RESUMO
Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR(+) mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Ductal Pancreático/patologia , Separação Celular/métodos , Citometria de Fluxo , Genômica , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias de Próstata Resistentes à Castração/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Análise Mutacional de DNA , Receptores ErbB/genética , Predisposição Genética para Doença , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Mutação , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fenótipo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismoRESUMO
New, and previously reported microsatellites, were characterized for a group of four cryptic sibling species in California (USA) in the subterranean termite genus Reticulitermes with the goal of finding loci appropriate for population and species level studies. Three new microsatellites were identified originating from R. hesperus, and 19 loci previously characterized in R. flavipes and R. santonensis were examined. Of the three loci specifically developed for R. hesperus, none amplify with the other species. Variation appropriate for population level studies was found in 4-13 loci depending on the species. Fifteen loci appeared to be appropriate for use at the species level. Unique or monomorphic alleles are found among the four species, indicating these loci will be taxonomically informative for this group.
Assuntos
Variação Genética , Isópteros/classificação , Isópteros/genética , Repetições de Microssatélites/genética , Animais , California , Classificação/métodos , Especificidade da EspécieRESUMO
Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.