Christopher M. Hoffmann

Distinct Types of Tumor-Initiating Cells Form Human Colon Cancer Tumors and Metastases

Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.

Specific Elimination of CD133+ Tumor Cells with Targeted Oncolytic Measles Virus

Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133(+) tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Notably, the CD133-targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our results indicate that CD133-targeted measles viruses selectively eliminate CD133(+) cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy.

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.

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.

Genetic subclone architecture of tumor clone-initiating cells in colorectal cancer

A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

Abstract 910: Genetic subclone heterogeneity of the human colon cancer initiating cell compartment

A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hubschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [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 910.

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 3043: Mesenchymal phenoptype of metastasizing primary human colorectal TIC is maintained through epigenetic silencing of miR-200

Metastasis formation is the most common cause of colorectal cancer (CRC) related death. How cancer cells disseminate from the primary tumor, gain access to blood and lymphatic vessels and seed to distant organs is not fully understood. To gain deeper mechanistic insights into metastasis formation, we developed a metastasis xenotransplantation model for primary human colorectal cancer cells. Patient (n=12) derived tumor-initiating cells (TIC) were enriched in spheroid cultures and subsequently transplanted into the kidney capsule of immunodeficient NOD/SCID-IL2RGnull (NSG) mice. Importantly, cultures derived from 3 distinct patients frequently metastasized into the murine liver and spleen, whereas spheroids derived from all other patients (n=9) never did. Compared to non-metastasizing spheroids, metastasizing spheroids strongly expressed mesenchymal markers (e.g. Vimentin and N-cadherin), whereas epithelial and colonic differentiation markers (e.g. E-cadherin, Trefoil factor 3, Defensin alpha 5, Keratin-8, -18 -20) were down-regulated suggesting that metastasizing primary patient derived CRC spheroids may have passed through an epithelial to mesenchymal transition (EMT). As the miR-200 family has been shown to support an epithelial phenotype and has been implicated in the process of EMT in tumor cells, we analyzed miRNA expression in metastasizing versus non-metastasizing spheroid cultures. Interestingly, metastasizing spheroids strongly down-regulated the miR-200 clusters miR-200a/-200b/-429 and miR-200c/-141 as well as miR-194 and miR-203. To further understand the mechanism of deregulated gene- and miRNA expression in metastasizing spheroids, we evaluated the DNA methylation status of miRNA promoters. In contrast to non-metastasizing spheroid cultures, metastasizing spheroids showed pronounced DNA methylation within the miR-200 promoter regions, indicating that miR-200 expression in primary metastasizing human colorectal cancer spheroids is suppressed through epigenetic mechanisms. Forced overexpression of both miR-200 clusters in metastasizing spheroids restored expression of epithelial genes like E-Cadherin and Keratin-8 and reduced expression of the mesenchymal markers Vimentin and N-cadherin. Moreover, incubation with low dose 5-aza-2′-deoxycytidine increased the expression of epithelial cell adhesion molecule (EpCAM) in metastasizing spheroids, indicating a partial reversion of the mesenchymal phenotype by demethylating agents. In summary, our findings indicate that metastasizing primary human CRC spheroids are epigenetically fixed in a mesenchymal state. This model allows further dissecting and understanding mechanisms of EMT and metastasis formation in human CRC thereby providing the basis for the development of future therapeutic intervention strategies against metastasizing human colorectal cancer. Citation Format: Christopher M. Hoffmann, Klara M. Giessler, Claudia R. Ball, Taronish D. Dubash, Sebastian M. Dieter, Sarah Bergmann, Wilko Weichert, Christof Von Kalle, Martin Schneider, Constance Baer, Christoph Plass, Manfred Schmidt, Hanno Glimm. Mesenchymal phenoptype of metastasizing primary human colorectal TIC is maintained through epigenetic silencing of miR-200. [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 3043. doi:10.1158/1538-7445.AM2014-3043