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Proteomics and phosphoproteomics data analyses II

Cell death analysis

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Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741–52. ©2017 AACR.

Tumor cell heterogeneity is a crucial characteristic of malignant brain tumors and underpins phenomena such as therapy resistance and tumor recurrence. Advances in single‐cell analysis have enabled the delineation of distinct cellular states of brain tumor cells, but the time‐dependent changes in such states remain poorly understood. Here, we construct quantitative models of the time‐dependent transcriptional variation of patient‐derived glioblastoma (GBM) cells. We build the models by sampling and profiling barcoded GBM cells and their progeny over the course of 3 weeks and by fitting a mathematical model to estimate changes in GBM cell states and their growth rates. Our model suggests a hierarchical yet plastic organization of GBM, where the rates and patterns of cell state switching are partly patient‐specific. Therapeutic interventions produce complex dynamic effects, including inhibition of specific states and altered differentiation. Our method provides a general strategy to uncover time‐dependent changes in cancer cells and offers a way to evaluate and predict how therapy affects cell state composition., A single cell‐based strategy that tracks and models time‐dependent changes in brain tumor cells indicates that patient‐derived glioblastoma cells follow a near‐hierarchical organisation that can be altered by therapeutic agents.

Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1β). IL1β induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called 'reactive GICs'. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Pharmacological treatment of complex diseases using more than two drugs is commonplace in the clinic due to better efficacy, decreased toxicity and reduced risk for developing resistance. However, many of these higher-order treatments have not undergone any detailed preceding in vitro evaluation that could support their therapeutic potential and reveal disease related insights. Despite the increased medical need for discovery and development of higher-order drug combinations, very few reports from systematic large-scale studies along this direction exist. A major reason is lack of computational tools that enable automated design and analysis of exhaustive drug combination experiments, where all possible subsets among a panel of pre-selected drugs have to be evaluated. Results Motivated by this, we developed COMBImage2, a parallel computational framework for higher-order drug combination analysis. COMBImage2 goes far beyond its predecessor COMBImage in many different ways. In particular, it offers automated 384-well plate design, as well as quality control that involves resampling statistics and inter-plate analyses. Moreover, it is equipped with a generic matched filter based object counting method that is currently designed for apoptotic-like cells. Furthermore, apart from higher-order synergy analyses, COMBImage2 introduces a novel data mining approach for identifying interesting temporal response patterns and disentangling higher- from lower- and single-drug effects. COMBImage2 was employed in the context of a small pilot study focused on the CUSP9v4 protocol, which is currently used in the clinic for treatment of recurrent glioblastoma. For the first time, all 246 possible combinations of order 4 or lower of the 9 single drugs consisting the CUSP9v4 cocktail, were evaluated on an in vitro clonal culture of glioma initiating cells. Conclusions COMBImage2 is able to automatically design and robustly analyze exhaustive and in general higher-order drug combination experiments. Such a versatile video microscopy oriented framework is likely to enable, guide and accelerate systematic large-scale drug combination studies not only for cancer but also other diseases. Electronic supplementary material The online version of this article (10.1186/s12859-019-2908-0) contains supplementary material, which is available to authorized users.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741–52. ©2017 AACR.

