Stable Cell Population Research Articles

Analysis of circulating CD14+/CD16+ monocyte-derived macrophages (MDMs) in the peripheral blood of patients with oral squamous cell carcinoma

Monocytes/macrophages are regarded as the first line of defense in tumors. Therefore, analyzing monocyte subtypes in oral squamous cell carcinoma (OSCC) may be of value in disease monitoring and to explore immunotherapeutic strategies for cancer patients. Circulating peripheral blood CD14+/CD16+ monocyte-derived macrophages (MDMs) were evaluated in OSCC patients with oral squamous cell carcinoma (n = 44) compared with controls (n = 85). Moreover, epitope detection in monocytes (EDIM) technology was used to detect biomarkers Apo10 and transketolase-like-1 in CD14+/CD16+ MDMs. Compared with controls, no significant (P = .3646) difference (control group 9.8%, OSCC group 8.8%) in CD14+/CD16+ MDM were noted in OSCC. However, EDIM-Apo10 and EDIM-TKTL1 scores detected in the CD14+/CD16+ MDMs were increased in OSCC compared with controls (P < .0001). Analyzing CD14+/CD16+ MDMs represents a stable cell population for detecting biomarkers in cancer disease monitoring.

Genetic analysis and preliminary function study of miR-423 in breast cancer.

Common genetic variants (single nucleotide polymorphisms SNPs) in microRNA (miRNA) genes may alter their maturation or expression and play a role in the formation of human cancer. Recently, the association between the SNP rs6505162 in pre-miR-423 and cancer risk has been frequently evaluated in diverse populations and in a range of cancers. In this study, we determined the genotypes of SNP rs6505162 in 5 matched cell lines (breast cancer cell lines and their corresponding peripheral blood cell lines) and 114 matched clinical specimens (clinical breast carcinoma specimens and their corresponding normal tissues), compared the processing efficiency of pri-miRNA to mature forms between pre-miR-423-12C (wild-type) and pre-miR-423-12A (mutant-type) expression vectors, and evaluated the function of miR-423 on cell proliferation. Our data showed that two out of five breast cancer cell lines and 8.77 % (10/114) of tumors underwent somatic mutations of the rs6505162 SNP, and somatic mutation state was significantly correlated with the expression of clinicopathologic variables, proliferating cell nuclear antigen (PCNA) and mutant p53. The pre-miR-423-12C SNP blocked the endogenous processing of pri-miR-423 to its two mature miRNAs. Interestingly, selected pre-miR-423-12C stable cell population had lower proliferation ability than pre-miR-423-12A stable cell population. Moreover, miR-423 promoted cell proliferation in breast cancer cell lines through its miR-423-3p strand, not miR-423-5p. Taken together, these results suggest that the SNP rs6505162 in pre-miR-423 affects the mature miR expression, and miR-423 plays a potentially oncogenic role in breast tumorigenesis.

Abstract 855: Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy

Abstract Introductory sentence: Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers. Recently, we reported that STAT3 expression correlated with resistance to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical relevance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. We therefore examined whether STAT3 contributes to resistance to chemoradiotherapy. Experimental procedures: STAT3 mRNA and protein expression levels were determined in 12 colorectal cancers cell lines. STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC) in the cell lines SW480 and SW837. Successful RNAi-mediated silencing of STAT3 or inhibition of phosphoSTAT3(Tyr705) was detected by Western blot and reduction of transcription factor activity was measured by a luciferase reporter assay. Additionally, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. To test the influence of STAT3 knock down or inhibition, clonogenic survival assays were performed. Therefore, RNAi or inhibitor treated cells were exposed to chemoradiotherapy using 3µM 5-FU and X-ray-irradiation at 1, 2, 4, 6, and 8 Gy. Finally, we tested the effect of a chemoradiotherapy combined with STATTIC treatment in a SW837 xenograft model in NMRI nude mice. To verify the sensitizing effect of STATTIC, tumor growth was recorded and growth delay assays were performed. Data: STAT3 was overexpressed in resistant cells at mRNA and protein level. siRNA transfected SW480, SW837, and SW480shRNA single cell clones showed a significant reduction of STAT3 protein and transcription factor activity after 96 hours. STATTIC inhibition led to a decreased phosphorylation of STAT3 after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.3 to 2.5 at a surviving fraction of 0.37. In vivo, additional STAT3 inhibition during chemoradiotherapy led to a profound chemoradiosensitization effect and a significant tumor growth delay in STATTIC treated mice. Survival of these mice was also enhanced, if compared to the control group. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing or inhibition of STAT3 leads to a significantly increased chemoradiosensitivity in vitro and in vivo. This highlights the potential relevance of STAT3 for mediating treatment resistance and provides a first proof of concept that STAT3 represents a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: Melanie Spitzner, Birte Roesler, Christian Bielfeld, Carolin Herzberg, Georg Emons, Jochen Gaedcke, Margret Rave-Fränk, Tim Beißbarth, Thomas Ried, B. Michael Ghadimi, Marian Grade. Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 855. doi:10.1158/1538-7445.AM2014-855

