Subpopulations Of Neoplastic Cells Research Articles

The role of OCT4 and CD44 in lower lip carcinogenesis.

Carcinogenesis is characterized by the transformation of normal cells into malignant cells. Concerning the lower lip, exposure to UV radiation is the main etiological factor associated with the development of epidermoid carcinomas and actinic cheilitis. According to the hierarchical model theory, cancer development is driven by populations of cancer stem cells. In this context, this study aimed to compare the expression of octamer-binding transcription factor 4 (OCT4) and CD44 in 40 lower lip epidermoid carcinoma (LLEC) and 40 actinic cheilitis (AC) cases. OCT4 and CD44 expressions were assessed semi-quantitatively according to the percentage of positive epithelial cells (PP) and intensity of expression (IE), resulting in a total immunolabeling score (PIT). No statistically significant differences were detected between OCT4 and CD44 immunoexpression and clinicopathological parameters, except for lymph node metastasis, in which a decrease in CD44 expression in the core tumor of cases with lymph node metastasis was observed. Furthermore, decreased CD44 expression was observed in LLEC cases when compared to AC cases. The findings reported herein suggest a higher participation of CD44 in early carcinogenesis stages. In addition, the imbalance between OCT4 and CD44 immunoexpressions suggests the presence of different neoplastic cell subpopulations.

Clinically unfavorable transcriptome subtypes of non-WNT/non-SHH medulloblastomas are associated with a predominance in proliferating and progenitor-like cell subpopulations.

The non-WNT/non-SHH (Grp3/Grp4) medulloblastomas (MBs) include eight second-generation subgroups (SGS; I-VIII) each with distinct molecular and clinical characteristics. Recently, we also identified two prognostically relevant transcriptome subtypes within each SGS MB, which are associated with unique gene expression signatures and signaling pathways. These prognostic subsets may be in connection to the intra-tumoral cell landscape that underlies SGS MB clinical-molecular diversity. Here, we performed a deconvolution analysis of the Grp3/Grp4 MB bulk RNA profiles using the previously identified single-cell RNA-seq reference dataset and focusing on variability in the cellular composition of SGS MB. RNA deconvolution analysis of the Grp3/Grp4 MB disclosed the subgroup-specific neoplastic cell subpopulations. Neuronally differentiated axodendritic GP3-C1 and glutamatergic GP4-C1 subpopulations were distributed within Grp3- and Grp4-associated SGS MB, respectively. Progenitor GP3-B2 subpopulation was prominent in aggressive SGS II MB, whereas photoreceptor/visual perception GP3/4-C2 cell content was typical for SGS III/IV MB. The current study also revealed significant variability in the proportions of cell subpopulations between clinically relevant SGS MB transcriptome subtypes, where unfavorable cohorts were enriched with cell cycle and progenitor-like cell subpopulations and, vice versa, favorable subtypes were composed of neuronally differentiated cell fractions predominantly. A higher than median proportion of proliferating and progenitor cell subpopulations conferred the shortest survival of the Grp3 and Grp 4MB, and similar survival associations were identified for all SGS MB except SGS IV MB. In summary, the recently identified clinically relevant Grp3/Grp4 MB transcriptome subtypes are composed of different cell populations. Future studies should aim to validate the prognostic and therapeutic role of the identified Grp3/Grp4 MB inter-tumoral cellular heterogeneity. The application of the single-cell techniques on each SGS MB separately could help to clarify the clinical significance of subgroup-specific variability in tumor cell content and its relation with prognostic transcriptome signatures identified before.

Matrix Gla Protein drives stemness and tumor initiation in ovarian cancer

Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking, and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings. Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.

Intratumoral Heterogeneity and Immune Modulation in Lung Adenocarcinoma in Female Smokers and Never Smokers.

Single-cell analysis of healthy lung tissue and lung cancer reveals distinct tumor cell populations, including cells with differential immune modulating capacity between smokers and never smokers, which could guide future therapeutic strategies.

Papillary thyroid carcinoma tall cell variant shares accumulation of mitochondria, mitochondrial DNA mutations, and loss of oxidative phosphorylation complex I integrity with oncocytic tumors.

