Putative Cancer Stem Cell Populations Research Articles

Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+ BRAF mutant MM cells. In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.

SummaryIntestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5−ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.

CD276 expression enables squamous cell carcinoma stem cells to evade immune surveillance.

Immunosurveillance is a critical mechanism guarding against tumor development and progression. Checkpoint inhibitors have shown significant success in cancer treatment, but expression of key factors such as PD-L1 in putative cancer stem cell (CSC) populations in squamous cell carcinoma has been inconclusive, suggesting that CSCs may have developed other mechanisms to escape immune surveillance. Here we show that CSCs upregulate the immune checkpoint molecule CD276 (B7-H3) to evade host immune responses. CD276 is highly expressed by CSCs in mouse and human head and neck squamous cell carcinoma (HNSCC) and can be used to prospectively isolate tumorigenic CSCs. Anti-CD276 antibodies eliminate CSCs in a CD8+ Tcell-dependent manner, inhibiting tumor growth and lymph node metastases in a mouse HNSCC model. Single-cell RNA sequencing (RNA-seq) showed that CD276 blockade remodels SCC heterogeneity and reduces epithelial-mesenchymal transition. These results show that CSCs utilize CD276 for immune escape and suggest that targeting CD276 may reduce CSCs in HNSCC.

Impact of in vitro driven expression signatures of CD133 stem cell marker and tumor stroma on clinical outcomes in gastric cancers

BackgroundThe CD133 transmembrane protein is a well-recognized stem cell marker that has been used to isolate putative cancer stem cell populations from gastric cancers (GCs). However, the molecular features or biomarkers underlying CD133 are largely unknown in GCs.MethodsWe performed gene expression profiling of CD133+ and CD133- cells sorted by flow cytometry from three GC cell lines to identify the CD133 expression signatures of GC. The CD133 expression signatures were investigated across publicly available expression profiles of multiple tumor types including GC and also for their relationship with patient survival.ResultsThe CD133 signature genes defined as 177 upregulated genes and 129 downregulated genes in CD133+ cells compared to CD133- cells were enriched with genes involving the cell cycle and cytoskeleton, implying that cancer stem cells with unlimited self-renewal play cancer-initiating roles. The CD133 expression signatures in GC expression profiles were positively correlated with those of brain tumors expressing CD133 and human embryonic stem cells, emphasizing the transcriptional similarities across stem cell-related expression signatures. We also found that these stem cell expression signatures were inversely correlated with those representing tumor infiltrating immune and stromal cells. Additionally, high CD133 expression signatures were found in intestinal subtypes and low tumor stage GCs as well as in those with microsatellite instabilities and high mutation burdens. As examined across 20 additional tumor types, both the expression signatures representing CD133 and stromal cells were unfavorable prognostic features; however, their impact were variable across tumor types.ConclusionsThe transcriptional activities of CD133 and those of stromal cells representing the activity of stem cells and level of epithelial-to-mesenchymal transition, respectively, may be inversely correlated with each other across multiple tumor types including GC. This relationship may be a confounding factor and should therefore be considered when evaluating the clinical relevance of stem cell-related markers.

Development of MGD007, a gpA33 x CD3-Bispecific DART Protein for T-Cell Immunotherapy of Metastatic Colorectal Cancer.

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761-72. ©2018 AACR.

Abstract 1772: Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action

Abstract Introduction MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic CRC, including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in non-human primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids. Method Redirected killing assays were performed using luciferase labelled gpA33+ve Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4+, CD127lo, CD25+) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively. Results MGD007 displays potent redirected T-cell killing of gpA33+ve CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33+ve tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33+ve target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33+ve target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007-mediated killing. Discussion MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients. Citation Format: Paul A. Moore, Yinhua Yang, Kalpana Shah, Daorong Liu, Vatana Long, Douglas H. Smith, Zhuangyu Pan, Ralph Alderson, Liqin Liu, Syd Johnson, Ezio Bonvini. Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1772.