Abstract 2413: Unstable genomes and the average cancer cell

Abstract The high degree of multi-level heterogeneity represents a key challenge for cancer research. Despite its importance, both the theoretical concepts and appropriate tools for proper quantitative analysis are underdeveloped. Specifically, the characterization of heterogeneity in cancer cell populations of different genomic stabilities, and its effect on systems dynamics are poorly understood. We use a single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth, and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. We conclude: 1) the karyotype cannot be cloned when genome instability is high, and caution is needed for single cell analysis for cell populations that are not genetically identical; 2) the “average” cancer cell does not exist, as the statistical mean does not accurately describe systems behavior, especially genome instability is high; and 3) single cell analysis is needed when cells are not genomically identical. Our results not only provide an explanation for population heterogeneity but also explain how genome-level heterogeneity contributes to drug resistance and cancer evolution. Note: This abstract was not presented at the meeting. Citation Format: Batoul Abdallah, Joshua B. Stevens, Steven Horne, Guo Liu, Henry HQ Heng. Unstable genomes and the average cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2413. doi:10.1158/1538-7445.AM2014-2413

ExoIN-Technologie — ein schneller Weg zu stabilen Assay-Zelllinien

Clonal cell lines stably expressing a target protein are a common tool in research, but the generation of such cell lines is laborious and time-consuming. In contrast, the novel ExoIN technology guarantees easy-to-handle and fast cell line generation. By expressing selection marker and target as a polyprotein that is efficiently cleaved at the ribosome, unmodified free protein is homogeneously expressed in all cells of the stable cell population.

Microbubble-mediated sonoporation for highly efficient transfection of recalcitrant human B- cell lines.

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult-to-transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B-cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP-positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP-positive cells, respectively. Here, we present sonoporation as a feasible non-viral strategy for stable and highly efficient heterologous transfection of recalcitrant B-cell lines. This is the first demonstration of a non-viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B-cell lines.

PD-1 and CTLA-4 signals are not important for the maintenance of tolerant self-reactive CD8 T cells in the small intestine (MUC8P.808)

Abstract Tolerance is an important mechanism by which the immune system distinguishes between self and non-self. Peripheral T cell tolerance is critical for the control of self-reactive cells that escape negative selection. Anergy is one mechanism that controls self-reactive cells in the periphery and prevents autoimmunity. Using a system that allows for the study self-specific CD8 T cell responses to a known intestinal auto-antigen, we determined that mucosal antigen signaling can lead to a long-term, stable population of cells in the intestine that have a classic anergic phenotype. These cells do not produce effector cytokines or induce intestinal damage. Inhibitory molecules, like PD-1 and CTLA-4, are believed to be important for both the induction and maintenance of T cell tolerance. We show that the induction phase of CD8 T cell tolerance to self was highly sensitive to control through both PD-1 and CTLA-4. However, once tolerance was established, the maintenance of tolerant CD8 T cells was independent of PD-1 or CTLA-4 signaling. Overall, this indicates that distinct mechanisms are responsible for the induction and maintenance of tolerant T cells. In addition, there may be many tolerant T cells resistant to immunotherapeutics targeting these pathways. Determining which subsets of self-reactive cells are susceptible to modulation through these pathways is important for understanding basic mechanisms tolerance and autoimmunity and for improving cancer therapeutics.

Pregnancy-induced maternal regulatory T cells, bona fide memory or maintenance by antigenic reminder from fetal cell microchimerism?

