జర్నల్ ఆఫ్ స్టెమ్ సెల్ రీసెర్చ్ & థెరపీ

నైరూప్య

TRA-1-60+, SSEA-4+, POU5F1+, SOX2+, NANOG+ Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Testes

Marek Malecki, Xenia Tombokan, Mark Anderson, Raf Malecki and Michael Beauchaine

Introduction: Cancer of the testes is currently the most frequent neoplasm and a leading cause of morbidity in men 15-35 years of age. Its incidence is increasing. Embryonal carcinoma is its most malignant form, which either may be resistant or may develop resistance to therapies, which results in relapses. Cancer stem cells are hypothesized to be drivers of these phenomena. Specific aim: The specific aim of this work was identification and isolation of spectra of single, living cancer stem cells, which were acquired directly from the patients’ biopsies, followed by testing of their pluripotency. Patients-methods: Biopsies were obtained from the patients with the clinical and histological diagnoses of the primary, pure embryonal carcinomas of the testes. Antibodies targeting TRA-1-60 and SSEA-4 were bioengineered to render magnetic or fluorescent. The cell surface display of the TRA-1-60 and SSEA-4 was analyzed by multiphoton fluorescence spectroscopy (MPFS), flow cytometry (FCM), immunoblotting (IB), nuclear magnetic resonance spectroscopy (NMRS), and total reflection x-ray spectroscopy (TRXFS). The single, living cells were isolated by magnetic or fluorescent sorting followed by their clonal expansion. The OCT4A, SOX2, and NANOG genes’ transcripts were analyzed by qRTPCR and products by IB and MPFS. Results: The clones of cells with the strong surface display of TRA-1-60 and SSEA-4 were identified and isolated directly from the biopsies acquired from the patients diagnosed with the pure embryonal carcinomas of the testes. These cells demonstrated high levels of transcription and translation of the pluripotency genes: OCT4A, SOX2, and NANOG. They formed embryoid bodies, which differentiated into ectoderm, mesoderm, and endoderm. Conclusion: In the pure embryonal carcinomas of the testes, acquired directly from the patients, we identified, isolated with high viability and selectivity, and profiled the clones of the pluripotent stem cells. These results may help in explaining therapy-resistance and relapses of these neoplasms, as well as, in designing targeted, personalized therapy.