Papillary thyroid carcinoma tall cell variant (PTC‐TCV), a form of PTC regarded as an aggressive subtype, shares several morphologic features with oncocytic tumors. Pathogenic homoplasmic/highly heteroplasmic somatic mitochondrial DNA (mtDNA) mutations, usually affecting oxidative phosphorylation (OXPHOS) complex I subunits, are hallmarks of oncocytic cells. To clarify the relationship between PTC‐TCV and oncocytic thyroid tumors, 17 PTC‐TCV and 16 PTC non‐TCV controls (cPTC) were subjected to: (1) transmission electron microscopy (TEM) to assess mitochondria accumulation, (2) next‐generation sequencing to analyze mtDNA and nuclear genes frequently mutated in thyroid carcinoma, and (3) immunohistochemistry (IHC) for prohibitin and complex I subunit NDUFS4 to evaluate OXPHOS integrity. TEM showed replacement of cytoplasm by mitochondria in PTC‐TCV but not in cPTC cells. All 17 PTC‐TCV had at least one mtDNA mutation, totaling 21 mutations; 3/16 cPTC (19%) had mtDNA mutations (p < 0.001). PTC‐TCV mtDNA mutations were homoplasmic/highly heteroplasmic, 16/21 (76%) mapping within mtDNA‐encoded complex I subunits. MtDNA mutations in cPTC were homoplasmic in 2 cases and at low heteroplasmy in the third case, 2/3 mapping to mtDNA‐encoded complex I subunits; 2/3 cPTC with mtDNA mutations had small tall cell subpopulations. PTC‐TCV showed strong prohibitin expression and cPTC low‐level expression, consistent with massive and limited mitochondrial content, respectively. All 17 PTC‐TCV showed NDUFS4 loss (partial or complete) and 3 of 16 cPTC (19%) had (partial) NDUFS4 loss, consistent with lack of complex I integrity in PTC‐TCV (p < 0.001). IHC loss of NDUFS4 was associated with mtDNA mutations (p < 0.001). Four BRAF V600E mutated PTCs had loss of NDUSF4 expression limited to neoplastic cell subpopulations with tall cell features, indicating that PTCs first acquire BRAF V600E and then mtDNA mutations. Similar to oncocytic thyroid tumors, PTC‐TCV is characterized by mtDNA mutations, massive accumulation of mitochondria, and loss of OXPHOS integrity. IHC loss of NDUFS‐4 can be used as a surrogate marker for OXPHOS disruption and to reliably diagnose PTC‐TCV.

Pancreatic Cancer Microenvironment and Cellular Composition: Current Understandings and Therapeutic Approaches.

Simple SummaryThere is currently no effective treatment for Pancreatic Ductal Adenocarcinoma (PDAC), and it is still the deadliest cancer, despite great research efforts in recent decades. The complexity in the tumor microenvironment (TME) is one of the main roots for the refractory nature of PDAC to treatment. Components of the PDAC TME are complex, dynamic, and closely reciprocate with each other from the early stages of pre-neoplastic lesion and during tumor progression. Detailed insight of the PDAC tumor microenvironment is needed for developing combined therapeutics that would target cancer cells and their supportive milieu for more efficient PDAC treatment.Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human solid tumors, despite great efforts in improving therapeutics over the past few decades. In PDAC, the distinct characteristic of the tumor microenvironment (TME) is the main barrier for developing effective treatments. PDAC TME is characterized by a dense stroma, cancer-associated fibroblasts, and immune cells populations that crosstalk to the subpopulations of neoplastic cells that include cancer stem cells (CSCs). The heterogeneity in TME is also exhibited in the diversity and dynamics of acellular components, including the Extracellular matrix (ECM), cytokines, growth factors, and secreted ligands to signaling pathways. These contribute to drug resistance, metastasis, and relapse in PDAC. However, clinical trials targeting TME components have often reported unexpected results and still have not benefited patients. The failures in those trials and various efforts to understand the PDAC biology demonstrate the highly heterogeneous and multi-faceted TME compositions and the complexity of their interplay within TME. Hence, further functional and mechanistic insight is needed. In this review, we will present a current understanding of PDAC biology with a focus on the heterogeneity in TME and crosstalk among its components. We also discuss clinical challenges and the arising therapeutic opportunities in PDAC research.

Doxorubicin treatment modulates chemoresistance and affects the cell cycle in two canine mammary tumour cell lines

BackgroundDoxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC).ResultsBoth cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis.ConclusionsDOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.

A possible interplay between HR-HPV and stemness in tumor development: an in vivo investigation of CD133 as a putative marker of cancer stem cell in HPV18-infected KB cell line.

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies.

Single-Cell RNA Sequencing of Childhood Ependymoma Reveals Neoplastic Cell Subpopulations That Impact Molecular Classification and Etiology

Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology.

