Felix Oppel

SOX2-RNAi attenuates S-phase entry and induces RhoA-dependent switch to protease-independent amoeboid migration in human glioma cells

BackgroundSOX2, a high mobility group (HMG)-box containing transcription factor, is a key regulator during development of the nervous system and a persistent marker of neural stem cells. Recent studies suggested a role of SOX2 in tumor progression. In our previous work we detected SOX2 in glioma cells and glioblastoma specimens. Herein, we aim to explore the role of SOX2 for glioma malignancy in particular its role in cell proliferation and migration.MethodsRetroviral shRNA-vectors were utilized to stably knockdown SOX2 in U343-MG and U373-MG cells. The resulting phenotype was investigated by Western blot, migration/invasion assays, RhoA G-LISA, time lapse video imaging, and orthotopic xenograft experiments.ResultsSOX2 depletion results in pleiotropic effects including attenuated cell proliferation caused by decreased levels of cyclinD1. Also an increased TCF/LEF-signaling and concomitant decrease in Oct4 and Nestin expression was noted. Furthermore, down-regulation of focal adhesion kinase (FAK) signaling and of downstream proteins such as HEF1/NEDD9, matrix metalloproteinases pro-MMP-1 and -2 impaired invasive proteolysis-dependent migration. Yet, cells with knockdown of SOX2 switched to a RhoA-dependent amoeboid-like migration mode which could be blocked by the ROCK inhibitor Y27632 downstream of RhoA-signaling. Orthotopic xenograft experiments revealed a higher tumorigenicity of U343-MG glioma cells transduced with shRNA targeting SOX2 which was characterized by increased dissemination of glioma cells.ConclusionOur findings suggest that SOX2 plays a role in the maintenance of a less differentiated glioma cell phenotype. In addition, the results indicate a critical role of SOX2 in adhesion and migration of malignant gliomas.

Succession of transiently active tumor‐initiating cell clones in human pancreatic cancer xenografts

Although tumor‐initiating cell (TIC) self‐renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long‐term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self‐renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady‐state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady‐state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification‐mediated‐polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV‐injected animals for up to 24 months, indicating stable marking of murine steady‐state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long‐term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short‐lived progenitor cells. Myelosuppressive treatment of mice prior to LV‐injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady‐state hematopoiesis. Stem Cells2012;30:1961–1970

Patient-derived xenografts of gastrointestinal cancers are susceptible to rapid and delayed B-lymphoproliferation

Patient‐derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV‐associated B‐lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV‐infected B‐lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B‐lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify “bona fide” carcinoma xenografts.

Phenotypic differentiation does not affect tumorigenicity of primary human colon cancer initiating cells

Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions. Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.

Abstract 1540: Creating faithful genetic zebrafish models of pediatric high grade gliomas and MPNSTs

Pediatric high-grade gliomas (HGGs) are the leading cause of cancer-related death in children. Despite a slight improvement of patient prognosis over the past decades pediatric HGGs remain largely incurable. Thus, new experimental models are needed to understand the mechanisms of the disease and find more effective treatment options. We previously reported a model of HGGs and malignant peripheral nerve sheath tumors (MPNSTs) which is based on the combined deficiencies in the tumor suppressor genes tp53 and nf1. However, HGG penetrance is very low in this line and most fish develop MPNSTs starting at about 3 months of age. On top of the existing model we used CRISPR/Cas9 to incorporate knock-out mutations in the tumor suppressor genes atrx or suz12 which are described to be involved in pediatric HGG biology. Heterozygous atrx loss-of-function (lof) did not impact tumor onset or penetrance of neither HGGs nor MPNSTs. Since a total loss of atrx was lethal in development, we re-injected effective atrx targeting gRNAs and Cas9 mRNA into the atrx+/- line to create a mosaic atrx-/- genotype. Surprisingly, despite a high mutation efficiency of the remaining atrx allele the re-injection strategy still did not alter tumor onset and penetrance in that model. This suggests that loss of atrx is only effective in HGGs in cooperation with additional hits other than tp53 and nf1. In contrast, loss of suz12 cooperated well with the tp53/nf1-deficient background. As nf1, suz12 is duplicated in zebrafish (suz12a and suz12b) resulting in 4 alleles of each gene per cell. When at least 2 out of 4 alleles of either suz12a or suz12b were lost, MPNST onset was accelerated. This effect was much stronger in tp53-/-, nf1a+/-, nf1b-/- fish (5-7 weeks) compared to tp53-/-, nf1a+/+, nf1b-/- siblings (3-4 weeks). This indicates that the tumor supporting effect of suz12 lof increases the more nf1 levels decrease. However, HGG onset still remained unchanged. We hypothesize that efficient onset of HGGs in our model requires the presence of an activated oncogene. Specific missense mutations in H3F3A are reported to be implicated in HGG progression in children and young adults. Thus, we overexpressed zebrafish h3f3a-K27M or -G34R mutant sequences in the tp53/nf1/atrx/suz12-deficient line and are currently investigating possible changes in tumor biology. Our zebrafish models of pediatric HGGs and MPNSTs will be useful to dissect the mechanisms underlying the cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies. Citation Format: Felix Oppel, Ting Tao, Shuning He, Mark W. Zimmerman, Dong H. Ki, Nina Weichert, A Thomas Look. Creating faithful genetic zebrafish models of pediatric high grade gliomas and MPNSTs [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 1540. doi:10.1158/1538-7445.AM2017-1540

