bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2993: Dll4 inhibition plus aflibercept markedly reduces ovarian tumor burden and ascites
Authors: Huang Jie , Hu Wei , Dalton Heather J. , Bottsford-Miller Justin , Coleman Robert L. , Jaffe Robert B. , Sood Anil K.
Published: May 2015

Purpose: To determine the effects of Dll4 blockade with and without aflibercept on ovarian tumor burden and ascites in preclinical models.

Methods: Using Dll4-Fc and anti-Dll4 antibodies, we evaluated the biological effects of Dll4 inhibition combined with aflibercept in vivo (orthotopic mouse models of ovarian cancer). Reverse phase protein array (RPPA) analysis was used to identify potential biomarkers of treatment response.

Results: To address the biological significance of Dll4 expression in tumor cells versus endothelial cells, we used human and mouse anti-Dll4 antibodies in vivo. In the A2780 model, treatment with mouse (REGN1035) or human (REGN421) anti-Dll4 antibodies reduced tumor weight by 61.5% and 82.4%, respectively, compared to the controls (P < 0.05 for both). Aflibercept alone reduced tumor weight by 90% (p < 0.05). The combinations of REGN1035 plus aflibercept, and REGN421 plus aflibercept resulted in the greatest inhibition of tumor growth, reducing respective tumor weight by 95.8% and 93.9%, compared to the controls (P < 0.05). The anti-tumor effects were related to decreased angiogenesis and increased apoptosis. RPPA analysis revealed that caspase3 and GATA3 were significantly increased; cyclin D1, Bcl-2 and pS6-s240_s244 were decreased in response to Dll4 Inhibition plus aflibercept. Among these, GATA3, a transcription factor for E-cadherin, was significantly induced by Dll4 Inhibition plus aflibercept, resulting in the up-regulation of E-cadherin expression in human cancer cells. Furthermore, GATA3 and E-cadherin expression might be useful as potential biomarkers of response.

Conclusions: Dual targeting of Dll4 and VEGF shows promise for inhibiting ovarian tumor growth and may have important clinical implications.

Citation Format: Jie Huang, Wei Hu, Heather J. Dalton, Justin Bottsford-Miller, Robert L. Coleman, Robert B. Jaffe, Anil K. Sood. Dll4 inhibition plus aflibercept markedly reduces ovarian tumor burden and ascites. [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 2993. doi:10.1158/1538-7445.AM2014-2993
Abstract 2995: Metronomic albumin-bound paclitaxel and topotecan has potent antitumor activity in ovarian cancer
Authors: Davis Ashley N. , Lin-Liu Yvonne G. , Previs Rebecca A. , Dalton Heather J. , Zand Behrouz , Bottsford-Miller Justin , Coleman Robert , Sood Anil K.
Published: May 2015

Objective: There is growing recognition of the important role of metronomic chemotherapy for cancer treatment. Based on their unique anti-angiogenic effects, we tested the efficacy of dual albumin-bound paclitaxel (ABP), which stimulates Thrombospondin-1, and topotecan, which inhibits Hypoxia-inducible factor 1-α, chemotherapy at metronomic dosing in ovarian carcinoma.

Methods: We examined the effects of ABP and topotecan in vitro (MTT) and in vivo using SKOV3ip1, HeyA8 and HeyA8-MDR orthotopic mouse models of ovarian cancer. We also examined effects on proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (MVD) in tumor samples obtained at necropsy.

Results: In vitro cytotoxicity assays revealed similar effects (IC50=20-60 nM) of maximum tolerated dosing (MTD) and metronomic dosing of ABP on HeyA8 tumor cells (p<0.05). In vivo therapy experiments using the SKOV3ip1 and HeyA8 tumor models demonstrated that treatment with metronomic ABP alone and in combination with metronomic topotecan resulted in significant reductions in tumor weight, 62% and 96% respectively as compared to controls (p<0.01). Combination treatment with metronomically dosed ABP and topotecan further reduced tumor growth by 82% compared to topotecan alone (p<0.01). In the chemoresistant tumor model, HeyA8-MDR, metronomically dosed monotherapy with either cytotoxic agent had a modest effect on tumor growth however, combination therapy decreased tumor burden by 74% when compared to controls (p<0.01). Metronomic ABP monotherapy resulted in reduced cell proliferation (p<0.001), reduced tumor angiogenesis (p≤0.01), and increased apoptosis (p<0.01).

Conclusions: Metronomically-dosed albumin-bound paclitaxel and topotecan offer a highly effective and novel therapeutic approach in ovarian carcinoma that merits further clinical development.