Single-Cell RNAseq of Childhood Ependymoma Reveals Distinct Neoplastic Cell Subpopulations that Impact Etiology, Molecular Classification and Outcome

Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in the majority of children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We therefore used single-cell RNA sequencing to catalog cellular heterogenity in childhood EPN. PFA subgroup EPNs concealed an undifferentiated progenitor subpopulation that either differentiated into subpopulations with ependymal cell characteristics, or transitioned into a mesenchymal subpopulation. Undifferentiated and mesenchymal PFA subpopulations were associated with a poor prognosis, providing new therapeutic targets. In conflict with current classification paradigms, relative PFA subpopulation proportions were shown to determine bulk-tumor assigned subgroups. This atlas of EPN cellular heterogeneity provides an important advance in our understanding of EPN biology.

Expression of microRNAs associated with apoptosis in neurospheres of glioblastoma cell line subjected to treatment with ionizing radiation and temozolomide.

IntroductionGlioblastomas (GBM) are the most common malignant primary tumors in the brain, and show high mortality rate. Despite the current advances in the therapy, the GBMs are extremely resistant to ionizing radiation and chemotherapy, and the number of relapse is high. Studies bring this eminent tumorigenic potential due to the presence of a subpopulation of neoplastic cells with characteristics of stem cells, called cancer stem cells (CSCs). In gliomas, the isolation of tumor stem cells have been done by antigenic markers and observing the culture conditions of normal neural stem cells in vitro. That is, the floating proliferation of tumor cells when cultured, are analogous to neurospheres derived from normal neural stem cells in defined culture conditions. It is believed that CSCs are responsible for the restoration of the tumor and the low efficiency of the treatment, as these cells demonstrate malignant properties as tumorigenesis, chemoresistance and radioresistance. The practical implication of this finding is that no current therapy is able to suppress or stop the proliferation of these cells. Several microRNAs have been linked to the development and proliferation of glioblastomas, associated with different molecular mechanisms, among them programmed cell death. Studies have shown that these microRNAs in brain tumors exhibit altered expression levels, one of the essential mechanisms for the regulation of tumor stem cells (CSCs).ObjectivesTo evaluate the effect of ionizing radiation and temozolomide, alone or associated, on the expression of miRNAs that act as tumor suppressors (miR‐15 and miR‐16) and as oncogene (miR‐21), and adhered cells in neurospheres in culture of glioblastoma cell line (U343‐MG).Materials and MethodsThe trypan blue was used to examine cell viability before and after treatment, and method of real time PCR to check the expression of microRNAs in time 0h, considered to be immediately after the treatments, and 48h after exposure to treatments.Results and ConclusionsThe presented microRNAs are differentially expressed when comparing the different treatment modalities and time. We also observed differences between adhered cells and neurospheres. The oncogene miR‐21 remained with higher expression on treatment with ionizing radiation in neurospheres compared with the group of adherent cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancing the Oncolytic Activity of CD133-Targeted Measles Virus: Receptor Extension or Chimerism with Vesicular Stomatitis Virus Are Most Effective.

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types.

Review on Research about Traditional Chinese Medicine in Cancer Stem Cell.

Cancer stem cells (CSCs) are small subpopulations of neoplastic cells within a tumor, which have self-renewal and differentiation abilities and could generate new tumors with few cells. Researches have showed that CSCs are considered the most likely reason for cancer recurrence and metastasis. Accumulating evidences have showed that traditional Chinese medicine (TCM) has significant effect on CSCs. It could inhibit the proliferation, self-renew, and multidifferentiation of CSCs. We aimed to summarize the theories of CSCs in TCM, the inhibitory effect, and the pathway on CSCs of TCM. This review will provide potential new strategies and alternative perspectives for CSCs treatments and basic research into complementary and alternative medicine.

Abstract 1113: Mir-194-3p suppresses tumorigenic potential of glioblastoma stem cells by targeting the canonical NF-kB pathway

