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Keywords:

  • Mesenchymal stroma cells;
  • Transformed cell line;
  • Cell interaction;
  • Xenotransplantation;
  • Metastasis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Mesenchymal stromal cells (MSCs) are able to influence the growth abilities of transformed cells. Here, we show that papillary thyroid cancer TPC1 and HEK 293T cells interact physically with human primary bone marrow-derived MSCs followed by evanescence of MSC cytoplasm. Interestingly, transformed cells were able to connect only to apoptotic MSCs that had lost their migration ability, whereas naïve MSCs avoided the direct contact. The interaction stimulated the proliferation of the cocultured transformed cells, activated mitogen and stress signaling, and increased resistance to cytotoxins. Consistent with in vitro data, the MSC interaction stimulated transformed cells had enhanced ability to grow and metastasize in vivo. The parental control cells showed mild tumorigenicity as compared to MSC interaction stimulated cells yielding measurable tumors in 31 days and 7 days, respectively. Our coculture model system describes how adjacent transformed cells absorb stromal cells thereby leading to the stroma-driven evolution of moderately carcinogenic cells to highly aggressive metastatic cells. STEM Cells 2013;31:1218–1223


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Mesenchymal stromal cells (MSCs) have been suggested to induce cellular growth by paracrine signaling, immunomodulation, and tumor-stroma coevolution involving energy metabolite transfer [1–5]. However, it is unclear how metabolic interaction and increased growth are causally linked. Since previous reports have suggested that improved survival and proliferation could be due to a direct contact between cells involving either mitochondrial transfer or cellular fusion [6–8], in this work, we studied the interaction of papillary thyroid cancer TPC1 and HEK 293T cells with MSCs and characterized the consequent impact on growth of the transformed cells in vitro and in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Cell Culture

Primary human MSC [9, 10], papillary thyroid cancer TPC1 [11], and HEK 293T cell culture conditions, growth analyses, and characterizations are described in extended Supporting Information Methods. Microsatellite analysis was done by IdentiCell (Århus University Hospital Skejby, Århus, Denmark).

Animals

HEK 293T (1 × 106), MSC (2 × 105), and MSC 293T (1 × 106, 1 × 104, 1 × 103, 1 × 102) cells were transplanted into BALB c/A nude mice (Taconic, Ejby, Denmark) according to License STH349A.

Statistical Analysis

The p-values were determined by one-way ANOVA with Tukey-Kramer multiple comparison postanalysis test.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

To study the interaction between human primary MSCs and transformed cells, we exposed stromal cells to conditioned serum-free medium collected from TPC1 or HEK 293T cells (Fig. 1A) to induce caspase 3-mediated apoptosis (Fig. 1B) [12]. After the exposure, MSCs were seeded with TPC1 or HEK 293T cells and cultured until the culture was free of stromal cells. TPC1 and HEK 293T cells cocultured with stromal cells are designated “TPC1 MSC” and “MSC 293T,” respectively.

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Figure 1. Interaction of MSCs with TPC1 and HEK 293T cells. (A): In vitro coculture model. (B): Conditioned medium pretreatment of MSCs increased caspase-3-mediated apoptosis. (C): Coculture of green fluorescent protein (GFP) transduced MSCs with unlabeled TPC1 cells resulted in GFP positivity in TPC1 cells (arrows). (D): Time-lapse imaging of conditioned medium treatment of MSCs with HEK 293T cells. Note the multinucleated MSCs surrounded by HEK 293T cells. Note that a large fragment of MSC cytoplasm disappears between 7 and 9 hours (white circles). Note the cell division at 17 hours time point (black arrow). Note the tube-like formations protruding toward MSCs (36-hour image white arrows). Magnification ×20. Abbreviation: MSC, mesenchymal stromal cell.

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Fluorescence microscopy during the coculture of green fluorescent protein (GFP) labeled MSCs with unlabeled TPC1 cancer cells showed temporal arise of GFP-positive TPC1 cells suggesting interaction between the stromal and cancer cells (Fig. 1C). To probe the interaction between MSC and transformed cells, we monitored the MSC-HEK 293T coculture by time-lapse imaging showing that in the untreated culture the cells maintained their normal growth behavior (Supporting Information Film 1), whereas in the treated culture MSCs interacted with transformed cells (Fig. 1D) (Supporting Information Film 2). The pretreatment with conditioned medium allowed a physical connection via membrane protrusions between the two cell populations followed by MSC atrophy and loss of cytoplasm (Fig. 1D, Supporting Information Films 2 and 3).

