Stem Cells 6: 1218-1223, 2013

Mesenchymal stromal cell atrophy in coculture increases aggressiveness of transformed cells.

Castellone MD, Laatikainen LE, Laurila JP, Langella A, Hematti P, Soricelli A, Salvatore M, Laukkanen MO.

 

Contact:

Mikko O. Laukkanen, PhD

SDN Foundation

Via Gianturco 111-113

80143 Naples, Italy

Email: mlaukkanen@sdn-napoli.it

Phone: +39 389 444 7260

 

Mesenchymal stem/stromal cells (MSC) affect tumor development1, 2, have tropism to tumors3, 4, and participate in stroma construction3, 5. MSCs have been suggested to support tumor growth by microenviromental paracrine signaling, immunomodulation, and tumor-stroma co-evolution involving energy metabolite transfer to cancer cells6-10. However, it is unclear how cellular interaction and increased carcinogenesis are causally linked. In our recent study published in Stem Cells11 we showed in vitro that tumorigenic cells interact physically with MSCs, and that this process is followed by evanescence of MSC cytoplasm. Thus, the interaction stimulated the proliferation of the co-cultured cancer cells, activated mitogen and stress signal transduction, increased resistance to cytotoxins, and enhanced the ability to grow and metastasize in vivo. Interestingly, cancer cells were able to connect only to damaged MSCs that had lost their migration ability, whereas untreated, low-passage MSCs avoided the contact.

Our data therefore describe how adjacent transformed cells absorb damaged stromal cells thereby leading to the stroma-driven evolution of moderately tumorigenic cells to highly aggressive metastatic cells.

Tumors are heterogenic cellular entities whose growth depends on mutual interaction between genetically altered neoplastic cells and non-neoplastic cells in the stromal microenviroment12-15. Bi-directional paracrine signals coordinately regulate tumorigenic cell populations, and tumorigenic cells in turn produce factors that attract and regulate the tumor stroma16-18. Stroma can promote tumor growth, however the mechanisms underlying this effect are not well understood.

To study the ability of MSCs exposed to non-autonomous paracrine signaling to affect the growth of transformed cells, we exposed bone marrow derived mesenchymal stem/stromal cells to conditioned serum-free medium collected from papillary thyroid cancer TPC1 and mildly tumorigenic HEK 293T cells (Fig. 1a) until stromal cells developed caspase 3 apoptosis (Fig. 1b). After exposure MSCs were seeded with 10-20 transformed cells and cultured for 25 additional passages. The TPC1 cell and HEK 293T cells co-cultured with stromal cells were designated “TPC1 MSC” and “MSC 293T cells”, respectively.

To probe the interaction we monitored the apoptotic MSC cultures seeded with HEK 293T cells by time-lapse imaging and observed a physical connection via membrane protrusions between the two cell populations; this was followed by MSC atrophy and loss of cytoplasm so that only a large fragmented nucleus remained (Fig. 1c). Interestingly, the interaction between transformed cells and MSCs resulted in significantly higher growth capacity and cytotoxin resistance of transformed cells in vitro analysis.

To confirm the in vitro data we evaluated whether MSC 293T can form tumors in vivo. The control mice transplanted with HEK 293T cells showed measurable tumor development at day 35, which indicates that HEK 293T cells are slightly tumorigenic. On the contrary, mice transplanted with stromal cells co-cultured MSC 293T cells were dramatically more tumorigenic. In fact, measurable tumors formed at day 7, which supports the in vitro data of increased proliferation of these cells (Fig 2).

Besides their increased tumorigenic potential, MSC 293T cells had a higher incidence of tumor initiation: 40% of transplantations resulted in tumor formation while only 10% of HEK 293T transplantations yielded detectable tumors. Further, the intravenous transplantation of stromal cell co-cultured MSC 293T cells modeling cancer cell extravasation and metastasis suggested much higher metastatic rate for MSC 293T cells as compared to controls.

In conclusion, our study demonstrated a direct in vitro interaction between tumorigenic cells and MSCs that lead to stromal cell cytoplasm evanescence with consequent increased cell growth, gain of cytotoxin resistance and increased tumorigenic potential. Although our data was produced in vitro the observed physical interaction of cancer and stromal cells with consequent evanescence of the MSC cytoplasm may be a mechanism whereby damaged/dying stromal cells are absorbed by neighboring tumor cells so leading to increased growth potential of the cancer cells. The mechanism underlying the direct physical contact between tumor cells and MSCs, with the consequent modification of both cell types, remains obscure. However, it is conceivable that emerging apoptosis and loss of motility of stromal cells make them vulnerable to cancer cell aggression.

Our results are consistent with a novel mechanism whereby a moderately carcinogenic cell line evolves to highly aggressive metastasizing cells. Elucidation of the networks responsible for cancer cell modifications is clinically relevant since it may lead to the identification of novel therapeutic targets and strategies able to overcome not only the progression of cancer but also the mechanisms underlying the development of resistance to anti-cancer treatments.

PMID: 23404893

 

FIGURE LEGENDSFigure 1

Figure 1. Interaction of MSCs with TPC1 and HEK 293T cells. a. In vitro co-culture model. b. Conditioned medium pre-treatment of MSCs increased caspase-3 mediated apoptosis. c. 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 7-9 h (white circles). Note the cell division at 17h time point (black arrow). Note the tube-like formations protruding towards MSCs (36-h image white arrows). d. Co-culture of GFP transduced stromal cells with unlabelled TPC1 cells resulted in temporal GFP positivity in TPC1 cells.

 

Figure 2Figure 2. In vivo analysis of MSC 293T growth characteristics. Transplantation of HEK 293T cells and stromal cell co-cultured MSC 293T showed mild tumorigencity for HEK 293T cells and robust tumor formation ability for MSC 293T cells.

 

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