Abstract Glioblastoma (GBM) is the most common tumor of the central nervous system, with its deadly nature owing to high rates of invasion and angiogenesis. A key element in its pathogenesis is a small subpopulation of neoplastic cells called glioma stem cells (GSCs) that are responsible for tumor initiation, maintenance and relapse. Approaches to target GSCs have mostly proved futile due to their chemo- and radio-resistance potential. Given that microRNAs carry a potential to affect both normal and stem cells, we employed a microRNA-based targeted approach to identify the subset of microRNAs that could distinctively affect the stemness of GSCs. Our study tests the hypothesis that GSCs will show an altered miRNA expression upon their differentiation, and their profiling may lead to identification of specific microRNAs that regulate the process. Our initial efforts resulted in identification of hsa-miR-194-3p as one of the potential candidates, which was consistently downregulated in multiple GSCs that we investigated. Stable cell lines overexpressing hsa-miR-194-3p showed a decreased proliferation rate and reduced sphere-forming ability. In silico analysis revealed TAB2, an upstream activator of NF-κB pathway to be the potential target of hsa-miR-194-3p, which was confirmed by luciferase assay and knockdown approaches. The regulatory role of hsa-miR-194-3p was substantiated by its ability to inhibit the NF-κB pathway and the corresponding downstream targets including IL-6 and STAT3. In vivo studies revealed a reduced tumor forming ability when the cell lines overexpressing hsa-miR-194-3p were injected intracranially in NOD/SCID mice, compared with the non-target control. Our results indicate that hsa-miR-194-3p blocked the canonical NF-κB signaling and negatively affected tumor formation. We have carried out this study this in a unique model system expecting it to uncover another layer of complexity that may add a new facet to meet the challenge imposed by the cancer stem cell paradigm. In conclusion, we accentuate the anti-tumorigenic role of hsa-miR-194-3p as a connecting link to both stemness and NF-κB signaling. Funding sources: This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC). Citation Format: Rajbir Singh, Kamalakannan Palanichamy, John R. Jacob, Arnab Chakravarti. Mir-194-3p suppresses tumorigenic potential of glioblastoma stem cells by targeting the canonical NF-kB pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1113.

LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway.

SummaryCancer stem cells (CSCs) are a subpopulation of neoplastic cells with self‐renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial‐mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non‐coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT‐associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist‐positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs‐associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh‐GLI1 pathway associated lncRNA‐Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere‐formation efficiency (MFE) and the self‐renewal capacity in vitro, and oncogenicity in vivo in Twist‐positive breast cancer cells are elevated. lncRNA‐Hh silence in Twist‐positive breast cells attenuates the activated Shh‐GLI1 signaling and decreases the CSC‐associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist‐induced EMT cells to gain the CSC‐like stemness properties. Stem Cells 2016;34:55–66

SciClone: inferring clonal architecture and tracking the spatial and temporal patterns of tumor evolution.

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.

Dynamic imbalance between cancer cell subpopulations induced by transforming growth factor beta (TGF-β) is associated with a DNA methylome switch.

BackgroundDistinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood.ResultsIn the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases.ConclusionsOur study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-435) contains supplementary material, which is available to authorized users.

High-sensitivity BRAF mutation analysis: BRAF V600E is acquired early during tumor development but is heterogeneously distributed in a subset of papillary thyroid carcinomas.

The homogeneous distribution of BRAF V600E in papillary thyroid carcinoma (PTC) has been called into question by recent reports. These studies claim that BRAF V600E is heterogeneous and is limited to tumor cell subsets in the majority of PTCs. The objective of the study was to understand the allele distribution of BRAF V600E by evaluating the percentage of mutated neoplastic cells in a group of PTCs using two different highly sensitive analytical approaches: allele-specific locked nucleic acid PCR and 454 next-generation sequencing targeted to BRAF exon 15. BRAF V600E was investigated using allele-specific locked nucleic acid PCR on 155 consecutive samples of PTC. Mutated cases were reanalyzed by 454 next-generation sequencing and immunohistochemistry. Because the evaluation of genetic heterogeneity in tumor samples can be profoundly biased by contamination with normal cells, all mutation frequency data were normalized to the real amount of neoplastic cells within each tumor. Eighty-five of 155 PTCs (54.8%) were BRAF V600E mutated. The distribution of mutated neoplastic cells within the tumor was as follows: greater than 80% in 37 of 85 (43.5%), 30-80% in 39 of 85 (45.9%), and less than 30% in 9 of 85 (10.6%). In most of the PTCs with less than 80% BRAF V600E-positive neoplastic cells, the mutation was present in large neoplastic cell subpopulations. Tumors with less than 30% mutated neoplastic cells were smaller than tumors with a percentage of mutated cells greater than 80% or between 30% and 80% (P < .05). BRAF V600E is heterogeneously distributed in some PTCs. The large BRAF V600E neoplastic cell subpopulations found in mutated cases is consistent with the view that the BRAF V600E is acquired early during PTC development.

Cytomorphology of giant cell tumor of bone in pleural fluid

Cytomorphology of giant cell tumor of bone in pleural fluid

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.