In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model.

The cancer stem cell (CSC) theory implicates a small subpopulation of cells with stem-like properties, which is responsible for tumour initiation, development and metastasis. The unique biological and functional characteristics of CSCs, widely associated with treatment resistance, indicate an association between metastasis and stemness. It was hypothesised that metastatic cell lines may be enriched in CSCs and that this would correlate with a more resistant tumour. In the present study, the SW480 and SW620 paired cell lines derived from a colon adenocarcinoma and its lymph node metastasis, respectively were compared as an in vitro model of cancer progression. Their chemosensitivity and CSC properties were investigated. A range of in vitro assays were performed, including the side population assay, ALDEFLUOR assay, tumoursphere assay and assessment of CSC-associated surface phenotypes. It was determined that the SW480 and SW620 cells exhibited similar growth rates, although the SW480 cells were more migratory in wound healing assays on collagen and fibronectin matrices. SW480 and SW620 cells displayed similar CSC profiles, however, SW480 cells demosntrated significantly greater tumoursphere forming efficiency over SW620 cells. Tumourspheres derived from SW480 and SW620 cells also displayed differential sensitivity to 5-fluorouracil, oxaliplatin, geldanamycin and novobiocin that was not apparent when cells were grown under adherent conditions. Taken together, these results suggest that although the two cell lines have similar levels of putative CSC populations, there are differences in their biology that cannot be explained by these CSC levels. To the best of our knowledge, this is the first study to conduct a detailed analysis of the CSC populations using multiple in vitro assays in a paired cell line model. These results have clinical relevance for the understanding of the differences between primary tumours and their metastatic counterparts.

Abstract 1922: S-SHIP promoter expression identifies a putative cancer stem cell population in C3(1)/Tag murine mammary tumors

Abstract Breast cancer is the most common cancer in women worldwide. The isolation and characterization of breast cancer stem cells (CSC) are crucial for understanding cancer biology and revealing potential therapeutic targets. One of the major issues in the study of CSC is the lack of reliable markers. A transgenic mouse model (Tg 11.5kb–GFP) was generated using the 11.5kb s-SHIP (stem-SH2-containing 5’-Inositol Phosphatase) promoter that specifically expressed enhanced green fluorescent protein (GFP) in embryonic and various tissue stem cells. In the mammary gland, previous experiments showed that GFP labels puberty cap cells and pregnancy basal alveolar bud cells, and it has been demonstrated that these mammary GFP+ cells are activated tissue stem cells. In order to determine if s-SHIP promoter expression could also mark mammary cancer stem cells, we generated a bi-transgenic mouse model by crossing Tg 11.5kb-GFP mice with Tg C3(1)/Tag mice. Tg C3(1)/Tag mice express SV40 T antigen under the regulatory control of the rat prostatic steroid binding protein C3(1) gene. In female mice, the transgene is expressed primarily in the mammary gland. Mice develop mammary hyperplasia by 3 months of age with subsequent development of mammary adenocarcinoma by 6 months of age. Here we show the presence of a rare population of GFP+ cells in mammary tumors of female bi-transgenic mice by using immunohistochemical and flow cytometry analysis. The GFP+ mammary cancer cells are also CD24+/CD49f+/CD29+. As compared to GFP- cells, GFP+ cells exhibit both a higher tumor sphere-forming potential, and a higher tumorigenicity when transplanted into SCID and FVB recipient mice. Moreover, upon subsequent transplantation, the GFP+ cells generated heterogeneous tumors that displayed properties similar to the primary tumor. Currently, we are conducting transcriptomic analysis of these GFP+ and GFP- cells in order to reveal differentially expressed genes. Altogether, these results suggest that s-SHIP promoter expression may be a new marker of mammary CSC that enables their identification and isolation via a single consistent parameter. Furthermore, our bi-transgenic C3(1)/Tag x 11.5kb-GFP mice provide a novel model for study of mammary cancer stem cells and investigation of potential therapeutic targets for breast cancer. Citation Format: Lu Tian, Chann Lagadec, Eric Adriaenssens, Emmanuel Bouchaert, Hélène Bauderlique-Le Roy, Xuefen LeBourhis, Roland P. Bourette. S-SHIP promoter expression identifies a putative cancer stem cell population in C3(1)/Tag murine mammary tumors [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 1922. doi:10.1158/1538-7445.AM2017-1922