LSD1 has emerged as a potential therapeutic target for a number of cancer types (e.g. AML, SCLC, colorectal, breast, liver, prostate, glioblastoma, Ewing sarcoma), as well as sickle cell anaemia and Alzheimer’s disease. Irreversible LSD1 catalytic inhibitors have shown limited clinical efficacy in AML and SCLC, while solid tumours are largely unaddressed. This is in contrast to LSD1 knockdown with impact on a wide range of cancers, indicating that LSD1 functions other than enzymatic should be targeted. We have developed novel small molecules that modulate LSD1 via an allosteric site - without inhibiting its enzymatic function - inducing a 60% reduction of nuclear LSD1 levels. The sensitivity profile in a cancer cell line panel is unique and dissimilar to >300 diverse reference compounds. In vitro efficacy is observed in glioma-initiating clones that are highly resistant to standard-of-care temozolimide as well as catalytic LSD1 inhibitors. Efficacy in the sub-µM range is observed with other solid tumour models, e.g. prostate cancer. The compounds exhibit synergy (Bliss independence >40%) with HDAC inhibitors as evaluated by viability in cellular cancer models, including lung, liver and glioblastoma. Pharmacokinetic studies show good blood-brain-barrier penetration and oral availability of the allosteric LSD1 modulator BEA-17. A repeat dose of 25 mg/kg was well tolerated by NOD SCID mice, leading to µM level accumulation in the brain. Results from orthotopic glioblastoma PDX models, and in vivo hollow-fiber models of other solid tumours will be presented, as well as mechanistic insights from biophysical assays and gene expression analysis. Citation Format: Wei B. Emond, Matthis Geitmann, Malin Jarvius, Konrad Koehler, Per Källblad, Mia Niklasson, Vendela Parrow, Rajiv Sawant, Maria Sjöberg, Johan Winquist, Anna Segerman, Ulf Bremberg. LSD1 modulation by allosteric ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3843.

Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.

Hematopoietic cells are attractive targets for gene therapy, but the conventional adenovirus (Ad) vectors, based on Ad5, transduce these cells inefficiently. One reason for low permissiveness of hematopoietic cells to infection by species C Ads appears to be inefficient attachment. Vectors pseudotyped with species B fibers are clearly more efficient at transducing hematopoietic cells than Ad5. To evaluate which Ad species B type(s) would be the most efficient vector(s) for primary T-cells, B-cells and monocytes, attachment to and entry of the species B1 serotypes 3p and 7p and the species B2 serotypes 11p and 35 into primary PBMCs was studied. Ad11p and Ad35 were the only serotypes to show efficient binding and for which uptake by PBMCs could be detected. Infection of PBMCs by Ad5, Ad11p and Ad35 was compared. Expression of Ad hexons was detected in stimulated PBMCs, most frequently in T-cells, and in unstimulated monocytes, although B-cells appear to be refractory to productive infection. Replication of Ad DNA was severely restricted in most PBMCs. Neither hexon expression nor genome replication could be detected in unstimulated lymphocytes, but FISH and a real-time PCR-based assay suggested that Ad11p and Ad35 DNA reach the nucleus. Activation thus appears to be required for T-cells to be permissive to Ad gene expression. In summary, there are substantial differences between Ad3p and Ad7p on the one hand and Ad11p and Ad35 on the other, in their ability to interact with PBMCs. Ad11p and Ad35 probably represent vectors of choice for these cell types.

Gene transfer into human hematopoietic stem cells using Ad5 is inefficient due to lack of the primary receptor CAR and the secondary receptors alphavbeta3 integrin and alphavbeta5 integrin, and due to the high seroprevalence of Ad5 antibodies in most adults, resulting in diminished gene transduction. In the present study, we screened six species (species A-F) of adenovirus, displaying different tropisms for interaction with CD34+ cells, at the level of virus attachment and expression. Virus particles were biotinylated and their binding capacity was determined by FACS analysis using streptavidin-FITC. Ad11p, Ad35, and Ad3 (species B) showed high binding affinity, while Ad7, Ad11a (species B), and Ad37 (species D) displayed intermediate affinity. Virions of Ad4 (species E), Ad5 (species C), Ad31 (species A), and Ad41 (species F) hardly bound to hematopoietic progenitor cells. Using a double-labeling system, we demonstrated that adenoviruses bind to quiescent CD34+ cells. Ad11p virions showed the highest affinity among the adenoviruses detected. We further confirmed that virus fiber-specific receptors were present on the hematopoietic progenitor cell surface, because both recombinant fiber of Ad11p and specific antiserum against rfiber could block virus attachment. The ability of the adenoviruses to infect hematopoietic cells was studied by immunofluorescence staining. The adenoviruses from species B and Ad37 showed higher infectivity than Ad31, Ad5, Ad4, and Ad41. Among the studied species B adenoviruses, Ad11p manifested a superior infectivity. Thus, we have confirmed that these cells have high-affinity receptors for species B:2 human adenovirus, Ad11p, and this virus may be used as candidate vector to target therapeutic genes to hematopoietic stem cells.