Abstract 1417: Clonal succession in pancreatic cancer progression is not driven by genetic instability

Our group has recently shown that human pancreatic cancer (PDAC) progression is driven by a succession of transiently active tumor initiating cell (TIC) clones during serial xenotransplantation. Genetic labeling demonstrated that serial PDAC xenograft tumors and even tumors of parallel mice transplanted with cells from the same donor xenografts harbored very little to no overlap of active TIC clones, indicating substantial changes in the proliferative activity of individual TIC predominantly producing progeny without detectable tumor-initiating activity. We now asked whether observed clonal activation and inactivation is caused by acquisition of de novo mutations during evolution of genetic subclones or by functional plasticity of genetically stable TIC clones. Therefore, we monitored somatic non-synonymous mutations in culture and during PDAC progression in genetically marked serial xenografts of two patients. DNA was isolated from xenografts, primary TIC cultures and corresponding normal pancreas or primary tumor tissue. Following paired-end exome sequencing, reads were aligned to a concatenated hs37d5 human and mm10 mouse genome assembly and human specific single nucleotide variants (SNVs) and small insertions/deletions (indels) were identified. We found a total of 45 altered gene coding genomic loci (P1 = 10; P2 = 35) not present in control tissue. Strikingly, most SNVs detected were present in all samples, only very few SNV were acquired during serial transplantation. In P1, 4 novel SNVs not present in the original patient tumor sample were detected within coding regions of TTC13, OR4K15, SSPO and TPGS1. Allele frequencies ranged from 2-27% in serial xenografts. In xenografts of P2 we detected 35 SNVs not present in healthy tissue. Of these, one mutation in the gene C10orf12 aroused after serial transplantation with a maximum altered allele frequency of 17%. None of these mutations is a known cancer driver or was found as recurrent in large scale cancer sequencing approaches. To evaluate whether the clonal TIC dynamics within established tumors are recapitulated in vitro, we analysed individual TIC clone kinetics in serially passaged cultures and in cultures derived from transduced xenografts. Strikingly, the kinetics in vitro were similar to those observed within serially transplanted xenografts. Every culture passage was formed by a distinct set of actively proliferating cell clones without significant overlap between individual serial passages indicating that clonal succession of TIC activity in PDAC is not dependent on the cellular context in tumors in vivo. The remarkable genetic stability of xenografts during serial transplantation strongly indicates that changes in the functional state of PDAC cells and not genetic instability drive clonal succession of TIC activity in PDAC. Citation Format: Karl Roland Ehrenberg, Claudia R. Ball, Felix Oppel, Naveed Ishaque, Taronish D. Dubash, Sebastian M. Dieter, Christopher M. Hoffmann, Ulrich Abel, Moritz Koch, Jens Werner, Frank Bergmann, Manfred Schmidt, Christof von Kalle, Wilko Weichert, Jurgen Weitz, Benedikt Brors, Hanno Glimm. Clonal succession in pancreatic cancer progression is not driven by genetic instability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1417. doi:10.1158/1538-7445.AM2015-1417