DOWN-REGULATION OF ALDH1A1 INCREASED EXPRESSION OF CARCINOGENESIS-RELATED GENES IN NON-SMALL CELL LUNG CANCER CELL LINE OF A549

Purpose: This study was aimed to isolate the putative cancer stem cell (CSC) populations from A549 lung cancer cell line and to evaluate the difference of carcinogenesis-related genes expression within parental population (A549) and isolated CSC populations (A549 CD166+ /EpCAM+ and CD166+ /CD44+). Methods: We performed flow cytometry analysis to sort out cell positive for these markers; CD166+ /EpCAM + and CD166+ /CD44+ from A549 cancer cell line. The isolated cells were tested for multipotent capacities by clonogenic and differentiation assays. Quantitative real time PCR was performed for both isolated CSC population and parental population to test expression of ALDH1A1 and 6 other genes that known to contribute to carcinogenesis; RARβ CYP24A1, BIRC5, EDN1, IL1β and PTGS2. Result(s): Both CD166+ /EpCAM+ and CD166+ /CD44+ have ability to form colonies and able to differentiate into adipocytes and osteocyte. Expression of ALDH1A1 was downregulated in all three cell populations (parental A549, A549 CD166+ /EpCAM+ and A549 CD166+ /CD44+) whereas the other 6 carcinogenesis-related genes were upregulated in all three cancer cell populations. There are no significant differences of gene expressions were detected among all three populations (p > 0.05). Conclusion(s): Downregulation of ALDH1A1 in all three cancer populations up-regulate the expression of other 6 carcinogenesis-related genes. Gene regulations between parental cancer cell (A549) and both putative CSC populations show no significant difference suggesting the existence of various CSC subpopulations reside within parental A549 population

Abstract A29: Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing

Abstract Most studies have mainly investigated clonal mutations due to the high background error frequencies of conventional next generation sequencing (NGS) methods. We have established a new method termed Duplex Sequencing (DS) that detects mutations with unprecedented accuracy and enables us to detect sub-clonal and rare mutations as well as clonal mutations. Using DS, we present our first genome-wide analysis of mitochondrial (mt) DNA mutations during the transformation of human normal breast stem cells to tumorigenic cells. We used a model system to dissect sequential steps in the progression of normal stem cells to tumorigenic cells. Significant differences have been found among normal stem cells, immortal cells, and tumorigenic cells. These cells differ in putative breast cancer stem cell (CSC) populations, mtDNA copy numbers, and the frequencies and types of mutations. We found that putative CSCs and mtDNA copy numbers increase as normal stem cells become tumorigenic cells. The vast majority of mutations identified in our study are stochastic (rare mutations) and the frequency of rare mutations is lower in transformed cells than in normal stem cells. This lower rare mutation load in transformed cells induces lower scores of predicted pathogenicity in transformed cells than in normal stem cells. C>T/G>A and A>G/T>C transitions are the major mutation types in normal stem cells, while only C>T/G>A are the major mutation types in transformed cells. Using DS, we detected a total of 1220 rare mutations and 678 of these variants have not been reported previously. The mtDNA mutation spectra of breast tumorigenic cells correlate with published nuclear DNA mutation signatures, which are found in the majority of tumors. We have identified mutations that can serve as potential biomarkers that indicate the different stages of breast carcinogenesis. These significant differences in mutation profiles could aid in characterizing different phenotypes between normal stem cells and transformed cells. Citation Format: Eun Hyun Ahn, Kensen Hirohata, Seung Hyuk Lee, Joon Yup Kim, Brendan F. Kohrn, Edward J. Fox, Chia-Cheng Chang, Lawrence A. Loeb. Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A29.

Abstract A46: The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo

Abstract Introduction: The Ets-transcription factor Etv1 is involved in epithelial-mesenchymal interactions during pancreatic development and shows enhanced expression in PanIN as well as in pancreatic ductal adenocarcinoma (PDAC). Therefore, we aimed to identify the mechanistic roles of Etv1 in EMT and invasion in PanIN and PDAC. Methods: Cells were isolated from Pdx1Cre;KrasG12D/+-mice (PanIN) and Pdx1Cre;KrasG12D/+;p53R175H/+-mice (PDAC) and lentiviral tranduction was used to induce overexpression or knockdown (shRNA) of Etv1, respectively. Cells were grown in 3D organotypic culture to analyze morphology and growth behavior. Invasion and sphere formation (self-renewal) assays were performed. FACS-sorting was used to isolate CD44+/CD24+ cells as a putative cancer stem cell population in PDAC and analyzed for Etv1 expression. To assess the role of Etv1 in PDAC in vivo, orthotopic pancreatic xenograft transplantation of Etv1 overexpressing PDAC cells was performed. Results: Etv1 overexpression in PanIN cells grown in 3D-organotypic culture induces a spindle shaped morphology and highly perturbed cyst architecture in contrast to control PanIN cells that form spheroid cysts that resemble normal pancreatic ductal structures. Concurrently and consistently, EMT-related genes (snail, twist, zeb1 and zeb2, as well as vimentin and N-cadherin) were found upregulated by >2-fold in PanIN-mEtv1 cells. Moreover, the expression of Mmp3 and Mmp9 was significantly increased. Functionally, the invasive capacity of PanIN-mEtv1 cells was more than twice that of control cells; knockdown of Etv1 in PDAC-cells significantly abrogated their invasive capacity. In sphere forming assays PanIN-mEtv1 cells revealed an increased self-renewal capacity, whereas sphere formation is reduced by knockdown of Etv1 in PanIN as well as in PDAC cells. FACS-sorting and subsequent expression analysis revealed increased Etv1 levels in the putative cancer stem cell population of CD44+/CD24+ PDAC-cells. Data from orthotopic xenografts showed significantly larger tumors, significantly increased stromal expansion measured by trichrome staining, and increased local tumor invasion in Etv1-overexpressing cells compared to controls. The increased stromal expansion significantly correlated to the increased tumor volume observed in Etv1 over-expressing xenografts. Conclusion: Our novel data indicate that Etv1 induces EMT as well as invasion both in vitro and in vivo and mediates self-renewal capacity of premalignant cells derived from PanIN- as well as PDAC-cells. Our in vivo data suggest a role for Etv1 in expanding the stromal compartment. These striking data suggest that Etv1 is biologically important in EMT. In vivo experiments with conditional knockout of Etv1 in Pdx1Cre;KrasG12D/+;p53R175H/+-mice are ongoing to further elucidate the role of Etv1 in PanIN and PDAC initiation and progression. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A46.

Treatment of biliary tract cancer cells with polycomb repressive complex 1 inhibitor PTC-209 reduces putative cancer stem cell population and causes cell cycle arrest

The polycomb repressive complex 1 (PRC1) is an epigenetic regulator that plays a crucial role in various types of cancer including biliary tract cancer (BTC). BTC is a fatal disease associated with poor outcome. Hence, new therapeutic strategies are needed. Recently, the first small-molecule PRC1 inhibitor called PTC-209 has been developed. In this study, we examined the effect of PTC-209 on eight different BTC cell lines.

Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.

