Else Munthe

Characterization of early stages of human B cell development by gene expression profiling.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population.

Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes

Background:Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations.Methods:To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling.Results:The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes—COL1A2, KYNU, ACTG2 and NPPB—were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease.Interpretation:The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines.

Characterization of liposarcoma cell lines for preclinical and biological studies

Liposarcoma cell lines represent in vitro models for studying disease mechanisms at the cellular level and for preclinical evaluation of novel drugs. To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacity in vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited for in vivo analyses (LPS141, SA-4, T778, SW872, and LISA-2), for testing novel drugs targeting cancer stem cells (LPS141) and for studying tumor progression and metastasis (T449 and T778).

The tankyrase‐specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

Wnt/β‐catenin is a major regulator of stem cell self‐renewal and differentiation and this signaling pathway is aberrantly activated in a several cancers, including osteosarcoma (OS). Attenuation of Wnt/β‐catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines (KPD, U2OS, and SaOS‐2) both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β‐catenin destruction complex, resulting in reduced levels of nuclear β‐catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase‐3‐mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let‐7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let‐7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS.

Liposarcoma cells with aldefluor and CD133 activity have a cancer stem cell potential

Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0, 1-1, 7% of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells.

Bone marrow stroma cells regulate TIEG1 expression in acute lymphoblastic leukemia cells: Role of TGFβ/BMP-6 and TIEG1 in chemotherapy escape

The bone marrow microenvironment regulates early B lymphopoiesis and protects leukemia cells against chemotherapy treatment, thus the microenvironment may serve as a sanctuary site for these cells. Yet, few factors that contribute to this process are known. We have explored the role of transforming growth factor β (TGFβ) and bone morphogenetic protein‐6 (BMP‐6) and one target gene, TGFβ inducible early gene 1 (TIEG1), in the communication between stroma cells and acute lymphoblastic leukemia (ALL) cell lines and their escape from chemotherapy. Here, we have demonstrated TIEG1 expression in both normal B progenitor cells and ALL cells, which increased rapidly upon TGFβ and BMP‐6 treatment. Stimulation with TGFβ or BMP‐6, as well as overexpression of TIEG1 inhibited proliferation. Furthermore, interaction with stroma cells induced TIEG1 expression in ALL cells, inhibited their proliferation and protected the cells against chemotherapeutic treatment. Similarly, treatment with TGFβ or BMP‐6, as well as overexpression of TIEG1, protected ALL cells against chemotherapy‐induced cell death. These data suggest that TGFβ and BMP‐6 in the bone marrow microenvironment allow leukemia cells to escape therapy. Further, the data indicate that TIEG1 might be involved in mediating this effect from the microenvironment onto the leukemia cells.

The Genomic landscape of pancreatic and periampullary Adenocarcinoma

Despite advances in diagnostics, less than 5% of patients with periampullary tumors experience an overall survival of five years or more. Periampullary tumors are neoplasms that arise in the vicinity of the ampulla of Vater, an enlargement of liver and pancreas ducts where they join and enter the small intestine. In this study, we analyzed copy number aberrations using Affymetrix SNP 6.0 arrays in 60 periampullary adenocarcinomas from Oslo University Hospital to identify genome-wide copy number aberrations, putative driver genes, deregulated pathways, and potential prognostic markers. Results were validated in a separate cohort derived from The Cancer Genome Atlas Consortium (n = 127). In contrast to many other solid tumors, periampullary adenocarcinomas exhibited more frequent genomic deletions than gains. Genes in the frequently codeleted region 17p13 and 18q21/22 were associated with cell cycle, apoptosis, and p53 and Wnt signaling. By integrating genomics and transcriptomics data from the same patients, we identified CCNE1 and ERBB2 as candidate driver genes. Morphologic subtypes of periampullary adenocarcinomas (i.e., pancreatobiliary or intestinal) harbor many common genomic aberrations. However, gain of 13q and 3q, and deletions of 5q were found specific to the intestinal subtype. Our study also implicated the use of the PAM50 classifier in identifying a subgroup of patients with a high proliferation rate, which had impaired survival. Furthermore, gain of 18p11 (18p11.21-23, 18p11.31-32) and 19q13 (19q13.2, 19q13.31-32) and subsequent overexpression of the genes in these loci were associated with impaired survival. Our work identifies potential prognostic markers for periampullary tumors, the genetic characterization of which has lagged. Cancer Res; 76(17); 5092-102. ©2016 AACR.

Preclinical evaluation of potential therapeutic targets in dedifferentiated liposarcoma

Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemotherapy and/or radiotherapy in combination with surgery, and would benefit from new personalized approaches. In this study we demonstrate the potential of combining personal genomic characterization of patient tumors to identify targetable mutations with in vitro testing of specific drugs in patient-derived cell lines. We have analyzed three metastases from a patient with high-grade metastatic dedifferentiated liposarcoma (DDLPS) by exome and transcriptome sequencing as well as DNA copy number analysis. Genomic aberrations of several potentially targetable genes, including amplification of KITLG and FRS2, in addition to amplification of CDK4 and MDM2, characteristic of this disease, were identified. We evaluated the efficacy of drugs targeting these aberrations or the corresponding signaling pathways in a cell line derived from the patient. Interestingly, the pan-FGFR inhibitor NVP-BGJ398, which targets FGFR upstream of FRS2, strongly inhibited cell proliferation in vitro and induced an accumulation of cells into the G0 phase of the cell cycle. This study indicates that FGFR inhibitors have therapeutic potential in the treatment of DDLPS with amplified FRS2.

Generation and characterisation of novel pancreatic adenocarcinoma xenograft models and corresponding primary cell lines

Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection in vitro, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding in vitro cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease.