Sorted Tumor Cells Research Articles

Functional features of cancer stem cells in melanoma cell lines

BackgroundRecent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features.MethodsNine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections.ResultsD10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected in xenografts and primary human melanoma sections using immunohistochemistry.ConclusionsEstablished melanoma cell lines exhibit, to variable extents, the typical features of CSCs. The tumorigenic cell line D10, expressing CD133 and growing in spheroids and might qualify as a potential model of melanoma CSCs.

Abstract 3262: Notch signaling pathway-targeted therapy suppresses tumor progression and prevents metastatic spread in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) has the highest mortality rate of any cancer type. The 5-year survival rate of 5% remained largely unchanged in the last three decades. The paucity of breakthroughs in treatment regimens and continued poor survival demonstrates an acute need for improvement in therapy for this lethal malignancy. Mounting evidence suggest that sustained re-activation of Notch signaling pathway contributes to the initiation, progression and maintenance of PDA. Recently, Notch signaling has been linked to the viability of putative tumor initiating cancer stem cell populations in pancreatic cancer. PF-03084014 is a selective γ-secretase inhibitor developed by Pfizer. The present study was conducted to determine the effect of PF-03084014 monotherapy and in combination with gemcitabine (GEM) to suppress tumor progression and prevent metastatic spread in preclinical models of pancreatic cancer. Methods: NOTCH-1 gene expression was determined in a panel of 30 human pancreatic cancer xenografts by Affymetrix gene array. We investigated the efficacy of PF-03084014 and in combination with GEM in subcutaneously (s.c) implanted as well as orthotopic models of pancreatic cancer. We also determined the treatment effect on putative pancreatic cancer stem cell (CSC) populations. TUNEL and Ki-67 staining were performed to determine whether the therapy induce apoptosis and suppresses tumor cell proliferation. Results: A combination of PF-03084014 and GEM is highly effective in producing durable tumor regression compared to GEM in subcutaneously implanted pancreatic cancer xenografts. Tumor re-growth curves plotted after 4 weeks of drug treatment demonstrate that the effect of the combination therapy is longer lasting than that of GEM. Notably, in a highly aggressive Panc265 orthotopic model, PF-03084014 and GEM combination was effective in controlling the primary tumor growth as well as preventing metastatic dissemination to organs as compared to GEM treatment. Flow cytometry of sorted tumor cells from Panc215 (after 4 weeks of treatment) showed that GEM treatment was not capable of diminishing the CSC pool as compared to the vehicle treated mice. However, PF-03084014 alone, and the combination of GEM with PF-03084014 resulted in a 3.72 and 3.04-fold decrease in CD24+CD44+ tumor cells compared with GEM treatment, respectively. TUNEL and Ki-67 staining demonstrated that combination therapy induced apoptosis and suppresses cell proliferation. Conclusions: The findings presented here provide strong rationale for the clinical investigation on notch inhibition as a chemotherapeutic strategy to combat pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3262. doi:1538-7445.AM2012-3262

In vitro models

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models.

Abstract LB-251: Isolation, expansion, and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer

Abstract The cancer stem cell (CSC) theory suggests that cancer is maintained by a subpopulation of cancer cells which possesses stem cell characteristics, including self renewal, tumor initiation and pluripotent differentiation abilities. A large series of CSC markers has been reported in various cancer types. CD133 is the most frequently used marker and has been applied to isolate cancer stem cells from cancers of the liver, brain, colon and lung, etc. CD44+/CD24-/low were used as CSC markers in breast cancers. CD34 and Sca-1 were used for identification of murine lung stem cells but Sca-1 is not expressed in human tissues. Expression of embryonic genes such as BMI1 and POU5F1 were found in CSC from different tissues and both proteins are key components in maintaining ‘stemness’ of embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells. It still remains unclear whether additional markers are needed to detect lung CSC. To our knowledge, limited studies using CSC markers on a panel of non-small cell lung cancer (NSCLC) cell lines including those raised from Chinese lung cancer patients have been documented. This study aimed to examine the expression profiles of 6 CSC markers (CD24, CD34, CD44, CD133, BMI1 and POU5F1) in a panel of 10 NSCLC cell lines, 163 NSCLC tumor specimens, fetal lung, reactive and regenerating lung tissues. NSCLC cell lines showed expression of CSC markers at various frequencies that ranged from 0-99.7%, suggesting absence of a single universal marker. Six cell lines expressed CD44 while only HCC1833 expressed CD133. Expression of CD44 and CD133 were mutually exclusive. The CD133+ and CD44+ populations showed distinct properties from CD133- and CD44- cells, respectively. Both CD133+ and CD44+ expanded into tumor-spheroids in vitro in non-adherent medium but not CD133- and CD44- cells. CD44+ H1299 cells expressed ‘stemness’ markers including Oct4, Nanog and Sox2 by RT-PCR and immunofluorescence but were lost after induced-differentiation. The clonogenicity of CD44+ cells is significantly higher than CD44- cells and self-renewal ability was maintained after serial passages. CD44+ cells were cisplatin-resistant and initiated tumor formation in nude mice. The xenograft tumors showed poor differentiation that resembled the parental cell line. Freshly sorted tumor cells showed both CD44+ and CD44- populations, suggesting in vivo differentiation. Immunohistochemistry (IHC) results showed CD44 expression in epithelia of developing airways and regenerating lung. In lung cancers, CD44 expression was associated with squamous cell carcinoma (SCC) (P=0.046). When only 96 adenocarcinomas (AD) were included, CD44 expression was associated with non-smokers (P=0.024). Overall, CD44+cells in NSCLC are capable of tumor-initiation and possess stem cell characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-251.

Identification of molecular targets associated with transformed diffuse large B cell lymphoma using highly purified tumor cells

Follicular lymphoma (FL) frequently transforms into the more aggressive diffuse large B cell lymphoma (DLBCL-tr), but no protein biomarkers have been identified for predictive or early diagnosis. Gene expression analyses have identified genes changing on transformation but have failed to be reproducible in different studies, reflecting the heterogeneity within the tumor tissue and between tumor samples. Gene expression analyses on Affymetrix Human Genome U133 Plus 2.0 arrays were performed, using flow cytometry sorted tumor cells derived from FL and transformed DLBCL. To identify molecular targets associated with the transformation, subsequent immunohistochemistry (IHC) analyses of the corresponding proteins were performed. Using highly purified cells, this study identified 163 genes, which were significantly deregulated during the transformation in a majority of cases. Among the upregulated transcripts, 13 genes were selected for validation using IHC, based on the availability of commercial antibodies, and galectin-3 and NEK2 proteins specifically identify DLBCL-tr, when compared with FL. We demonstrate that by purifying tumor cells through cell sorting, thereby reducing the heterogeneity due to infiltrating cells, it was possible to identify distinct differences between tumor entities rather than variations due to cellular composition. Galectin-3 and NEK2 both identified a subgroup of DLBCL-tr, and the function of these protein markers also suggests a biological role in the transformation process.

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

Abstract 4997: Prospective identification, tracking, and targeting of cancer-initiating cells in breast cancer and glioma via low proteasome activity

Abstract Cancer initiating cells (CICs) in breast cancer and glioma are resistant to radiation and chemotherapy. Complete elimination of these cells from the tumor mass determines therapeutic success. Thus, prospective identification of this cell population is key to developing novel targeting strategies for CICs. The current surface marker combinations employed in identifying CICs have several shortcomings. Here we report a novel marker for CICs that allows identification, tracking, and targeting of CICs in-vitro and in-vivo. Proteasome activities of human (U87 and U87viii) and murine (GL261) glioma cells, as well as human (MCF-7 and MDA-MB-231) and murine (67NR) breast cancer cells were analyzed using fluorogenic peptide substrates. Stable cell lines were generated with retroviruses coding for fusion proteins of ZsGreen and the degron of murine ornithine decarboxylase (ODC), or in combimation with thymidine kinase (TK). Accumulation of ZsGreen was monitored by fluorescence microscopy and flow cytometry. Self-renewal capacity was assessed in sphere forming assays, and tumorgenicity was determined by injection of sorted tumor cells into nude mice. Cell populations enriched for CICs demonstrated significantly reduced activities of the 26S proteasome and showed differential expression of proteasome subunits. Accumulation of ZsGreen corresponded with the CD24-/low/CD44+ phenotype in MCF-7 cells, and glioma ZsGreen-positive cells expressed high levels of the stem cell marker, nestin. Cells with low proteasome function exhibited a 5-fold increased sphere forming capacity, and 3-log increased tumorgenicity. Histologically, ZsGreen-positive U87 cells resided in a perivascular niche, and stained negative for Ki67. Optical in-vivo imaging revealed a significant increase in the number of ZsGreen-positive cells after treatment with sublethal doses of radiation. Fractionated sublethal irradiation of these xenografts caused induction of jagged-1 and notch-1 expression, and induced Ki67 expression in the zsGreen-positive population. Treatment of TK-ZsGreenODC-expressing U87 cells with ganciclovir eliminated cells with low proteasome activity, and subsequently prevented sphere formation in-vitro, and caused complete regression of tumors in-vivo. We conclude that low proteasome activity is a unique characteristic of CICs. Cell lines infected with reporter constructs for proteasome activity can be utilized to identify CICs and track their response to cancer treatments. Most importantly, this feature of CICs can be exploited as a specific target for selective elimination of CICs, leading to improved tumor control.

The Gene Expression Profile of Nodal T-Cell Lymphomas Identifies a Molecular Link between Angioimmunoblastic T-Cell Lymphoma (AITL) and Follicular Helper T Cells (TFH), and between CD30+ Peripheral T-Cell Lymphoma and ALK-Negative Anaplastic Large Cell Lymphoma (ALCL).

AITL and PTCL-U, the two most common forms of T-cell lymphomas in western countries, usually present as nodal disease and pursue an aggressive clinical course. AITL is commonly associated with a constellation of clinical symptoms and distinct pathological features. Conversely, PTCL-U lacks precise diagnostic criteria, and by default comprises cases not fulfilling criteria for other entities, including tumors with borderline features to ALCL and AITL. The genetic alterations and pathogenic mechanisms underlying AITL and PTCL-U are largely unknown. To determine whether the molecular signature of AITL and PTCL-U could help in distinguishing both entities and in understanding ther ontogeny, we performed gene expression profile (GEP) analysis of 15 PTCL-U tissue samples (6 CD30+ and 9 CD30−) and 19 AITL samples (including 2 sorted tumor cell suspensions) using Affymetrix HG-U133A Plus2.0 pan-genomic oligonucleotide microarrays, with comparison to that of previously published normal T-cell subsets (J Immunol 173:68; J Immunol 175: 7837; Blood 104: 1952). Principle component analysis (PCA, accumulated variance 95%) of all 33 tissue samples yielded three groups of tumors: one group of 12 AITLs, one group of 10 PTCLs-U and one mixed group comprising 5 AITLs (some with features borderline to PTCL-U) and 6 PTCLs-U (including 5 of 6 CD30+ tumors). The AITL molecular signature consisted of 442 genes with increased levels of expression in AITL compared to PTCL-U (t test, p<0.002), including genes encoding cell adhesion molecules, immune receptors, extracellular matrix components and several chemokines, B-cell-related and follicular dendritic cell-related genes, genes involved in endothelial and vascular biology, and several genes reported to belong to the gene expression signature of normal TFH cells (CXCL13, BCL6, PDCD1, CD40L, CD200). To specifically address the question of a molecular link beween AITL and TFH cells, we performed gene set enrichment analysis (GSEA) of our dataset using published gene sets specific of distinct normal T-cell subsets (TFH, TH1, TH2). Compared to that of PTCL-U, the molecular signature of AITL was significantly enriched in TFH-specific genes, and the enrichment was even higher for sorted AITL cells compared to AITL tissues. GSEA failed to disclose a molecular link between PTCL-U and known T-cell subsets (TH1, TH2, TFH). Compared to CD30− PTCL-U, CD30+ PTCL-U had lower expression of genes involved in TCR signalling (t test, p<0.002), and showed molecular similarities with ALK-negative ALCL. In conclusion, GEP of non-anaplastic nodal PTCL (1) segregates AITL and PTCL-U, supporting the basis for histotyping; (2) shows molecular analogies between AITL and TFH cells, strongly supporting the hypothesis of a histogenetic link; (3) suggests molecular analogies between CD30+ PTCL-U and ALK-negative ALCL.

