ABSTRACT Introduction: Mesenchymal stem cells (MSCs) are defined as a heterogeneous population of stromal stem cells and can be isolated from many adult tissues like bone marrow or adipose tissue. Many studies have shown that MSCs play a key role in tumour progression. MSCs recruited in tumour sites can stimulate tumour cell progression and mobilization, especially in breast cancer. However it is still unclear the molecular mechanism used by MSCs to promote angiogenesis, metastasis and tumour progression. Recently, studies demonstrated that P2X7 purinergic receptor plays a key role in stimulating tumour cell mobilization and metastatization. In fact, they have proposed that TGF-β stimulation promotes ATP release from cancer cells that stimulates P2X7 receptor activation. This activation stimulates neoplastic cells migration and then promoting tumour metastatization. Moreover, tumour cells that over-express P2X7 receptor grow faster than other cells and in vivo generate larger tumours. In these animals, the use of oATP (a P2X7 inhibitor) reduces tumour dimensions. The aim of this study is to confirm MSCs role in tumour progression and to clarify the involvement of P2X7 receptor as a key mediator of MSC-mediated induction of tumor growth. Mat & met: We used in vitro and in vivo optical imaging to study the influence of MSCs in tumour progression. Murine (EMT-6 and MET-1 cell lines) and human (SUM159) mammary carcinoma cells were infected with a lentiviral construct expressing mCherry or Luciferase as a constitutive reporter gene. These cells were used for in vitro and in vivo studies. Tumour cells were cultured in the presence or absence of MSCs for 48h with or without an inhibitor: oATP (100 µM). In in vitro studies cell viability was monitored by Trypan blue exclusion test, and by reporter gene activity. To perform in vivo studies, tumor cells (EMT-6, MET-1 and SUM159) were injected into mammary fat pad of nude mice. mCherry or Luciferase activity was used to assess tumour growth over time by whole body optical imaging. MSC injection and the treatment with the inhibitors started after tumour establishment, and the longitudinal imaging was further performed twice a week. At the endpoint, tumors were explanted and tumour weight and volume were evaluated for each experimental group. Results: In vitro studies showed that MSC migration ability significantly increases after treatment with tumour conditioned culture medium. Moreover, tumor cells proliferation was increased by MSCs co-culture as demonstrated by cell count and by reporter photon emission observed by optical imaging and biochemical analysis in cell lysates. This effect was prevented by treatment with the inhibitor oATP. In vivo data confirmed in vitro observations, in fact, a significant increase of tumour dimension at the endpoint was detected in animals injected with MSCs. The evaluation of tumour growth by optical imaging of reporter activity over time, demonstrated that the presence of MSCs significantly stimulated the proliferation of mammary carcinoma cells whereas the treatment with oATP prevented this phenomenon. These results were validated by ex vivo analysis. In comparison to control group, tumour weight and volume were higher in the MSC treated group, while they were similar to controls for the inhibitor treated ones. The inhibitory effect of oATP underlines a key role of P2X7 receptor in tumor growth and a correlation between MSCs tumour stimulation and P2X7 activity. Conclusions: These data demonstrated the capability of stem cells to stimulate the rate of breast cancer growth. These preliminary studies pave the way for the understanding of the molecular mechanism of MSC-mediated induction of tumor cell proliferation, proposing the involvement of the purinergic receptor pathway. In the TME, mesenchymal cells release a large amount of TGF-β that could support tumour progression and metastatizzation by indirectly activating P2X7 receptors. In this scenario, the recruitment of endogenous MSCs to tumour sites acquires important implications. In fact, it could be useful to understand the rate of endogenous MSC migration to tumour sites to evaluate if an inhibition of MSC-tumor cross talk or MSC-mediated immunomodulation could help in increasing anti-neoplastic treatment efficacy.
|Titolo:||STUDIO NON INVASIVO DEL RUOLO DI CELLULE MESENCHIMALI STAMINALI NELLA PROGRESSIONE DI MODELLI DI CARCINOMA MAMMARIO|
|Supervisori e coordinatori interni:||CLERICI, MARIO SALVATORE|
|Data di pubblicazione:||17-dic-2015|
|Settore Scientifico Disciplinare:||Settore MED/36 - Diagnostica per Immagini e Radioterapia|
|Citazione:||STUDIO NON INVASIVO DEL RUOLO DI CELLULE MESENCHIMALI STAMINALI NELLA PROGRESSIONE DI MODELLI DI CARCINOMA MAMMARIO ; tutor: L.Ottobrini ; direttore del dottorato: M. Clerici. - Milano : Università degli studi di Milano. DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2015 Dec 17. ((28. ciclo, Anno Accademico 2015.|
|Digital Object Identifier (DOI):||10.13130/diceglie-cecilia_phd2015-12-17|
|Appare nelle tipologie:||Tesi di dottorato|