Abstract 669: Development of MGD007, a gpA33 x CD3 bi-specific DART for T-cell immunotherapy of metastatic colorectal cancer

Abstract Introduction: Encouraging clinical responses have been observed through various strategies designed to harness T-cells for their anti-tumor properties, including immune checkpoint inhibition, chimeric antigen receptor-expressing T-cells or bispecific molecules designed to co-engage T-cells and cancer cells. Furthermore, a positive correlation has been observed in colorectal cancer between the degree and type of T-cell infiltration and prognosis suggesting that T-cell recruitment strategies may be particularly advantageous in this type of cancer. We have developed MGD007, a Dual-Affinity Re-Targeting (DART®) protein designed to redirect T-cells to target gpA33 expressing colon cancer. MGD007 has enhanced pharmacokinetic properties via incorporation of a neonatal FcR-binding Fc domain. The gpA33 target was selected based on its ubiquitous expression in colorectal cancer including reactivity with putative cancer stem cell populations (Li 2013, AACR #3763). To enable preclinical toxicokinetics and dose optimization, MGD007 was designed to cross-react with non-human primates. Methods: MGD007 was stably expressed in CHO cells and purified to homogeneity via a standard antibody-purification platform; in vitro functional studies were performed with a range of colorectal cancer cell lines and primary human T-cells; tumor growth inhibition studies were performed in NOD-SCID mice co-implanted with Colo205 and human T-cells (1:1 E:T ratio) and treated IV with MGD007; pharmacokinetic analyses were performed in cynomolgus monkeys. Results: MGD007 displays the anticipated bispecific binding properties and mediates potent lysis of gpA33-positive - but not gpA33-negative cancer cell lines through recruitment of either human or cynomolgus monkey T-cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Both CD8 and CD4 T-cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme levels. Xenograft studies showed tumor growth inhibition at doses as low as 4ug/kg. In cynomologus monkeys, 4 weekly doses of 200ug/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Conclusions: MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design. Furthermore it displays favorable PK in cynomolgus monkeys supporting convenient dosing. Taken together, these data support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Citation Format: Paul A. Moore, Ralph Alderson, Kalpana Shah, Yinhua Yang, Steve Burke, Hua Li, Valentine Ciccarone, Ezio Bonvini, Syd Johnson. Development of MGD007, a gpA33 x CD3 bi-specific DART for T-cell immunotherapy of metastatic colorectal cancer. [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 669. doi:10.1158/1538-7445.AM2014-669

Increased breast cancer cell toxicity by palladination of the polyamine analogue N 1,N 11-bis(ethyl)norspermine

Breast cancer is one of the most common malignant tumor forms among women and many women succumb to their disease. Thus, new anticancer agents that can efficiently improve patient survival are of the utmost importance. In this study, the effects of the polyamine analogues N 1,N 11-bis(ethyl)norspermine (BENSpm) and N 1-cyclo-propylmethyl-N 11-ethylnorspermine (CPENSpm) and the synthesized dinuclear complexes Pd2BENSpm (Pd-BENSpm), Pt2CPENSpm (Pt-CPENSpm) and Pd2Spm (Pd-Spm) were investigated in normal-like breast epithelial MCF-10A cells and the breast cancer cell lines JIMT-1 and L56BR-C1. The overall data show that palladination of BENSpm resulted in enhanced cytotoxicity, in contrast to platination of CPENSpm that reduced cytotoxicity, which might be explained by differences in the cellular uptake of Pd-BENSpm and Pt-CPENSpm. BENSpm and Pd-BENSpm treatment reduced the CD44+CD24− putative cancer stem cell population, evaluated by flow cytometry. Furthermore, Pd-BENSpm was the most efficient compound regarding induction of DNA damage and decrease in colony formation in soft agar. Pt-CPENSpm and Pd-Spm, on the other hand, were shown to be the least toxic compounds of all tested. Pd-Spm efficiently reduced the cellular glutathione levels, which probably was a consequence of its metabolic inactivation by conjugation to this endogenous thiol. The normal-like cells were found to be less sensitive to the agents than the breast cancer cells. Our findings show that Pd-BENSpm exhibits promising anticancer effects which render it suitable for further optimization to develop a new metal-based chemotherapeutic drug for breast cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-013-1621-y) contains supplementary material, which is available to authorized users.