A randomized phase I trial of melphalan + bortezomib as conditioning for autologous transplant for myeloma

17550 High dose chemotherapy and autologous transplant (HDT) clearly benefits most patients with myeloma, but the addition of chemotherapy or TBI to melphalan (M) does not improve outcomes. Bortezomib (B) is a proteasome inhibitor which synergizes with chemotherapy due to its effects on DNA repair enzymes. Recent data has shown that B upregulates the anti-apoptotic protein MCL-1, which would suggest that B followed by M is inferior to M followed by B. We designed a randomized phase I trial combining B and Melphalan 200 mg/m2 (Mel200) in order to determine the toxicity, optimal dose and sequence of administration. Patients were randomized to receive either B 24 hours before Mel 200 or B 24 hours after Mel 200. Standard transplant criteria were used with the addition of requiring measurable numbers of plasma cells at the time of transplant. Enrolled patients underwent BM aspirate on day -4 (before B) and day 0 (before PBSC infusion). Bone marrows were tested for apoptosis, and myeloma cells were sorted for protein analysis. Age range was 48–74 years. Prior therapy has included Velcade (n = 2, both responsive) thalidomide (n = 2) and prior melphalan in 2 (1 HDT). Time to WBC and Plt engraftment were not different from historical cohorts receiving MEL 200 alone. Four patients have been randomized to date with 3 in the B “before“ arm, and 1 in the B “after” arm. Of the 3 patients who received B before M, day 0 bone marrow revealed minimal changes in marrow myeloma cell apoptosis in two patients, and in one patient a &gt;20 fold increase in myeloma cell apoptosis on the day 0 marrow sample when compared to the day - 4 marrow. In the patient enrolled in the B after M arm, there was a 10 fold increase in the day 0 marrow myeloma cell apoptosis. Western blot analysis for DNA repair enzymes and MCL-1 on sorted tumor cells are planned, as is ELISA for secreted cytokines before and after therapy. Toxicity and response data with an update of the molecular correlates will be presented. The combination of B and M as conditioning for HDT is feasible. The optimal dose and sequence of administration remains unknown. Accrual continues. [Table: see text]

Minimal Disease Detection and Confirmation in Hematologic Malignancies: Combining Cell Sorting with Clonality Profiling

In this study we demonstrate the technical application of flow cytometry and cell sorting combined with gene-rearrangement clonality profiling to detect and confirm minimal disease in 2 leukemia and 2 lymphoma cases. Specimens with low percentages (0.05%-5%) of abnormal lymphoid populations were identified by flow cytometry. The abnormal lymphoid populations were sorted by flow cytometry, and the purified tumor populations along with unsorted fractions were subsequently analyzed for the presence of clonal gene rearrangements by PCR and fluorescence-based capillary electrophoresis fragment analysis. In 3 cases, distinct clonality profiles could be detected in the purified tumor cell fraction, and suspicious amplicons of identical sizes were detected among the polyclonal backgrounds in the unsorted specimens. For 1 patient, a monoclonal signal was detected in the sorted tumor cell fraction but not in the unseparated bone marrow specimen containing 0.05% abnormal lymphoblasts. A subsequent bone marrow specimen containing 4.8% recurring leukemia cells tested positive with a clonality profile that matched the previous profile in the sorted cell population. The described method integrating 2 technologies allows genotypic confirmation of an aberrant population detected by immunophenotype to increase diagnostic certainty. This strategy provides a sensitive tool for disease monitoring without the need for patient-specific primer design and assay optimization required for quantitative PCR analysis.

Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation

A method for labelling the intracellular ras oncogene product, p21, with a monoclonal antibody, in B16BL6 mouse melanoma cells for subsequent flow cytometric analysis and viable cell sorting is described. Permeabilization of the cells for introduction of labelled antibody was attempted using (1) lysolecithin treatment, and (2) electroporation, a much more highly controllable technique. Permeabilization was assessed using propidium iodide or calcofluor white M2R staining, while short-term cellular viability was determined using fluorescein diacetate staining and long-term viability by reculturing the sorted cells. We successfully introduced labelled antibody into the cells with both permeabilization techniques. Insufficient numbers of viable permeabilized cells were obtained with lysolecithin treatment to warrant an attempt at viable cell sorting. On the other hand, good numbers of viable, permeabilized cells were obtained using electroporation and we successfully sorted viable tumor cell populations based on the intensity of their anti-p21 ras staining. These sorted tumor cells retained their characteristic anti-p21 ras staining intensity for at least 2 weeks of propagation in culture.