
      • Sasaki M.
      • Kai K.
      • Sugihara E.
      • Abe E.
      • Yoshida A.
      • Mikami S.
      • Nakamura S.
      • Saya H.
      Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer.
      We tested the ability of pluripotent differentiation by inducing differentiation to osteogenic and adipose cells (Figure 4, B and C, respectively). Osteogenic differentiation of mesenchymal cells produced ALP-positive aggregates, which stained black after 3 weeks' induction in osteogenesis medium (Figure 4B). For adipogenic differentiation, cells staining positively for oil red were obtained after 3 weeks' induction in adipogenesis medium (Figure 4C).
      Figure thumbnail gr4
      Figure 4Human cervical cancer–derived MSCs are isolated successfully and co-express NANOG, CD44, and CD105. A: Human cervical cancer–derived MSCs (hMSCs) demonstrate fibroblast-like morphological characteristics. Original magnification, ×200. B: Osteoblast formation is induced by culturing hMSCs in osteogenesis medium, and ALP is detected by Gomori's assay. The ALP-rich osteoblasts show as strong black plaques. Original magnification, ×400. C: Adipocytes are induced by culturing hMSCs in adiopogenesis medium and identified with oil red O staining. Original magnification, ×400. D: Flow cytometric analysis of surface marker expression in cervical cancer–derived mesenchymal cells. Staining for CD44, CD90, and CD105 is positive, whereas staining for the hematopoietic markers CD31, CD34, and CD45 is negative, suggesting that the cervical cancer–derived MSCs were successfully isolated. E: Confocal microscopy shows that NANOG and CD44 colocalize in cervical cancer–derived MSCs, in which CD44 and CD105 expression appeared as green on the membrane and in the cytoplasm and NANOG appeared as red in the cytoplasm. Original magnification, ×630. F: RT-PCR shows NANOG transcription in cervical cancer–derived MSCs. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
      Flow cytometry confirmed a phenotype consistent with the published cell surface molecule expression profile for MSCs.
      • Mathieu J.
      • Zhang Z.
      • Zhou W.
      • Wang A.J.
      • Heddleston J.M.
      • Pinna C.M.
      • Hubaud A.
      • Stadler B.
      • Choi M.
      • Bar M.
      • Tewari M.
      • Liu A.
      • Vessella R.
      • Rostomily R.
      • Born D.
      • Horwitz M.
      • Ware C.
      • Blau C.A.
      • Cleary M.A.
      • Rich J.N.
      • Ruohola-Baker H.
      HIF induces human embryonic stem cell markers in cancer cells.
      The MSCs lacked expression of hematopoietic lineage markers (CD34 and CD45) and of endothelial marker (CD31), but expressed CD105 (SH2), CD90 (Thy-1), and CD44 (Figure 4D). Taken together, this evidence further verified that we had successfully isolated cervical cancer–derived MSCs. Most important, we reconfirmed that NANOG and CD44 or CD105 were colocalized in the cervical cancer–derived MSCs using confocal microscopy (Figure 4E). From the RT-PCR results, we concluded that NANOG and NANOGP8L1 genes were the sources of NANOG transcription in cervical cancer–derived MSCs (Figure 4F). In conclusion, cervical cancer–derived MSCs expressed cytoplasmic NANOG, and they were one type of cytoplasmic NANOG-positive cells in the cervical cancer stroma.