Abstract 3763: Identification of gpA33 as a marker expressed on colon cancer stem cell lines.

Abstract Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [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 3763. doi:10.1158/1538-7445.AM2013-3763

Abstract 5218: Development of Anti-CD47 therapy for pediatric brain tumors.

Abstract Introduction: Extensive molecular characterization of Brain tumors done in recent years has proven valuable for tumor classification, risk stratification and outcome prediction for current treatment modalities. However the current standard of care has not improved the prognosis and carries an increased risk of cognitive impairments for children with brain tumors. A characteristic feature of tumor progression and recurrence is its ability to evade the immune system. Our hypothesis is that by modulating the innate immune system we can enhance the ability of macrophages to ‘eat and kill’ brain cancer cells. Recent data suggests that the interaction between the cell surface antigen CD47 and its binding partner SIRPα is a mechanism by which non-solid and solid tumors can evade the innate immune system. To see if anti-CD47 therapy is a potential therapy in malignant pediatric brain tumors we first looked at CD47 expression in freshly isolated patient and postmortem samples from 4 different tumor types; Diffused Intrinsic Pontine Glioma, Medulloblastoma, Ependymoma and ATRTs. We tested the hypothesis that blocking CD47- SIRPα interaction induced phagocytosis in an in-vitro phagocytosis assay. We next established orthotopic xenografts models in immune compromised mice and treated them with anti-CD47 humanized antibody, which is currently being developed for clinical trials in hematopoietic and non-CNS malignancies. Result: CD47 expression was upregulated in all tumor types and was present in >90% of the cells in high grade tumors. Increased CD47 expression was observed in CD15+ and CD133+ putative cancer stem cell population. Blocking the CD47- SIRPα interaction increases tumor phagocytosis by macrophages in-vitro. Systemic treatment with anti-CD47 antibody significantly reduced tumor burden in an orthotopic xenograft setting. Conclusion: Anti-CD47 therapy is a viable and effective treatment modality for pediatric high grade brain tumors. Citation Format: Sharareh Gholamin, Siddhartha S. Mitra, Chase Elliott Richard, Achal Achrol, Doosik Kong, Jun Jae Shin, Michelle Monje-Deisseroth, Yoon-Jae Cho, Irving Weissman, Samuel H. Cheshier. Development of Anti-CD47 therapy for pediatric brain tumors. [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 5218. doi:10.1158/1538-7445.AM2013-5218

Animal Models of Cancer Stem Cells: What are They Really Telling Us?

The cancer stem cell hypothesis suggests that a subpopulation of tumor cells exhibits stem cell properties such as self-renewal capacity and multilineage differentiation. The modern day cancer stem cell model emerged following initial work in leukemia and subsequent identification of putative cancer stem cell populations in a variety of solid tumors. These studies have been largely based on transplantation assays in immunodeficient mice and have revolutionized how we view tumor cell heterogeneity. However, recent studies with both genetically engineered and more highly immunocompromised animal models have challenged the notion that cancer stem cells are a rare subpopulation. Issues with the cell surface markers used to enrich for cancer stem cells and the hierarchical organization within tumors have also come to light and remain unresolved. As we continue to optimize in vivo models and understand microenvironmental influences, our view of cancer stem cells within heterogeneous tumors will likely become even more complex.

