RESUMO
BACKGROUND: According to the cancer stem cell hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor-like cells. Self-renewal and high clonogenic potential are characteristics shared by normal stem and neoplastic stem/progenitor-like cells. We investigated whether human prostate cancer specimens contain cells with these properties. METHODS: Self-renewal and clonogenic potential were assessed by serial passaging of spheres and colony formation, respectively. Gene expression was analyzed by real time PCR. Protein expression was detected by immunocytochemistry. The neoplastic nature of the cells was verified by detection of the TMPRSS2/ERG gene fusion expression. RESULTS: The epithelial fraction isolated from surgical specimens generated colonies in 68% (19/28) of the patients. Laminin adhesion selected for cells with high clonogenic potential. The epithelial fraction from 85% (42/49) of the patients generated primary prostaspheres. Serial passaging of prostaspheres demonstrated their self-renewal capacity, which is also supported by their expression of the stem cell markers Oct-4, Nanog, Bmi-1, and Jagged-1 mRNA. Cells derived from prostaspheres were more clonogenic than the parental epithelial fraction. The pattern of mRNA expression in prostaspheres resembled that of the basal compartment of the prostate (CK5(+)/CK14(+)/CK19(high)/CK18(-/low)/c-met(+)/AR(-/low)/PSA(-/low)), but also included stem cell markers (CD49b(+)/CD49f(+)/CD44(+)/DeltaNp63(+)/Nestin(+)/CD133(+)). The distribution of marker expression in prostaspheres suggests their heterogeneous cell composition. Prostaspheres expressed significantly higher PSCA mRNA levels than the epithelial fraction. CONCLUSION: Human prostate cancer specimens contain neoplastic cells with self-renewal and clonogenic potential, which can be enriched and perpetuated in prostaspheres. Prostaspheres should prove valuable for the identification of prostate cancer stem/progenitor-like cells.
Assuntos
Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Neoplasias , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Processos de Crescimento Celular/fisiologia , Células Clonais/patologia , Proteínas Ligadas por GPI , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/genética , Proteína Jagged-1 , Queratina-14/biossíntese , Queratina-14/genética , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteína Homeobox Nanog , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Nestina , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-JaggedRESUMO
Carbonic anhydrase-IXG250/MN (CA9) is a renal cell carcinoma (RCC)-associated antigen ubiquitously expressed in the clear-cell subtype of RCC. Two CA9-derived peptides have been identified defining a cytotoxic T-lymphocyte epitope and human leukocyte antigen (HLA)-DR epitope, able to induce T-cell responses in vitro. A phase I clinical trial was performed with CA9-peptide-loaded dendritic cells (DCs) in patients with progressive, cytokine-refractory metastatic RCC to assess the safety, toxicity, and induction of CA9-specific immunity. Patients with objective progressive metastatic RCC received 5 vaccinations of mature DCs pulsed with the CA9-derived peptides and keyhole limpet hemocyanine (KLH). Peripheral blood was collected at regular intervals, delayed-type hypersensitivity (DTH) was tested at baseline and after the last vaccination, and skin biopsies of positive DTH sites were collected for immunomonitoring purposes. Patients were also monitored for clinical responses. No significant toxicity was observed. All patients developed humoral responses against KLH, and demonstrated DTH conversion. Evaluation of biopsy material suggested an increased influx of T-helper cells. In none of the immunomonitoring assays was evidence for the induction of CA9-peptide-specific immunity observed. No clinical responses were observed. The vaccination of DCs pulsed with KLH and 2 CA9-derived peptides was well tolerated. The lack of induction of CA9-peptide-specific immune responses indicates that this particular vaccine regimen is poor in inducing CA9-peptide-specific immune responses.
Assuntos
Antígenos de Neoplasias/imunologia , Anidrases Carbônicas/imunologia , Carcinoma de Células Renais/terapia , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Sequência de Aminoácidos , Formação de Anticorpos/imunologia , Anidrase Carbônica IX , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/imunologia , Humanos , Hipersensibilidade Tardia/sangue , Hipersensibilidade Tardia/imunologia , Imunidade Celular/imunologia , Imunoterapia Adotiva/efeitos adversos , Neoplasias Renais/sangue , Neoplasias Renais/imunologia , Dados de Sequência MolecularRESUMO
Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.