      Cervical Cancer–Derived MSCs Promote Tumor Progression in Vitro and in Vivo

      The MSCs either promoted or suppressed cancer growth, depending on the tumor type and the experimental conditions.
      • Shi C.J.
      • Gao J.
      • Wang M.
      • Wang X.
      • Tian R.
      • Zhu F.
      • Shen M.
      • Qin R.Y.
      CD133(+) gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity.
      • Fareh M.
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      • Virolle V.
      • Debruyne D.
      • Almairac F.
      • de-la-Forest Divonne S.
      • Paquis P.
      • Preynat-Seauve O.
      • Krause K.H.
      • Chneiweiss H.
      • Virolle T.
      The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.
      • Looijenga L.H.
      • Gillis A.J.
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      Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance.
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      Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function.
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      Correlation between cancer stem cells and circulating tumor cells and their value.
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      Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model.
      We next sought to analyze the impact of cervical cancer–derived NANOG-positive MSCs on tumor growth, including in vitro and in vivo assays. For the in vitro study, we performed a clonogenic experiment. SiHa, MSCs, and SiHa + MSCs were plated separately. After 14 days of culture, the SiHa + MSC group showed the formation of tight holoclones, whereas the SiHa group generally formed middle-sized clones and the MSC group did not form standard clones. With respect to cloning numbers, the SiHa + MSC group showed more clones than the SiHa group (P = 0.028, Figure 5A), suggesting that cervical cancer–derived MSCs facilitated cell proliferation and colony formation in vitro.
      Figure thumbnail gr5
      Figure 5Human cervical cancer–derived MSCs promote tumor progression in vitro and in vivo. A: Colony formation of SiHa and SiHa + MSCs cells. Data represent mean ± SEM. *P < 0.05. B: Tumor formation (top panel) and tumor growth curves (bottom panel) of SiHa (blue), MSCs (green), and SiHa + MSCs (red), with or without Matrigel, in female BALB/c nude mice. Data represent mean ± SEM tumor volume. *P < 0.05. C: IHC staining of mouse xenografts, followed by image capturing. Original magnification, ×1000.
      Furthermore, for the in vivo study, we assessed the growth of 1.8 × 106 SiHa cells injected s.c. into nude mice (n = 3) that were or were not combined with 5 × 105 MSCs (n = 3) in two independent experiments, which did or did not contain Matrigel. We observed that the MSC group alone did not form any tumors, regardless of Matrigel's presence. On the other hand, a high tumor incidence was found in the SiHa + MSC group compared with the group of only SiHa cells (P < 0.05, Figure 5B). This result was repeatable in an additional experiment with five mice, and the weight of the tumors was higher in the SiHa + MSC group (P = 0.0049; see Supplemental Figure S2, A–D, at http://ajp.amjpathol.org). The tumor nodules were first formed approximately 14 days after s.c. injection in the group that was treated with Matrigel, but they first appeared approximately 30 days in the group without Matrigel incorporation (Figure 5B), suggesting that Matrigel could accelerate the speed of tumor formation and increase the size of the tumor. This finding strongly implied that the tumor environment, including the stromal cells and the matrix, was important for tumor formation. The study by Fareh et al
      • Fareh M.
      • Turchi L.
      • Virolle V.
      • Debruyne D.
      • Almairac F.
      • de-la-Forest Divonne S.
      • Paquis P.
      • Preynat-Seauve O.
      • Krause K.H.
      • Chneiweiss H.
      • Virolle T.
      The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.
      also demonstrated that carcinoma-associated MSCs promoted tumor proliferation by increasing the number of cancer stem cells, but not MSCs.
      The IHC analysis of these tumors was performed to determine the potential mechanism for the increased tumor growth seen in NANOG-positive MSCs compared with that of controls. The IHC analysis of vascular CD34 expression demonstrated that there was a slight increased microvascular density of tumors with MSCs, compared with tumors without MSCs. Matrix factors (MMP2 and MMP9) and epithelial-mesenchymal transition (EMT)–associated markers (TWIST, SNAI1, and plakoglobin) were also positive in the SiHa + MSC group, but not in the SiHa group (Figure 5C). Taken together, our results demonstrated that cervical cancer–derived NANOG-positive MSCs promoted tumor formation in vitro and in vivo; this may be related to angiogenesis and EMT mechanisms.