Abstract 3057: Unstable phenotype of human colon cancer tumor initiating cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have previously shown that long term progression of human colorectal cancer in serial xenotransplantation is driven by a small sub-fraction of all tumor initiating cells (TIC). These long-term TIC (LT-TIC) self-renew extensively in vivo and are exclusively able to seed metastases in distant organs. Defined phenotypic surface markers of LT-TIC would be crucial to develop effective antibody-based targeting strategies. However, it remains elusive whether a fixed phenotype of long-term TIC is associated with their tumorigenicity thereby allowing prospective isolation of this most relevant cancer cell fraction. To address this question, three dimensional spheroid cultures in serum free medium supplemented with FGF and EGF were generated from primary human colon cancer specimens to enrich for TIC. To calculate the frequency of TIC in spheroid cultures, cells were transplanted in limiting dilution into cohorts of Nod/SCID-IL2RG-/- (NSG) mice. TIC frequency varied from 1 in 22 to 1 in 2x104 spheroid cells, depending on the respective patient sample. When the spheroid cells were transferred to culture conditions that favor their differentiation (gelatin coated plates in the presence of serum and withdrawal of cytokines) the phenotype changed dramatically. The primary colon cancer cells did no longer grow as spheroids but formed an adherent cell layer and up-regulated the colon epithelial differentiation markers CDX2, DEFA5, KRT80, Muc20 and TFF2. In addition, CD133, a widely used marker to enrich for TICs in various solid cancers, was strongly down-regulated upon serum treatment. Strikingly, when xenotransplantated into NSG mice, cells cultured under spheroid and differentiation conditions equally formed serially transplantable tumors, demonstrating that tumor initiating and self-renewal capacity of TIC was not restricted to phenotypically immature spheroid cells. Moreover, CD133 expression did not predict successful tumor formation in vivo. Sorted CD133+ and CD133- cells from 3 individual patients formed tumors with equal efficiency and regenerated both, CD133+ and CD133- cells in vivo. Importantly, clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in TIC frequency, demonstrating that phenotypic differentiation does not lead to quantitative elimination of TIC clones. Our results demonstrate that phenotypic differentiation does not eliminate the tumor-initiating potential of human colorectal TIC. Moreover, expression of CD133 does not predict their tumor forming and self-renewal potential. This pronounced phenotypic plasticity of human colon cancer TIC poses a grave challenge for surface-targeted elimination of TIC in colorectal cancer. Citation Format: Taronish D. Dubash, Christopher M. Hoffmann, Felix Oppel, Klara Giessler, Sarah Bergmann, Sebastian M. Dieter, Wilko Weichert, Martin Schneider, Manfred Schmidt, Christof von Kalle, Hanno Glimm, Claudia R. Ball. Unstable phenotype of human colon cancer tumor initiating cells. [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 3057. doi:10.1158/1538-7445.AM2014-3057

Abstract 2295: Succession of transiently active TIC clones drives long-term human pancreatic cancer progression .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite accumulating evidence indicating a pivotal role of tumor-initiating cells (TIC) in tumor progression and metastases formation of pancreatic cancer (PC), very little is known about the clonal TIC dynamics within PC tumors in vivo. It is unclear whether the high aggressiveness of PC is driven by a homogeneous population of self-renewing TIC or whether distinct classes or activation states of TIC drive long-term disease progression. To address this we tracked the clonal contribution of individual TIC to tumor formation in vivo. Serum-free adherent cultures were established allowing enrichment of PC TIC without restriction to a certain phenotype. These cultures grow as 3-dimensional epithelial colonies with tight cell-to-cell contacts und reliably form subcutaneous tumors in NSG mice. To induce differentiation of TIC, FBS was added and FGF2, FGF10 and Nodal were withdrawn. Under these conditions, irregular shaped cells form a monolayer and down regulate markers previously described for TIC or normal pancreatic progenitors. Strikingly, despite profound changes in morphology and marker expression tumor initiation and self-renewal of TIC in serial transplantation were unchanged. Moreover, sorted CD133+ and CD133− cells equally efficient formed tumors containing similar proportions of CD133+ in vivo. Together these data indicate an unexpected phenotypic plasticity of TIC in PC. To determine the clonal kinetics of individual self-renewing TIC in vivo, early passage serum-free cultures from 3 patients were transduced with a lentiviral vector (LV) and serially transplanted. LV stably integrate into the host cell genome resulting in TIC clone specific fusion sequences which were identified by highly sensitive LAM-PCR and high throughput sequencing. In primary mice, 0.003-0.113% of all transduced cells contributed actively to tumor formation. Unexpectedly, in subsequent generations tumor formation was mainly driven by distinct TIC clones not detectable in earlier but recruited to tumor formation in later generations. Moreover, individual primary xenografts generated pairs of secondary tumors with very little overlap between clonal compositions. Mathematical modeling indicates strong changes in proliferative activity of individual, otherwise homogenous TIC which predominantly produce non-tumorigenic progeny with very limited self-renewal. These data indicate that long-term progression of PC is driven by a succession of transiently active TIC generating tumor cells in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation points to a context-dependent regulation of TIC activity within the growing tumor. Further understanding the mechanisms regulating the balance between activated and resting states of TIC may allow developing specific treatment strategies targeting this most relevant cell population in PC. Citation Format: Felix Oppel, Claudia R. Ball, Christopher M. Hoffmann, Sebastian M. Dieter, Taronish D. Dubash, Moritz Koch, Jurgen Weitz, Frank Bergmann, Manfred Schmidt, Ulrich Abel, Christof von Kalle, Hanno Glimm. Succession of transiently active TIC clones drives long-term human pancreatic cancer progression . [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 2295. doi:10.1158/1538-7445.AM2013-2295