Calcein-effluxing human colon cancer cells are enriched for self-renewal capacity and depend on β-catenin

Putative cancer stem cells are a subpopulation of cancer cells that give rise to chemotherapy resistance and are therefore of prognostic and therapeutic interest, though their identification remains elusive in colon cancer due to lack of reliable and accurate markers. We previously identified a p53-dependent putative cancer stem cell population, the calcein low population (C(lo)P), based on their exclusive efflux of the fluorescent dye Calcein. This functional identification method enables comparative live cell studies of subpopulations without differential toxicity that occurs with traditional Hoechst methods, which has confounded conclusions and limited the utility of this cancer stem cell marker. In this study, we examined the cancer stem cell-like properties of the C(lo)P population in vivo in comparison with the parental and calcein-high population (C(hi)P) in human colon cancer xenografts. Serial dilution xenograft experiments in NOD/SCID mice revealed that the C(lo)P is only marginally more tumorigenic compared to the C(hi)P or parental cells. However, serial passage of these tumors revealed that the C(lo)P is uniquely enriched for self-renewal capacity in vivo compared to the other populations. Immunohistochemical analysis of these tumors revealed that the C(lo)P possesses increased levels of nuclear β-catenin and furthermore, siRNA-mediated knockdown of β-catenin significantly reduced the C(lo)P population. These findings highlight the C(lo)P as an important subpopulation of tumor cells that are exclusively endowed with the ability to self-renew and propagate tumors. The dependency of the C(lo)P on β-catenin provides a molecular explanation for this ability and suggests that this population can and should be therapeutically targeted by inhibition of Wnt signaling.

Abstract 3262: Notch signaling pathway-targeted therapy suppresses tumor progression and prevents metastatic spread in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) has the highest mortality rate of any cancer type. The 5-year survival rate of 5% remained largely unchanged in the last three decades. The paucity of breakthroughs in treatment regimens and continued poor survival demonstrates an acute need for improvement in therapy for this lethal malignancy. Mounting evidence suggest that sustained re-activation of Notch signaling pathway contributes to the initiation, progression and maintenance of PDA. Recently, Notch signaling has been linked to the viability of putative tumor initiating cancer stem cell populations in pancreatic cancer. PF-03084014 is a selective γ-secretase inhibitor developed by Pfizer. The present study was conducted to determine the effect of PF-03084014 monotherapy and in combination with gemcitabine (GEM) to suppress tumor progression and prevent metastatic spread in preclinical models of pancreatic cancer. Methods: NOTCH-1 gene expression was determined in a panel of 30 human pancreatic cancer xenografts by Affymetrix gene array. We investigated the efficacy of PF-03084014 and in combination with GEM in subcutaneously (s.c) implanted as well as orthotopic models of pancreatic cancer. We also determined the treatment effect on putative pancreatic cancer stem cell (CSC) populations. TUNEL and Ki-67 staining were performed to determine whether the therapy induce apoptosis and suppresses tumor cell proliferation. Results: A combination of PF-03084014 and GEM is highly effective in producing durable tumor regression compared to GEM in subcutaneously implanted pancreatic cancer xenografts. Tumor re-growth curves plotted after 4 weeks of drug treatment demonstrate that the effect of the combination therapy is longer lasting than that of GEM. Notably, in a highly aggressive Panc265 orthotopic model, PF-03084014 and GEM combination was effective in controlling the primary tumor growth as well as preventing metastatic dissemination to organs as compared to GEM treatment. Flow cytometry of sorted tumor cells from Panc215 (after 4 weeks of treatment) showed that GEM treatment was not capable of diminishing the CSC pool as compared to the vehicle treated mice. However, PF-03084014 alone, and the combination of GEM with PF-03084014 resulted in a 3.72 and 3.04-fold decrease in CD24+CD44+ tumor cells compared with GEM treatment, respectively. TUNEL and Ki-67 staining demonstrated that combination therapy induced apoptosis and suppresses cell proliferation. Conclusions: The findings presented here provide strong rationale for the clinical investigation on notch inhibition as a chemotherapeutic strategy to combat pancreatic cancer. 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 3262. doi:1538-7445.AM2012-3262