      Discussion

      In this study, we demonstrate that the cellular location of NANOG is related to cell type and tumor stage, and that stromal cytoplasmic NANOG expression is associated with the progression of cervical cancer. In addition, we reveal that MSCs are one of the sources of NANOG-positive stromal cells in cervical cancer and have the potential to promote tumorigenesis and tumor progression in vitro and in vivo.
      The expression of NANOG has been observed in various human tumors, such as breast cancer, prostate cancer, embryonic carcinoma, metastatic germ cell tumor, lung cancer, colon cancer, and ovarian cancer.
      • Zhang S.
      • Balch C.
      • Chan M.W.
      • Lai H.C.
      • Matei D.
      • Schilder J.M.
      • Yan P.S.
      • Huang T.H.
      • Nephew K.P.
      Identification and characterization of ovarian cancer-initiating cells from primary human tumors.
      • Nirasawa S.
      • Kobayashi D.
      • Tsuji N.
      • Kuribayashi K.
      • Watanabe N.
      Diagnostic relevance of overexpressed Nanog gene in early lung cancers.
      • Gu G.
      • Yuan J.
      • Wills M.
      • Kasper S.
      Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo.
      • Latifi A.
      • Abubaker K.
      • Castrechini N.
      • Ward A.C.
      • Liongue C.
      • Dobill F.
      • Kumar J.
      • Thompson E.W.
      • Quinn M.A.
      • Findlay J.K.
      • Ahmed N.
      Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile.
      • Mimeault M.
      • Batra S.K.
      Frequent gene products and molecular pathways altered in prostate cancer- and metastasis-initiating cells and their progenies and novel promising multitargeted therapies.
      • Walter D.
      • Satheesha S.
      • Albrecht P.
      • Bornhauser B.C.
      • D'Alessandro V.
      • Oesch S.M.
      • Rehrauer H.
      • Leuschner I.
      • Koscielniak E.
      • Gengler C.
      • Moch H.
      • Bernasconi M.
      • Niggli F.K.
      • Schafer B.W.
      CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres.
      Elevated expression of NANOG was positively associated with late-stage progression and poorer prognosis for patients with oral cancer.
      • Chiou S.H.
      • Yu C.C.
      • Huang C.Y.
      • Lin S.C.
      • Liu C.J.
      • Tsai T.H.
      • Chou S.H.
      • Chien C.S.
      • Ku H.H.
      • Lo J.F.
      Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma.
      From IHC staining of 95 samples, we identified that stromal NANOG expression is related to cervical cancer progression, which depends more on the frequency of stromal NANOG expression, rather than its intensity. Based on our results, stromal NANOG expression could be a sign for the progression of cervical cancer in future clinical studies.
      NANOG and its isoforms derived from NANOG-like genes may function as transcription factors in a cell type–specific manner.
      • Ambady S.
      • Malcuit C.
      • Kashpur O.
      • Kole D.
      • Holmes W.F.
      • Hedblom E.
      • Page R.L.
      • Dominko T.
      Expression of NANOG and NANOGP8 in a variety of undifferentiated and differentiated human cells.
      The study by Zhang et al
      • Zhang J.
      • Wang X.
      • Li M.
      • Han J.
      • Chen B.
      • Wang B.
      • Dai J.
      NANOGP8 is a retrogene expressed in cancers.
      showed that NANOGP8, but not the NANOG transcript, was translated and functioned in tumorigenesis in human osteosarcoma cell line OS732. Although our study shows that seven cervical tissues express NANOGP8 and that Tera-1, C-33 A, two cervical tissues, and cervical cancer–derived MSCs express NANOGP8L1, the transcription levels of these NANOG variants were low compared with the transcription levels of the main NANOG gene. Taken together, our results strongly imply that NANOG transcription plays a governing role in cervical cancer.
      As stem cell marker, NANOG is a homeobox transcription factor essential to the self-renewal of stem cells. However, the role of NANOG signaling in tumorigenesis is still under exploration. NANOG is expressed in different tumor variants. Recently, in vitro cell systems and animal model studies have supplied functional evidence that NANOG is a key factor regulating human tumor development.
      • Barrett J.M.
      • Parham K.A.
      • Pippal J.B.