Long-term maintenance of immune components with defined specificity, without antigen is the hallmark feature of immunological memory. However, there are fundamental differences in how memory CD8(+) compared with CD4(+) T cells are maintained. After complete antigen elimination, CD8(+) T cells can persist as a self-renewing numerically stable cell population, and therefore satisfy the most stringent definition of "memory." Comparatively, CD4(+) T cell maintenance is considerably less stable, often requiring low-level antigen persistence or antigenic reminders. Recent studies show these basic memory features, classically ascribed to effector CD8(+) and CD4(+) T cells, extend to immune suppressive Foxp3(+) regulatory CD4(+) T cells (Tregs). In particular, gestational expansion and postpartum retention of maternal Tregs with fetal specificity may explain the protective benefits of primary pregnancy on complications in subsequent pregnancy. Herein, the possibility of ongoing antigenic reminders from fetal cell microchimerism in postpartum maintenance of maternal Tregs with fetal specificity is considered.

Single cell heterogeneity

Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution.

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

Characterization of the cytokine and transcription factor profile of CD4+ T cells from conditional LATY136F mice (P4133)

Abstract LAT is an adaptor protein that is required for TCR signaling and T-cell activation. LATY136F mice have a point mutation at tyrosine 136 of LAT (Latm/m) whichprevents LAT from interacting with PLC-γ1 resulting in defective calcium flux and ERK activation upon TCR stimulation. Our lab created conditional LATY136F mice (LATf/m) in which the floxed allele can be deleted upon tamoxifen treatment. T cells from these mice undergo normal thymic development yet when thewildtype LAT allele is deleted in the periphery, develop a lymphoproliferative disease similar to LATY136F mice. Using these mice we were able to characterize transcription factor and cytokine production over the course of disease development in isolation from defective thymic development. We observed similar symptoms of disease to LATY136F mice includingsplenomegly and lymphadenopathy. Unlike CD4 cells from LATm/m mice, we observed, using immunostainingand qRT-PCR, elevated expression of Th1 cytokines and transcription factors during disease development that then declined by week 5. We also observed a stable population of cells that expressed both Th1 and Th2 cytokines. Finally we observed a transient decline in the pro-survival transcription factor BCL-2 and a steady decline of the pro-survival factor MCL-1. From this data we conclude that disease development in theperiphery of LATf/m mice is dynamic and differs from disease development in LATm/m mice.

Abstract 68: Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy.

Abstract Introductory sentence: Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers. Recently, we reported that STAT3 expression correlated with resistance to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical relevance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. We therefore examined whether STAT3 contributes to resistance to chemoradiotherapy. Experimental procedures: STAT3 mRNA and protein expression levels were determined in 12 colorectal cancers cell lines. STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC) in the cell lines SW480 and SW837. Successful RNAi-mediated silencing of STAT3 or inhibition of phosphoSTAT3(Tyr705) was detected by Western blot and reduction of transcription factor activity was measured by a luciferase reporter assay. Additionally, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. To test the influence of STAT3 knock down or inhibition, clonogenic survival assays were performed. Therefore, RNAi or inhibitor treated cells were exposed to chemoradiotherapy using 3μM 5-FU and X-ray-irradiation at 1, 2, 4, 6, and 8 Gy. Finally, we tested the effect of a chemoradiotherapy combined with STATTIC treatment in a SW837 xenograft model in NMRI nude mice. To verify the sensitizing effect of STATTIC, tumor growth was recorded and growth delay assays were performed. Data: STAT3 was overexpressed in resistant cells at mRNA and protein level. siRNA transfected SW480, SW837, and SW480shRNA single cell clones showed a significant reduction of STAT3 protein and transcription factor activity after 96 hours. STATTIC inhibition led to a decreased phosphorylation of STAT3 after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.3 to 2.5 at a surviving fraction of 0.37. In vivo, additional STAT3 inhibition during chemoradiotherapy led to a profound chemoradiosensitization effect and a significant tumor growth delay in STATTIC treated mice. Survival of these mice was also enhanced, if compared to the control group. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing or inhibition of STAT3 leads to a significantly increased chemoradiosensitivity in vitro and in vivo. This highlights the potential relevance of STAT3 for mediating treatment resistance and provides a first proof of concept that STAT3 represents a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: Melanie Spitzner, Birte Roesler, Christian Bielfeld, Carolin Herzberg, Georg Emons, Jochen Gaedcke, Margret Rave-Fränk, Tim Beißbarth, Thomas Ried, B. Michael Ghadimi, Marian Grade. Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2013-68

Cav1 Suppresses Tumor Growth and Metastasis in a Murine Model of Cutaneous SCC through Modulation of MAPK/AP-1 Activation

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Evolutionary Approaches to Prolong Progression-Free Survival in Breast Cancer