The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.

Unlike most adenovirus (Ad) serotypes, the species B Ads do not use the coxsackie-adenovirus receptor as an attachment receptor. The species B attachment receptor(s) has not yet been identified and is also poorly characterized. Species B Ads can be further divided into species B1 and B2 Ads, and these display different organ tropisms, suggesting a difference in receptor usage. We have studied the receptor interactions of the species B1 serotypes 3p and 7p and the species B2 serotypes 11p and 35 and characterized the properties of the species B receptor(s). Reciprocal blocking experiments using unlabeled Ad11p or Ad3p virions to block the binding to A549 cells of 35 S-labeled 3p, 7p, 11p, and 35 showed that only Ad11p virions efficiently blocked the binding of all the species B Ads studied (≥70%). Thus, there is apparently a common species B Ad receptor (sBAR). However, Ad3p virions only partially (≤30%) blocked the binding of Ad11p and Ad35 to A549 cells. Binding experiments after trypsin treatment of the cells confirmed that the species B2 serotypes address at least two different receptors on A549 and J82 cells, since sBAR is trypsin sensitive but the species B2 Ad receptor (sB2AR) is not. Both receptors are proteins or glycoproteins, since binding of all species B serotypes was abolished after proteinase K or subtilisin treatment of A549 or J82 cells. Furthermore, binding of the species B serotypes to sBAR was abolished with EDTA and restored with Ca 2+ , whereas the binding of Ad11p and Ad35 to SB2AR was independent of divalent cations.

The overall aim of this study was to find ways to sensitize treatment resistant glioma-initiating cell (GIC) clones to conventional therapy. Heterogeneity is substantial in glioblastoma multiforme (GBM) and through establishment of clonal GIC cultures from patient biopsies we have demonstrated a wide variety in the responses to drugs and radiation. A multitherapy resistance phenotype was linked to proneural-mesenchymal transition (PMT) in the transcriptome. The variety in therapy response was observed as a continuum of phenotypes. The distribution of phenotypes resembled a normal distribution and multitherapy resistance was associated with low DNA methylation grade in promoter regions of mesenchymal master regulators (FOSL2, RUNX1). Our data thus implied that the transition is bi-directional and epigenetically regulated (Segerman et al, Cell Reports - accepted in principle). To investigate if spontaneous changes in drug and radiation response occur, we have derived subclones from a resistant clone. Both subclones with higher and lower therapy resistance than the parental clone were generated. Also molecularly the subclones largely reconstituted the original clonal variation. PMT shows similarities to epithelial-mesenchymal transition (EMT), which is induced by extrinsic factors. We therefore specifically analyzed the gene expression data for signaling receptors differentially expressed in resistant vs. sensitive clones as well as cognate ligands. To estimate the importance of a particular signaling pathway, expression of co-receptors and ligands was taken into account. We found several cases of coherent upregulation of receptor and ligand indicative of autocrine loops. Regarding pathways that appeared to be overactive in resistant vs. sensitive clones, there was a prominent overlap with EMT. It was also apparent that several pathways were activated concomitantly. We are currently focusing on identifying combinations of drugs (and antibodies) that sensitize resistant clones to conventional treatment through modulation of cell signaling patterns. In the initial screen temozolomide (TMZ) response is used as an indicator of achieved sensitization. The strategy is to iteratively combine primarily antagonists of signaling receptors connected to resistance. We are also exploring the effect of stimulating pathways with apparently higher activity in sensitive clones (e.g. addition of ligands).The concept of sensitizing glioma and other types of cancer cells by targeting the mesenchymal character through usage of e.g. signaling receptor inhibitors is not new and has shown promise. Indeed, our preliminary data look encouraging. In conclusion, our data show that multitherapy resistance is connected to a plastic cell-state. Also, receptors and ligands that are differentially expressed in resistant and sensitive clones engage pathways regulating EMT. Citation Format: Mia Niklasson, Malin Jarvius, Caroline Haglund, Efthymia Chantzi, Tobias Bergström, Frida Nyberg, Annika Hermansson, Mårten Fryknäs, Mats Gustafsson, Bo Segerman, Rolf Larsson, Bengt Westermark, Anna Segerman. Targeting of a mesenchymal profile in order to sensitize multitherapy resistant glioblastoma clones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2017-4175

Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP, brain lipid-binding protein) in this extended analysis. In summary, NSC-associated NES+/FABP7(+)/GRIA1(+) GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells.

Glioblastoma multiforme (GBM) remains in the group of incurable malignancies, with a median survival of 15 months. Intratumoral heterogeneity is a key factor driving relapse by providing the basis for escape of therapy-resistant cells. While the heterogeneous genomic and transcriptomic landscape of cancers is currently thoroughly characterized, phenotypic heterogeneity within tumors is less well understood. The overall goal in this study was to identify pre-existing treatment resistant clones with disease relapse potential and biomarkers linked to resistance vs sensitivity. To enable functional studies of isolated tumor clones we adopted an in vitro subcloning strategy. An adherent glioma neural stem cell culturing protocol was used to enrich for cells with stem-like properties and to permit efficient phenotypic screening. We established six libraries of clonal cell cultures from fresh GBM surgical specimens, corresponding to five different patient tumors. In all 708 clonal cultures were expanded. These were considered representatives of the self-renewing compartment within the tumor cell population. We found a remarkable variation in drug and radiation response within the clone libraries. A striking observation was that clones resistant to one drug, also tended to be resistant to most of the drugs we used, regardless of the drug's mechanism of action. This indicates that resistance in large parts is mediated by a general mechanism. Most clones carried genetic aberrations that were unique or found only in a few clones but it was primarily the transcriptome data that demonstrated a clear link to the phenotypic data. A continuous gradient between multi-resistance and sensitivity was found to be connected to a gradual transition between a mesenchymal (MES), in resistant clones, and a more proneural (PN) and proliferative character in sensitive clones. The continuous distributions and lack of discrete groups in clonal response phenotypes and in linked signatures points to a general cell-state related resistance mechanism. We also found that resistant clones had a lower methylation level in promoters of known master regulators of MES subtype associated genes. This indicates that the phenotypic heterogeneity is driven by fluctuations in the epigenetic status. Taken together, our findings imply that intratumoral heterogeneity in GBM includes general clonal resistance mechanisms among glioma- initiating cells driven by epigenetic mechanisms. This evokes hope that new therapeutic approaches involving epigenetic reprogramming can be applied to sensitize cells toward conventional treatment. Citation Format: Anna Segerman, Maria Niklasson, Caroline Haglund, Tobias Bergstrom, Malin Jarvius, Yuan Xie, Demet Caglayan Simov, Annika Hermansson, Marianne Kastemar, Zeinab Naimaie-Ali, Malin Berglund, Ann Westermark, Magnus Sundstrom, Goran Hesselager, Lene Uhrbom, Mats Gustafsson, Rolf Larsson, Marten Fryknas, Bo Segerman, Bengt Westermark. GBM exhibits phenotypic microheterogeneity and harbors pre-existing multi- resistant clones with a mesenchymal transition signature. [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 2415.

The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Glu substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46.