      • Cockshell M.P.
      • Moretti P.A.
      • Brice S.L.
      • Pitson S.M.
      • Bonder C.S.
      Over-expression of sphingosine kinase-1 enhances a progenitor phenotype in human endothelial cells.
      Meng et al
      • Meng H.M.
      • Zheng P.
      • Wang X.Y.
      • Liu C.
      • Sui H.M.
      • Wu S.J.
      • Zhou J.
      • Ding Y.Q.
      • Li J.M.
      Overexpression of nanog predicts tumor progression and poor prognosis in colorectal cancer.
      demonstrated that NANOG protein was functionally important in regulating tumor proliferation, invasion, and motility and contributed to the EMT process. In our study, we systematically demonstrate that NANOG protein is generally present in cervical tissues and cancer cell lines, which is consistent with the studies by Ezeh et al
      • Ezeh U.I.
      • Turek P.J.
      • Reijo R.A.
      • Clark A.T.
      Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma.
      and Ye et al.
      • Ye F.
      • Zhou C.
      • Cheng Q.
      • Shen J.
      • Chen H.
      Stem-cell-abundant proteins Nanog, Nucleostemin and Musashi1 are highly expressed in malignant cervical epithelial cells.
      However, to our surprise, we noticed expression of NANOG protein in the cytoplasm of stromal cells and increasing stromal distribution during tumor progression. The cellular translocation of NANOG from nucleus to cytoplasm could be related to the molecular characteristics of the human NANOG protein. There are six amino acids in the homeodomain (136YKQVKT141) for NANOG nuclear localization and at least four tryptophan-rich regions (W) for NANOG nuclear export,
      • Chang D.F.
      • Tsai S.C.
      • Wang X.C.
      • Xia P.
      • Senadheera D.
      • Lutzko C.
      Molecular characterization of the human NANOG protein.
      suggesting a possible cellular shuttling-behavior mechanism. In addition, our Western blot analysis results showed different sizes of NANOG protein, depending on the antibody (ie, 42 kDa with the Cell Signaling antibody and 50 kDa with the Santa Cruz Biotechnology and Epitomics antibodies) (data not shown). Taken together, we speculate that protein modification and/or spatial structure changes could be one of the factors involved in cellular translocation and differential distribution of NANOG. The increasing distribution of NANOG-positive stromal cells during tumor progression reinforces the notion that stromal components are involved in tumor progression. The tumor is composed of an expanding population of transformed cells that are supported by a surrounding stromal microenvironment. The tumor stroma consists of fibroblasts, MSCs, and vascular and immune cells, along with the extracellular matrix and extracellular molecules, and is regarded as playing a central role in tumor initiation, progression, metastasis, and resistance.
      • Li H.
      • Fan X.
      • Houghton J.
      Tumor microenvironment: the role of the tumor stroma in cancer.
      • Zigrino P.
      • Loffek S.
      • Mauch C.
      Tumor-stroma interactions: their role in the control of tumor cell invasion.
      • Miyamoto H.
      • Murakami T.
      • Tsuchida K.
      • Sugino H.
      • Miyake H.
      • Tashiro S.
      Tumor-stroma interaction of human pancreatic cancer: acquired resistance to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins.
      As an important part of the extracellular matrix, human MSCs first expressed NANOG in the cytoplasm in the study by Ambady et al.
      • Ambady S.
      • Malcuit C.
      • Kashpur O.
      • Kole D.
      • Holmes W.F.
      • Hedblom E.
      • Page R.L.
      • Dominko T.
      Expression of NANOG and NANOGP8 in a variety of undifferentiated and differentiated human cells.
      However, little is known about solid cancer-associated MSCs. The findings of our study show in detail that MSCs are universally present in human cervical cancer and demonstrate great multipotent differentiation capacity. This is consistent with reports of the presence of many MSCs in ovarian, gastric, and lung cancers.
      • Fareh M.
      • Turchi L.
      • Virolle V.
      • Debruyne D.
      • Almairac F.
      • de-la-Forest Divonne S.
      • Paquis P.
      • Preynat-Seauve O.
      • Krause K.H.
      • Chneiweiss H.
      • Virolle T.
      The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.