To characterize the MSC 293T cells generated by MSC-HEK 293T coculture, we analyzed the expression of CD29, CD44, CD73, CD90, and CD105 markers of MSCs and cancer stem cell [13–16]. The flow cytometry suggested a close relationship between MSC 293T and HEK 293T cells, lack of cancer stem cell markers, and clearance of stromal cells from the coculture (Supporting Information Fig. S1A). We then confirmed the common origin of the cells by genomic PCR that revealed the presence of large T antigen, used to create HEK 293T cells, both in HEK 293T and MSC 293T cells (Supporting Information Fig. S1B). As a final identification, we analyzed microsatellite (i.e., short tandem repeat; STR) structure. Interestingly, as compared to HEK 293T among the 10 STRs screened, the structure of six sequences (CSF1PO, D13S317, D16S539, TH01, TPOX, and vWA) was modified in MSC 293T (Supporting Information Fig. S1C). To exclude in vitro contamination and to determine the stability of the microsatellites, we analyzed the STR markers from MSC 293T xenograft tumors. The analysis revealed additional plasticity in CSF1PO, D13S317, D16S539, TH01, TPOX, and vWA confirming STR fluctuation (Supporting Information Fig. S1C) being in line with previous reports showing that the microenvironment induces instability of microsatellites [17, 18].

The analysis of growth characteristics of TPC1 MSC and MSC 293T cells suggested significantly increased proliferation capacity compared to parental control cells (Fig. 2A, 2B). In line with these data, in soft agar, TPC1 MSC and MSC 293T cells formed significantly more colonies than parental cells (Fig. 2C) indicating increased anchorage-independent growth, which is associated with the capacity of cells to migrate and survive in circulation and to metastasize to distant tissues [19]. We then exposed the MSC 293T and parental control cells to etoposide or cisplatin at concentrations between 1 mM and 32 mM for 24 hours that showed significantly higher cytotoxin resistance and survival for MSC 293T than controls (Fig. 2D). In summary, our in vitro data suggested increased DNA replication, proliferation, anchorage-independent growth, and resistance to cytotoxins caused by direct MSC interaction.

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Figure 2. In vitro growth characteristics of TPC1 MSC and MSC 293T. (A): Cocultured cells had a significantly increased DNA replication as compared to parental controls. ×40 magnification. (B): The growth curve analysis confirmed the BrdU incorporation data. (C): Anchorage-independent growth assay showed significantly increased colony formation by cocultured cells. ×10 magnification. (D): MSC 293T had higher resistance to cytotoxins than parental HEK 293T. The p-values correspond to p < .05 (*), p < .01 (**), p < .001 (***) as compared to HEK 293T controls. Abbreviation: MSC, mesenchymal stromal cell.

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To enforce the data showing increased growth potential, we analyzed the mitochondrial activity. TPC1 MSCs had significantly elongate mitochondria as compared to controls suggesting increased energy production (Fig. 3A, 3B) [20]. We then studied the growth-related signaling by phosphokinase array (Supporting Information Fig. S2A, S2B) and Western blotting. Compared with parental control cells, TPC1 MSC and MSC 293T displayed a higher expression and phosphorylation of epithelial growth factor receptor, Mek1/2, Erk1/2, and p38 MAPK, ribosomal S6 kinase, mitogen/stress-activated kinase, oncogenic cJun, and cell cycle-related cyclin D1 suggesting overall activation of the mitogen and stress signaling pathways (Fig. 3C, 3D).

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Figure 3. The effect on mitochondrial activity and signal transduction. (A): TPC1 MSC cells had significantly (p < .001) elongated mitochondria as compared to parental TPC1 cells. (B): Mitochondrial staining. (C): Western blot analysis showing coculture-related increased signal transduction. (D): Schematic presentation of signal transduction. Abbreviation: MSC, mesenchymal stromal cell.

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The ability to form tumors in vivo is a characteristic of a cancer cell line [21]. Even among highly carcinogenic cells, there could be a small subset of tumor-initiating cells that often have stem cell-like characteristics such as tumorigenicity in limiting dilution transplantations [22]. To evaluate whether MSC 293T can form tumors in vivo, we injected 1 × 106 cells subcutaneously in BALB c/A nude mice using untreated MSC (2 × 105) and parental HEK 293T cells (1 × 106) as controls (Fig. 4A, 4B). The MSC control mice generated a bioluminescent signal that lasted up to day 7 without regrowth of the graft. The HEK 293T control mice graft development indicated slight tumorigenicity. On the contrary, MSC 293T transplantation resulted in measurable tumors at day 7 supporting the in vitro data. To verify the increased aggressiveness, we transplanted MSC 293T cells cultured for additional 2 months that yielded similar robust tumorigenesis (Fig. 4B). This suggests that the increased aggressiveness was prolonged even though it may not indicate permanently increased carcinogenesis of the parental cells. Besides their increased tumorigenic potential, MSC 293T had a higher incidence of tumor initiation: 10% (four out of 40 injections) of HEK 293T transplantations resulted in detectable tumors, whereas 43% (29 out of 68 injections) of MSC 293T transplantations yielded tumors.

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Figure 4. In vivo analysis of MSC 293T growth characteristics. (A): Transplantation of MSC, HEK 293T cells, and MSC 293T cells for bioluminescence imaging (BLI) analysis showed no tumorigenicity for MSCs, mild tumorigenicity for HEK 293T cells, and robust tumor formation ability for MSC 293T cells. (B): Measurement of tumor growth kinetics confirmed BLI data and indicated prolonged tumorigenicity (2 months cultured cells) for MSC 293T cells. (C, D): MSC 293T cells have a higher metastasis rate than HEK 293T cells. Abbreviation: MSC, mesenchymal stromal cell.

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To examine the cancer stem cell characteristics of MSC 293T, we performed limiting dilution transplantations in BALB c/A nude mice by injecting 100, 1,000, and 10,000 cells subcutaneously. None of the injections resulted in a tumor (Supporting Information Fig. S3) suggesting, in line with the flow cytometry data (Supporting Information Fig. S1), that the tumors did not evolve from a few cells with stem cell characteristics.

Finally, since the in vitro data showed that MSC 293T cells are endowed with increased anchorage-independent growth ability (Fig. 2C), we transplanted intravenously 1 × 106 HEK 293T or MSC 293T cells in BALB c/A nude mice. The bioluminescence analysis and autopsy 2 months after transplantation suggested higher metastatic rate in MSC 293T than in HEK 293T mice (Fig. 4C, 4D).

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

In conclusion, our study demonstrates a direct in vitro interaction between transformed cells and MSCs leading to stromal cell cytoplasm evanescence, microsatellite instability, increased growth and signaling, gain of cytotoxin resistance, and increased tumorigenicity. The physical interaction of transformed and stromal cells with consequent evanescence of the MSC cytoplasm may be a mechanism whereby damaged/dying stromal cells are absorbed by neighboring cells so leading to increased growth potential.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

We would like to thank professor Massimo Santoro for his comments and suggestions to improve the work. We thank Jean Ann Gilder (Scientific Communication srl) for editing the text.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0952_sm_SupplFigure1.pdf464KSupplementary figure S1. A. Flow cytometry analysis. MSC 293T cells have similar CD markers as HEK 293T and lack cancer stem cell phenotype. Importantly, MSC 293T cell population is free of mesenchymal stromal cells. B. Large-T antigen genomic PCR suggests that MSC 293T cells develop from HEK 293T cells. The analysis resulted in a 180-bp amplicon, which indicates the large-T antigen in control HEK 293T and MSC 293T cells. Mesenchymal stromal cells lacked the large-T PCR amplicon. C. The co-culture induced microsatellite instability. Microsatellite analysis of parental MSC, HEK 293T cells and nine MSC 293T cell subclones. Note that the genomic DNA isolated from the tumor refers to the microsatellite structure of in vivo transplanted MSC 293T cells. The microsatellite structure of clones 2 (D13S317), clone 6 (CSF1PO), clone 7 (D165539, vWA), and clone 10 (vWA) differed from that of STR profiles in the DSMZ database (Deutsche Sammlung von Mikroorganismen und zellkulturen), which was detected also in in vivo transplanted tumor-derived genomic DNA. Clone 3 was lost during the isolation procedure.
sc-12-0952_sm_SupplFigure2.pdf250KSupplementary figure S2 A,B. Phosphokinase array of MSC 293T showed significantly increased mitogen activation and stress signaling pathways as compared to parental HEK 293T cells. The p-values correspond to p<0.05 (*), p<0.01 (**), p<0.001 (***) as compared to HEK 293T controls.
sc-12-0952_sm_SupplFigure3.pdf296KSupplementary figure S3. Limiting dilution transplantation showed inability of a low number of grafted MSC 293T cells to form tumors thereby suggesting the lack of cancer stem cell characteristics. Mice were injected subcutaneously with 100, 1000 or 10,000 cells. Red circles, the location of the graft, black arrows, the site of a signal.
sc-12-0952_sm_SupplFilm1.mpg4993KSupplementary film 1. Co-culture of untreated MSCs with HEK 293T cells in fresh 10% FBS β-medium. Note the remarkable motility of MSCs in eluding contact with HEK 293T cells that generated membrane protrusions towards MSCs. HEK 293T cells showed a high number of cell divisions thereby reducing the possibility for MSC to migrate.
sc-12-0952_sm_SupplFilm2.mpg21804KSupplementary film 2. Co-culture of MSCs pre-treated with HEK 293T conditioned serum-free medium with HEK 293T cells. Note that the MSCs incubated in these conditions did not move and were surrounded by HEK 293T cells that formed a physical connection with MSC. Consequently, the MSC lost their cytoplasm leaving only a fragmented nucleus.
sc-12-0952_sm_SupplFilm3.mpg11094KSupplementary film 3. The final stages of the co-culture showing the almost complete destruction of MSC.
sc-12-0952_sm_SupplMethods.pdf28KSupplementary Data

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