Hematopoietic cells are attractive targets for gene therapy. However, no satisfactory vectors are currently available. A major problem with the most commonly used adenovirus vectors, based on adenovirus type 2 (Ad2) or Ad5, is their low binding efficiency for hematopoietic cells. In this study we identify two adenovirus serotypes with high affinity for hematopoietic cells. The binding efficiency of prototype serotypes Ad4p, Ad11p, and Ad35p for different committed hematopoietic cell lines representing T cells (Jurkat), B cells (DG75), monocytes (U937-2), myeloblasts (K562), and granulocytes (HL-60) was evaluated and compared to that of Ad5v, the commonly used adenovirus vector, using flow cytometry. In contrast to Ad5v, which bound to less than 10% of the cells in all experiments, Ad11p and Ad35p showed high binding efficiency for all of the different hematopoietic cell lines. Ad4p bound to the lymphocytic cell lines to some extent but less well to the myelomonocytic cell lines. The abilities of the different serotypes to infect, replicate, and form complete infectious particles in the hematopoietic cell lines were also investigated by immunostaining, 35 S labeling of viral proteins, and titrations of cell lysates. Ad11p and Ad35p infected the highest proportion of cells, and Ad11p infected all of the cell lines investigated. The Ad11p hexon was expressed equally well in K562 and A549 cells. Jurkat cells also showed high levels of expression of Ad11p hexons, but the production of infectious particles was low. The binding properties of virions were correlated to their ability to infect and be expressed.

Glioblastoma (or WHO grade IV glioma) is the most common and most malignant primary brain tumor, in adults. Despite gradual optimization of the conventional treatment protocol, the prognosis for these patients is still poor. The tumors are very heterogeneous and division into four main groups has been suggested. Although intra-tumor heterogeneity has long been regarded a major cause for treatment failure the extent of glioma intra- tumor heterogeneity at the cellular level is not well known. Heterogeneity can arise by two parallel but interdependent mechanisms. (i) Sequential accumulation of mutations as a consequence of genetic instability. (ii) Hierarchical organization of glioma cells. High grade gliomas contain subpopulations of tumor cells with stem-like properties with a unique ability to reconstitute the original tumor when xenografted and have been referred to as glioma initiating cells (GICs). GICs resist conventional treatment and the intense interest is based on the assumption that accurate targeting of these cells will cure glioma patients. Separation and direct comparision of GICs and non-GICs is difficult since universal and exclusive GIC (or non-GIC) markers are missing. GICs are instead defined by functional criteria, self-renewal under neural-stem cell (NSC) culture conditions, multipotency and most importantly, ability to reconstitute the morphology (and expression pattern) of the original tumor when xenografted to immunocompromised mice. In vitro culturing under NSC conditions (as spheres or adherent to laminin) strongly selects for GICs. Conversely, the tumor forming ability of human glioma cells have repeatedly been observed to be repressed by serum or BMP exposure which also slow or cease proliferation in NSC and induce expression of markers associated with glial/neuronal differentiation. Thus, the malignant phenotype of glioma cells can be modulated by external factors. We are here setting up a marker independent separation method based on functional properties of GICs and non-GICs, respectively. Glioma cells exposed to serum, i.e. an induced mixed population of GICs and non-GICs, were transferred to NSC and probed with CMFDA to follow proliferation rates. After 12-14 days in NSC culture, two subpopulations of U2987MG cells were visible, one highly proliferative (HP) and one low proliferative (LP). Immunostainings for stemness and glial lineage markers identified GFAP when combined with S100 or MAP-2 as robust markers for the LP group of cells. The HP and LP subgroups as well as a GFAP+ clone with LP properties were orthotopically xenografted to SCID mice. HP cells gave rise to big, high grade tumors whereas injection of the GFAP+ clone with LP properties resulted in very small non-invasive tumors. Our preliminary data thus suggests that this is a useful approach. We are now analyzing these subgroups using transcriptome sequencing. 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 5356. doi:1538-7445.AM2012-5356

