RESUMEN

Despite improvements in early detection and advances in treatment, patients with prostate cancer continue to die from their disease. Minimal residual disease after primary definitive treatment can lead to relapse and distant metastases, and increasing evidence suggests that circulating tumour cells (CTCs) and bone marrow-derived disseminated tumour cells (BM-DTCs) can offer clinically relevant biological insights into prostate cancer dissemination and metastasis. Using epithelial markers to accurately detect CTCs and BM-DTCs is associated with difficulties, and prostate-specific markers are needed for the detection of these cells using rare cell assays. Putative prostate-specific markers have been identified, and an optimized strategy for staining rare cancer cells from liquid biopsies using these markers is required. The ideal prostate-specific marker will be expressed on every CTC or BM-DTC throughout disease progression (giving high sensitivity) and will not be expressed on non-prostate-cancer cells in the sample (giving high specificity). Some markers might not be specific enough to the prostate to be used as individual markers of prostate cancer cells, whereas others could be truly prostate-specific and would make ideal markers for use in rare cell assays. The goal of future studies is to use sensitive and specific prostate markers to consistently and reliably identify rare cancer cells.

Asunto(s)

Biomarcadores de Tumor/análisis , Células de la Médula Ósea/patología , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/química , Neoplasias de la Próstata/patología , Biomarcadores de Tumor/biosíntesis , Humanos , Biopsia Líquida , Masculino , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas

RESUMEN

Approximately 29 000 men die of prostate cancer (PCa) each year in the United States, and 90% to 100% of them are due to incurable bone metastasis. It is difficult to determine (1) when PCa disseminates in the natural history of the disease; (2) where cancer cell disseminates before becoming overt metastatic lesions; and (3) which tumors are aggressive and which are indolent. Tumor tissue and liquid (blood and bone marrow) biopsies provide important information to answer these questions, but significant limitations exist for immunostaining strategies that assess protein expression in these tissues. Classic immunohistochemistry (IHC) assays can typically assess expression of one or two proteins per tissue section. We have developed a novel immunofluorescence staining protocol to detect a panel of seven proteins on PCa tissue from primary tumor biopsies and metastatic lesion autopsy tissue, as well as cancer cells from liquid biopsies. We used a tyramide-based system to amplify the true signal and optimized the protocol to reduce background signal, thereby boosting the signal-to-noise ratio. Any protein-specific antibody in this protocol can be exchanged for a different validated antibody. This protocol therefore, represents a highly informative and flexible assay that can be used to provide important information about cancer tissue for the purpose of improving detection, diagnosis, and treatment.

Asunto(s)

Biotina/análogos & derivados , Neoplasias Óseas/diagnóstico , Técnica del Anticuerpo Fluorescente/métodos , Neoplasias de la Próstata/diagnóstico , Coloración y Etiquetado/métodos , Tiramina/análogos & derivados , Animales , Antígenos de Superficie/metabolismo , Biotina/metabolismo , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Línea Celular Tumoral , Colorantes Fluorescentes/metabolismo , Glutamato Carboxipeptidasa II/metabolismo , Xenoinjertos , Humanos , Indoles/metabolismo , Calicreínas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfoproteínas/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas de Unión al ARN/metabolismo , Racemasas y Epimerasas/metabolismo , Receptores Androgénicos/metabolismo , Tiramina/metabolismo , Nucleolina

RESUMEN

Prostate cancer bone metastasis remains lethal and incurable, and often arises years after elimination of the primary tumor. It is unclear what underlies the decades-long clinical latency before recurrence, but evidence points to the existence of dormant residual tumor cells that disseminated before the primary tumor was eliminated. To design therapies to prevent progression of disseminated tumor cells (DTC) into lethal metastases, it is crucial to understand the mechanism(s) underlying this dormancy. The current study functionally validated our previous observation that implicated the GAS6/AXL axis in mediating DTC dormancy in the bone marrow. AXL-null and AXL-overexpressing prostate cancer cell lines were generated to determine if AXL was necessary and/or sufficient for dormancy. Characterization of these cells in vitro and using in vivo mouse models of DTC growth demonstrated that AXL was indeed sufficient to induce dormancy, but was unable to maintain it long-term and was not absolutely required for a dormancy period. Clinically, AXL expression correlated with longer survival in prostate cancer patients, and AXL was not expressed by cancer cells in primary or metastatic tissue. These data point to a tumor-suppressive role for AXL in prostate cancer, and future work is required to determine if AXL is expressed on human bone marrow DTCs. IMPLICATIONS: The ability of AXL to initiate but not maintain dormancy, coupled with its dispensability, suggests that targeting AXL alone will not prevent lethal metastatic outgrowth, and likely a cooperative network of factors exists to mediate long-term cellular dormancy.

Asunto(s)

Genes Supresores de Tumor , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Análisis de Matrices Tisulares , Tirosina Quinasa del Receptor Axl

RESUMEN

BACKGROUND: Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. RESULTS: We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. CONCLUSIONS: Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.