Many cancers adapt to chemotherapeutic agents by upregulating membrane efflux pumps that export drugs from the cytoplasm, but this response comes at an energetic cost. In breast cancer patients, expression of these pumps is low in tumors before therapy but increases after treatment. While the evolution of therapeutic resistance is virtually inevitable, proliferation of resistant clones is not, suggesting strategies of adaptive therapy. Chemoresistant cells must consume excess resources to maintain resistance mechanisms, so adaptive therapy strategies explicitly aim to maintain a stable population of therapy-sensitive cells to suppress growth of resistant phenotypes through intratumoral competition. We used computational models parameterized by in vitro experiments to illustrate the efficacy of such approaches. Here, we show that low doses of verapamil and 2-deoxyglucose, to accentuate the cost of resistance and to decrease energy production, respectively, could suppress the proliferation of drug-resistant clones in vivo. Compared with standard high-dose-density treatment, the novel treatment we developed achieved a 2-fold to 10-fold increase in time to progression in tumor models. Our findings challenge the existing flawed paradigm of maximum dose treatment, a strategy that inevitably produces drug resistance that can be avoided by the adaptive therapy strategies we describe.

Th17 cells are restrained by Treg cells via the inhibition of interleukin-6 in patients with rheumatoid arthritis responding to anti-tumor necrosis factor antibody therapy.

The importance of interleukin-17 (IL-17) is underscored both by its resistance to control by Treg cells and the propensity of Treg cells to produce this highly inflammatory cytokine. This study sought to address whether Th17 cells are inhibited by Treg cells in rheumatoid arthritis (RA) patients responding to anti-tumor necrosis factor (anti-TNF) therapy, and if so defining the underlying mechanisms of suppression. Inhibition of Th17 cell responses was determined by Treg cell suppression assays. The Treg cell phenotype was analyzed using flow cytometry and enzyme-linked immunosorbent assay. Mechanisms of suppression were tested by cytokine addition or antibody blockade. Th17 responses were inhibited by Treg cells from RA patients responding to the anti-TNF antibody adalimumab (Treg(ada) ), but not by Treg cells from healthy individuals or patients with active RA. Furthermore, Treg(ada) cells secreted less IL-17, even when exposed to proinflammatory monocytes from patients with active RA. Treg(ada) cells suppressed Th17 cells through the inhibition of monocyte-derived IL-6, but this effect was independent of IL-10 and transforming growth factor β, which mediated the suppression of Th1 responses. Adalimumab therapy led to a reduction in retinoic acid receptor-related orphan nuclear receptor C-positive Th17 cells and an increase in FoxP3+ Treg cells lacking expression of the transcription factor Helios. However, this acquisition of IL-17-suppressor function was not observed in RA patients responding to treatment with etanercept, a modified TNF receptor-Fc fusion protein. Indeed, there was no alteration in Treg cell number, function, or phenotype in etanercept-treated patients, and Th17 responses remained unchecked. Th1 and Th17 responses are controlled through distinct mechanisms by Treg cells from patients responding to anti-TNF antibody therapy. Adalimumab therapy, but not etanercept therapy, induces a potent and stable Treg cell population with the potential to restrain the progression of IL-17-associated inflammation in RA via regulation of monocyte-derived IL-6.

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4(+) Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4(+) T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP(+) LV-IL-10-transduced human CD4(+) T (CD4(LV-IL-10)) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4(LV-IL-10) T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.

Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria

Genome inversions are ubiquitous in organisms ranging from prokaryotes to eukaryotes. Typical examples can be identified by comparing the genomes of two or more closely related organisms, where genome inversion footprints are clearly visible. Although the evolutionary implications of this phenomenon are huge, little is known about the function and biological meaning of this process. Here, we report our findings on a bacterium that generates a reversible, large-scale inversion of its chromosome (about half of its total genome) at high frequencies of up to once every four generations. This inversion switches on or off bacterial phenotypes, including colony morphology, antibiotic susceptibility, hemolytic activity, and expression of dozens of genes. Quantitative measurements and mathematical analyses indicate that this reversible switching is stochastic but self-organized so as to maintain two forms of stable cell populations (i.e., small colony variant, normal colony variant) as a bet-hedging strategy. Thus, this heritable and reversible genome fluctuation seems to govern the bacterial life cycle; it has a profound impact on the course and outcomes of bacterial infections.