Top of pageAbstract Background: Adenoviruses of species A, B, C, D, E and F differ in tropism. In order to address the molecular determinants of adenovirus tropism the distribution of adenovirus receptors on cells of hematopoietic and endothelial origin, and the expression in these cells was assessed. Results: The receptors for species B adenoviruses were most abundantly expressed. CAR binding amino acid residues are not conserved in their fiber knobs consequently CAR can not be used as a receptor. Virions of Ad11p, Ad35 & Ad3 (species B) showed high- affinity to CD34(+) cells, Ad7, Ad11a (species B) and Ad37 (species D) showed intermediate affinity whereas Ad4 (species E), Ad5 (species C), Ad31 (species A) and Ad41 (species F) hardly bound to CD34 hematopoietic progenitor cells. Only species B and species D adenovirus were expressed in these cells. Based on DNA homology two groups are defined within species B. Ad3, 7, 16, 21 (B:1), and Ad11, 14, 34, 35 and 50 (B:2). Ad3 and Ad7 cause acute respiratory tract infections, whereas Ad11, 34 and 35 cause persistent infections of the urinary tract. We suggest two different receptors for species B adenoviruses: sBAR common to species B members and sB2AR unique for Ad11 and Ad35 members of species B:2. Ad11 virions blocked the binding of Ad3 or Ad7 species B to 70%, whereas Ad3 blocked the binding of Ad11 and Ad35 only partially 30%. sBAR is sensitive to trypsin and EDTA treatment of A549 cells or J82 cells whereas sB2AR is resistent. The binding of Ad3 and Ad7 was restored with Ca++. Non permissive chinese hamster ovary (CHO) cells were transfected with the BC1 or BC2 isoforms of CD46 |[ndash]| cDNA. Ad11 virions did but Ad5 virions did not bind to CD46 transfected CHO cells. Adenovirus 11 and 35 can attach and infect primary lymphocytes and monocytes but hexon expression in T- cells required prior activation, whereas neither Ad3, Ad7 nor Ad5 were expressed in the primary PBMCs. Assessment of the transduction ability of established hematopoietic cell lines by a species B vector revealed that Jurkat, U937 and K562 were more efficiently transduced than DG75 cells. EKC causing Ad37 (species D) uses 2-3 linked sialic acid as a primarily functional receptor. The structure of the fiber knob of Ad37 and Ad19p was resolved at 1.5-2|[Aring]| by W. Burmeister using X-ray crystallography. The binding site for sialyl lactose was identified on the top of the knob close to the 3-fold axis of the trimer. NB Ad37 infects CD34+ cells Conclusion: Species B and D adenovirus using CD46 and 2-3- linked sialic acid as receptors, respectively, infected hematopoietic progenitor cells. Ad11 and Ad35 were more efficient than all other assessed adenoviruses.

Gene transfer into human hematopoietic stem cells using Ad5 has been shown to be inefficient due to lack of the primary receptor CAR and the secondary receptors avβ3 integrin and avβ5 integrin, and due to the high seroprevalence of Ad5 antibodies in most adults, resulting in diminished gene transduction. In the present study, we screened 6 species (species A-F) of adenovirus, displaying different tropisms for interaction with CD34+ cells, at the level of virus attachment and expression. Virus particles were biotinylated and their binding capacity was determined by FACS analysis using streptavidin-FITC. Ad11p, Ad35 and Ad3 (species B) showed high binding affinity, while Ad7, Ad11a (species B) and Ad37 (species D) displayed intermediate affinity. Virions of Ad4 (species E), Ad5 (species C), Ad31(species A) and Ad41(species F) hardly bound to hematopoietic progenitor cells. By using a double-labeling system, we demonstrated that adenoviruses did bind to quiescent CD34+ cells. Ad11p virions showed the highest affinity. We further confirmed that virus fiber-specific receptors were present on the hematopoietic progenitor cell surface, since both recombinant fiber of Ad11p and specific antiserum against rfiber could block virus attachment. The ability of Ad11p, Ad35 and Ad5 to infect hematopoietic cells was studied by immunofluorescence staining. Ad11p and Ad35 showed higher infectivity than Ad5. Thus, we have confirmed that these cells have high affinity receptors for species B:2 human adenoviruses Ad11p and Ad35, and these viruses may be used as candidate vectors to target therapeutic genes to hematopoietic stem cells.