Gene expression analysis of tumor sorted fractions demarcating cancer stem cell hierarchy and DNA content based ploidy
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE74853 | GEO | 2016/01/01
SECONDARY ACCESSION(S): PRJNA301675
REPOSITORIES: GEO
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-07-03 | E-GEOD-74853 | biostudies-arrayexpress
Project description:Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37ËC followed by pyronin Y for 45min at 37ËC and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid. Euploid and aneuploid were sorted with 99% purity and 1x105 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4Euploid and A4Aneuploid. Three samples of each were chosen for hybridization.
2016-07-03 | E-GEOD-74852 | biostudies-arrayexpress
Project description:Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37˚C followed by pyronin Y for 45min at 37˚C and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid.
2016-01-01 | GSE74852 | GEO
Project description:The origins of the complex process of intratumoral heterogeneity have been highly debated and different cellular mechanisms have been hypothesized to account for the diversity within a tumor. The clonal evolution and cancer stem cell (CSC) models have been proposed as drivers of this heterogeneity. However, the concept of cancer stem cell plasticity and bidirectional conversion between stem and non-stem cells has added additional complexity to these highly studied paradigms and may help explain the tumor heterogeneity observed in solid tumors. The process of cancer stem cell plasticity in which cancer cells harbor the dynamic ability of shifting from a non-CSC state to a CSC state and vice versa may be modulated by specific microenvironmental signals and cellular interactions arising in the tumor niche. In addition to promoting CSC plasticity, these interactions may contribute to the cellular transformation of tumor cells and affect response to chemotherapeutic and radiation treatments by providing CSCs protection from these agents. Herein, we review the literature in support of this dynamic CSC state, discuss the effectors of plasticity, and examine their role in the development and treatment of cancer.
| S-EPMC4300934 | biostudies-other
Project description:Our earlier report states isolation of ovarian cancer stem cell heirarchy using membrane labeling PKH26/67 dyes (Kusumbe and Bapat Cancer Research. 2009) in a xenograft model. We earlier reported that ovarian cancer is a stem cell disease with isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). A4T cells were labelled with PKH26 membrane labeling dye, flourescence intensity was quantified and set as 100% dye retaining cells (PKHhi) with flow cytometery. 2.5x10^6 P65 A4PKH26 labeled cells were injected s.c in NOD/SCID mice, while unlabeled cells were also injected for generation of control tumors . Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Tumor cells were acquired on BD FACS Aria II across PE channel of blue laser. Cells retaining dye intensity as that of preinjected cells were gated as PKHhi while cells exhibiting complete dye depletion were gated as PKHneg using unlabeled tumor cells as control; while cells showing intermediate label intensity were gated as PKHlo. Details of the flow cytometry analysis and sorting are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009). PKHhi PKHlo and PKHneg were sorted with 99% purity and 1x10^5 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4PKHhi, A4PKHlo and A4PKHneg. Three samples of each were chosen for hybridization. Custom Agilent Human whole Gene Expression Microarray 8x60K designed by Genotypic Technology Private Limited.(AMADID: 27114);Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
2016-07-03 | E-GEOD-74851 | biostudies-arrayexpress
Project description:Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.
| S-EPMC3114859 | biostudies-literature
Project description:Our earlier report states isolation of ovarian cancer stem cell heirarchy using membrane labeling PKH26/67 dyes (Kusumbe and Bapat Cancer Research. 2009) in a xenograft model. We earlier reported that ovarian cancer is a stem cell disease with isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). A4T cells were labelled with PKH26 membrane labeling dye, flourescence intensity was quantified and set as 100% dye retaining cells (PKHhi) with flow cytometery. 2.5x10^6 P65 A4PKH26 labeled cells were injected s.c in NOD/SCID mice, while unlabeled cells were also injected for generation of control tumors . Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Tumor cells were acquired on BD FACS Aria II across PE channel of blue laser. Cells retaining dye intensity as that of preinjected cells were gated as PKHhi while cells exhibiting complete dye depletion were gated as PKHneg using unlabeled tumor cells as control; while cells showing intermediate label intensity were gated as PKHlo. Details of the flow cytometry analysis and sorting are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009).
2016-01-01 | GSE74851 | GEO
Project description:Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
| S-EPMC9006079 | biostudies-literature
Project description:Glioblastomas are the most prevalent and lethal primary brain tumor and are comprised of hierarchies with self-renewing cancer stem cells (CSCs) at the apex. Like neural stem cells (NSCs), CSCs reside in functional niches that provide essential cues to maintain the cellular hierarchy. Bone morphogenetic proteins (BMPs) instruct NSCs to adopt an astrocyte fate and are proposed as anti-CSC therapies to induce differentiation, but, paradoxically, tumors express high levels of BMPs. Here we demonstrate that the BMP antagonist Gremlin1 is specifically expressed by CSCs as protection from endogenous BMPs. Gremlin1 colocalizes with CSCs in vitro and in vivo. Furthermore, Gremlin1 blocks prodifferentiation effects of BMPs, and overexpression of Gremlin1 in non-CSCs decreases their endogenous BMP signaling to promote stem-like features. Consequently, Gremlin1-overexpressing cells display increased growth and tumor formation abilities. Targeting Gremlin1 in CSCs results in impaired growth and self-renewal. Transcriptional profiling demonstrated that Gremlin1 effects were associated with inhibition of p21(WAF1/CIP1), a key CSC signaling node. This study establishes CSC-derived Gremlin1 as a driving force in maintaining glioblastoma tumor proliferation and glioblastoma hierarchies through the modulation of endogenous prodifferentiation signals.
| S-EPMC4035537 | biostudies-literature
Project description:BackgroundSmall and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient.MethodsThree Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored.ResultsThe small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample.ConclusionWe demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80-90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.
| S-EPMC8500090 | biostudies-literature