Abstract SY28-02: Interactions between cancer stem cells and their niche govern metastatic colonization

Abstract Metastatic growth in distant organs is the major cause of cancer mortality. Formation of metastasis is a multi-step process with several rate-limiting steps. While dissemination of tumour cells appears to be an early and frequent event, successful initiation of metastatic growth, a process termed “metastatic colonization,” is rather inefficient for many cancer types and accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic for many tumour entities. The inefficiency of the metastatic process suggests two possibilities: a differential potential of the disseminated cancer cells to grow at the secondary sites or an inadequate support by distant tissues. We aimed at exploring limiting factors that determine metastatic success using the MMTVPyMT mouse breast cancer model which spontaneously metastasizes to the lungs. Considering that the disseminated cancer cells might have a differential metastatic potential, we investigated whether the recently identified cancer stem cells (CSCs, also termed tumour initiating cells), might be also relevant for the metastatic process. We found that a small population of cancer stem cells (CSCs) is critical for metastatic colonization. These CSCs represent a stable population of tumour cells which selectively survive and proliferate upon metastatic seeding and thereby drive the initial expansion of cancer cells at the secondary site. In contrast, nonCSCs fail to grow, are rapidly lost and do not de-differentiate into CSCs. Yet, the numbers of CSCs appear to be too high to fully explain the inefficiency of metastatic nodule formation, indicating that additional factors restrict successful metastatic colonization. Stem cells are suspected to rely on signals from their stromal environment such as localized growth factors that can affect stem cell maintenance and proliferation. In this study, we find stromal niche signals to be crucial for this process. We identify the extracellular matrix (ECM) component POSTN to be expressed by fibroblasts in the normal tissue exclusively in response to tumour cell infiltration. Infiltrating tumour cells induce stromal POSTN expression in the secondary target organ via TGFb3 secretion. Importantly the induction of POSTN at the target site is essential for the metastatic process. Indeed, generation of a POSTN null mouse revealed that the metastatic process is heavily compromised in the absence of POSTN. Similarly, cancer cells unable to induce POSTN expression at the target site due to blocking of TGFb3 signaling show poor metastatic activity. We further show that this ECM protein is required to allow maintenance of cancer stem cells: the cells driving the metastatic process. To understand how POSTN connects to the mechanisms that control stem cell maintenance we characterize its binding partners. We found that POSTN is able to bind Wnt ligands and therefore increase Wnt signaling. In vivo we observed Wnt signaling activity selectively in the CSCs population and this activity is abrogated in absence of POSTN. Finally, the metastatic activity of tumour cells with constitutively active Wnt signaling is independent of POSTN presence. Thus, POSTN acts as a niche component that can promote stem cell maintenance and metastatic colonization by augmenting Wnt signaling. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de-novo niche formation may represent a novel treatment strategy against metastatic disease. 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 SY28-02. doi:1538-7445.AM2012-SY28-02

Abstract 3446: Stat3 is a potential molecular target for chemoradiosensitization of colorectal cancer cells

Abstract Introductory sentence: Resistance to preoperative chemoradiotherapy represents a major clinical problem in the treatment of patients with locally advanced rectal cancer. Therefore, the identification of novel molecular targets that are differently expressed in responsive and resistant tumors is exceedingly important. Furthermore, modulating target gene expression may sensitize a priori resistant tumors to multimodal therapy. Experimental procedures: Previously, we established an in vitro model for chemoradiotherapy in 12 colorectal cancer cell lines, and correlated differences in treatment sensitivity with pretherapeutic gene expression profiles. Within our signature we identified the signal transducer and activator of transcription 3 (STAT3), which was significantly correlated with treatment resistance. To test the functional relevance of the observed over-expression of STAT3, we first determined STAT3 mRNA and protein expression levels in all 12 cell lines. Subsequently, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. The induced single cell clones and shNEG were treated with 3µM 5-FU and exposed to 1, 2, 4, 6, and 8 Gy of X-rays. In addition, STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC). Successful RNAi-mediated silencing of STAT3 and inhibitor treatment was detected by Western blot. Data: STAT3 was significantly overexpressed in resistant cells at mRNA and protein level. In stable STAT3 knockdown single cell clones and transiently siRNA transfected SW480 and SW837 cells, successful silencing of the STAT3 protein was detected after 72 hours, and STATTIC-induced inhibition of STAT3-phosphorylation after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.8 to 2. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing of STAT3 leads to a significantly increased chemoradiosensitivity. This highlights the potential relevance of STAT3 as a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. 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 3446. doi:1538-7445.AM2012-3446

Abstract SY35-02: Phenotypic heterogeneity and instability of human serous ovarian cancer tumor-initiating cells

Abstract SY35-02: Phenotypic heterogeneity and instability of human serous ovarian cancer tumor-initiating cells