      • Oskarsson T.
      • Acharyya S.
      • Zhang X.H.
      • Vanharanta S.
      • Tavazoie S.F.
      • Morris P.G.
      • Downey R.J.
      • Manova-Todorova K.
      • Brogi E.
      • Massague J.
      Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs.
      • Kohsaka S.
      • Sasai K.
      • Takahashi K.
      • Akagi T.
      • Tanino M.
      • Kimura T.
      • Nishihara H.
      • Tanaka S.
      A population of BJ fibroblasts escaped from Ras-induced senescence susceptible to transformation.
      Moreover, we successfully proved that NANOG-positive stromal cervical cancer cells are MSCs. In vitro and in vivo experiments support the view that NANOG-positive cervical cancer–derived MSCs promote tumor proliferation and invasion. Also, the xenograft IHC staining demonstrated angiogenesis and EMT marker expression, suggesting that, in the tumor microenvironment, MSCs are co-opted and recruited by the tumor to promote its growth. Consistent with these results, MSCs have promoted tumor progression or invasion by changing their expression pattern, secreting stimulating factors, and increasing the number of cancer stem cells.
      • Pirozzi G.
      • Tirino V.
      • Camerlingo R.
      • Franco R.
      • La Rocca A.
      • Liguori E.
      • Martucci N.
      • Paino F.
      • Normanno N.
      • Rocco G.
      Epithelial to mesenchymal transition by TGFbeta-1 induction increases stemness characteristics in primary non small cell lung cancer cell line.
      • Shi C.J.
      • Gao J.
      • Wang M.
      • Wang X.
      • Tian R.
      • Zhu F.
      • Shen M.
      • Qin R.Y.
      CD133(+) gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity.
      • Fareh M.
      • Turchi L.
      • Virolle V.
      • Debruyne D.
      • Almairac F.
      • de-la-Forest Divonne S.
      • Paquis P.
      • Preynat-Seauve O.
      • Krause K.H.
      • Chneiweiss H.
      • Virolle T.
      The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network.
      • O'Connor M.D.
      • Wederell E.
      • Robertson G.
      • Delaney A.
      • Morozova O.
      • Poon S.S.
      • Yap D.
      • Fee J.
      • Zhao Y.
      • McDonald H.
      • Zeng T.
      • Hirst M.
      • Marra M.A.
      • Aparicio S.A.
      • Eaves C.J.
      Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance.
      Therefore, we surmise that MSCs are one source of cytoplasmic NANOG-positive cells in the cervical cancer stroma that participate in tumorigenesis and tumor progression.
      To conclude, we showed that the stem cell–associated gene NANOG is a tumorigenesis marker that is found in cervical cancer stroma. Our data show, for the first time to our knowledge, that MSCs are one of the sources of stromal cells that express NANOG in the cytoplasm, highlighting the role of NANOG in carcinogenesis and emphasizing the potential importance of NANOG and NANOG-like genes in tumor progression. Our study provides evidence that NANOG is a cervical cancer progression marker and serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and stromal multifunctionality.

      Supplementary data

      • Supplemental Figure S1

        NANOG expression in cervical cancer cell lines and tissues, visualized using different antibodies. A: Immunocytochemistry staining with three anti-NANOG antibodies. Original magnification, ×1000. B: Western blot analysis with antibody from Santa Cruz Biotechnology. C: IHC shows anti-NANOG antibody staining in NC, CIS, and SCC samples. Original magnification, ×200 and ×1000. D–F: IHC (D) and average IRS values of epithelia (E) and stroma (F) in NC, CIS, and SCC samples for anti-NANOG antibody bought from Epitomics. Original magnification, ×200 and ×1000. G–I: IHC (G) and average IRS values of epithelia (H) and stroma (I) in NC, CIS, and SCC samples for anti-NANOG antibody bought from Santa Cruz Biotechnology. Original magnification, ×200 and ×1000.

      • Supplemental Figure S2

        Human cervical cancer–derived MSCs promote tumor progression in vivo. A: Tumor growth curves of SiHa (blue) and SiHa + MSCs (red) with Matrigel in female BALB/c nude mice. B: Mice with xenografts of SiHa and SiHa + MSCs. C and D: Removed xenografts, with measurement results (C) and photographs (D) shown.

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