Citation Format: Ashley N. Davis, Yvonne G. Lin-Liu, Rebecca A. Previs, Heather J. Dalton, Behrouz Zand, Justin Bottsford-Miller, Robert Coleman, Anil K. Sood. Metronomic albumin-bound paclitaxel and topotecan has potent antitumor activity in ovarian 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 2995. doi:10.1158/1538-7445.AM2014-2995
Abstract 3: Imaging of stromal cells during hepatocellular metastasis formation in the RFP-MUP-uPA mouse
Authors: Suetsugu Atsushi , Ogata Hisanobu , Hiroshima Yukihiko , Momiyama Masashi , Osawa Yosuke , Moriwaki Hisataka , Saji Shigetoyo , Karin Michael , Hoffman Robert M.
Published: May 2015

Major urinary protein-urokinase type plasminogen activator (MUP-uPA) transgenic mice develop liver disease. We have developed the RFP-MUP-uPA mice, by crossing MUP-uPA mice with transgenic mice ubiquitously-expressing RFP. Hepatocyte RFP fluorescence in this model is very strong. In addition, RFP fluorescence of stromal cells is very bright enabling the portal vein and sinusoid structures to be clearly visualized. Huh7 liver cancer cells expressing green fluorescent protein (GFP) were injected in the spleen of MUP-uPA transgenic mice. The vessels of the MUP-uPA liver are filled with Huh7-GFP cells as imaged in live mice 3h after cell injection. Huh7-GFP cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. The Huh7-GFP cells were visualized in the liver with the Olympus FV1000 confocal microscope. In large metastatic liver tumors in RFP-MUP-uPA mice, stroma cells derived from the host animal appeared around and into the edge of Huh7-GFP tumors. In contrast, in small metastatic liver tumors, there were no stromal cells observed. The stromal cells were observed in the spleen primary tumor, as well as kidney and the lung metastasis in addition to the large liver tumors. This model enables imaging the effects of uPA on liver metastasis and stromal recruitment enabling the metastasis to progress.

Citation Format: Atsushi Suetsugu, Hisanobu Ogata, Yukihiko Hiroshima, Masashi Momiyama, Yosuke Osawa, Hisataka Moriwaki, Shigetoyo Saji, Michael Karin, Robert M. Hoffman. Imaging of stromal cells during hepatocellular metastasis formation in the RFP-MUP-uPA mouse. [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 3. doi:10.1158/1538-7445.AM2014-3
Abstract 3002: ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions
Authors: Felty Quentin H. , Das Jayanta K.
Published: May 2015

Higher levels of Id3 expression have been reported in tumor-infiltrating blood vessels from various tumors, but the exact role of Id3 in vascular lesion formation is poorly understood. This study aims at identifying the status of Id3 expression and distribution in benign vascular lesions and attempts to correlate this information with the progression of vascular lesion. In this study we investigated the expression pattern of Id3 in malignant, benign, and normal human vascular tissue specimens. Expression analysis of Id3 was carried out using immunohistochemical analysis in 48 sporadic samples of a heterogeneous group of benign and malignant vascular tissue. Immunohistochemical analysis confirmed that many of the malignant vascular lesions exhibited strong Id3 expression that correlated positively with increased 8-hydroxydeoxyguanosine (8-OHdG) a marker of oxidative DNA damage. In contrast, in normal human vascular tissue, Id3 expression and 8-OHdG was low. In malignant vascular tissue, Id3 protein displayed a stronger nuclear localization when compared to the cytoplasm. Id3 expression levels in benign vascular lesions while still expressing Id3 were to a lesser extent when compared to malignant vascular tissue. To our knowledge, this is the first study to compare benign and malignant vascular lesions to assess the role of Id3 and 8-OHdG. Our data reveals Id3 expression and oxidative DNA damage to be highly associated in malignant cells and to a lesser extent in their benign counterparts, indicating that the growth of vascular lesions may require oxidative stress signaling converging on Id3. The results also support the involvement of Id3 in tumor angiogenesis a process that critically influences the malignant behavior of cancer cells.

Citation Format: Quentin H. Felty, Jayanta K. Das. ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions. [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 3002. doi:10.1158/1538-7445.AM2014-3002
Abstract 3010: The role of NF-kappaB in mammary tumor initiation
Authors: Barham Whitney , Tikhomirov Oleg , Chen Lianyi , Ortega Ryan , Onishko Halina , Connelly Linda , Yull Fiona E.
Published: May 2015

Nuclear factor kappa B signaling is accepted as a major component of pro-survival signaling within breast cancer cells. However, less is known about how NF-kappaB could contribute to breast tumor initiation. Inflammatory signaling can be activated within the breast by such common factors as obesity, involution following lactation, aging, and psychological stress. Thus, understanding how activated NF-kappaB could lead to the initiation of breast tumors would provide opportunities to develop prevention strategies. We generated a doxycycline-inducible mouse model to study the effects of constitutively active IKK2 on ductal morphogenesis. In this model, Tet-o-cIKK2 mice are crossed with MMTV-rtTA transgenics to target inducible expression specifically to the mammary epithelium. Expression of cIKK2 in transplanted mammary tissue results in enlarged terminal end-buds, increased lateral branching, and intraductal hyperplasia. Transgene induction for only 3 days in a 6 week old female induces filling of ductal lumens, hypercellularity and loss of strict basal/luminal structure. Quantitative PCR of mammary tissue reveals up-regulated markers of macrophage infiltration such as CCL2 and F4/80 and markers of inflammation including Cox-2, TNF-alpha, and CXCL1. Interestingly, constitutive IKK2 activation also results in a decrease in hormone receptor levels (ERα and PR). Immunofluorescent staining shows that activation of classical NF-kappaB increases nuclear p65, as expected. However, nuclear p52 is also increased in transgene positive cells, suggesting that a subset of the effects resulting from activation of classical NF-kappaB are mediated by cross talk with the alternative pathway. Taken together, we find that activation of NF-kappaB in mammary epithelium is sufficient to induce hyperplastic growth and loss of ductal structure. Ongoing studies seek to define what additional factors, such as the right hormonal context, will cause the lesions to progress to malignancy.

Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Halina Onishko, Linda Connelly, Fiona E. Yull. The role of NF-kappaB in mammary tumor initiation. [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 3010. doi:10.1158/1538-7445.AM2014-3010
Abstract 3013: Different tumor-initiating cells from a IDH1 wt glioblastoma patient
Authors: Soeda Akio , Hara Akira , Kunisada Takahiro , Iwama Toru
Published: May 2015

Glioblastoma multiforme, as its name suggests, composes pathologically heterogeneous mixed cells, such as variable degree of cellular and nuclear polymorphism, or so called “gemistocytes” or “giant cells”. Cancer cell regulates dynamic cell-cell interactions, thus, cell-cell dynamic heterogeneity is fundamental questions to know which cells are origin, therapeutic targets or source of recurrences for these decades. Therefore, functional analysis of each heterogenous cells and their role in tumor pathogenesis are critical. To investigate the cellular origin of population diversity, we established 4 subclones from a IDH1 wild type GBM patient. These subclones were subsequently propagated and analyzed. All clones expressed stem cell makers (nestin, sox2, musashi) heterogeneously. The morphology, self-renewal and proliferative capacities of the subclones were varying. FACS analysis showed that high CD133 expression clones tended to have high proliferation and invasive capacities. Most of these clones’ cells expressed high level of CD44, an adhesion molecule in cancer stem cells regulating redox status in cancer cells. Surface expression of CD24 and CD56 showed statistically different pattern. However, these 2 did not enhance their morphology, proliferation capacities and tumorigenesis. On the other hand, cDNA analysis with using 58721 probes showed that the level of COL1A1 and IGFBP7 were matched their morphology and tumorigenesis character. In animal models, xenografts from subclones, tumor-progression/invasion and animal survivals were also different. Some clones invaded contra-lateral hemisphere via tract fiber. Our observations suggest that single cell derived subclones from a patient is capable of producing phenotypically heterogeneous self-renewing progenies in an in vitro and in vivo setting. The functional analysis of the heterogeneous GSC clones may provide a better understanding of GSC biology and novel means for testing of new treatment strategies that focus on the eradication of the true GSCs.

Citation Format: Akio Soeda, Akira Hara, Takahiro Kunisada, Toru Iwama. Different tumor-initiating cells from a IDH1 wt glioblastoma patient. [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 3013. doi:10.1158/1538-7445.AM2014-3013
Abstract 3026: Cancer stem cells maintain a hierarchy of differentiation by creating their niche
Authors: Mizutani Akifumi , Matsuda Shuichi , Yan Ting , Prieto-Vila Marta , Chen Ling , Satoh Ayano , Kasai Tomonari , Masuda Junko , Kudoh Takyuki , Murakami Hiroshi , Fu Li , Salomon David S. , Seno Masaharu
Published: May 2015

Cancer stem cells (CSCs) are unique subpopulation of tumor cells that possess self-renewal and differentiation capacity, and can give rise to the entire heterogeneous population of the tumor tissue. The self-renewal and differentiation properties of CSCs are regulated and maintained by the CSC niche. However, the mechanism of this maintenance, especially the contribution of differentiated cancer cells, remains to be fully elucidated. It has been shown that vascular endothelial cells can activate the Notch signaling pathway in glioblastoma (GMB) stem-like cells, and that was required for self-renewal of GBM stem-like cells. Given the fact that GBM stem-like cells differentiate into vascular endothelial cells, it raises the possibility that these differentiated cells that arise from CSCs might generate a CSC niche. In this study, we addressed this hypothesis using a model of CSCs, miPS-LLCcm, derived from mouse induced pluripotent stem cells (miPSCs) we have established recently.

We found in vitro cultured miPS-LLCcm cells were autonomously balanced with stem-like cells and differentiated cells. The analysis of VEGFR2 and VE-cadherin expression, and in vitro tube formation assay indicated the differentiation capacity of miPS-LLCcm into vascular endothelial cells. In the presence of the conditioned medium from bulk population of miPS-LLCcm cells, self-renewal capacity of CSC population of miPS-LLCcm, which was evaluated by spheroid formation in suspension culture, was enhanced, partly in Notch depending manner. In addition, we found that the ligands for Notch activation were secreted from the differentiated tumor cells including vascular endothelial cells. In addition, the secreted factor(s) from differentiated cells appeared to regulate the differentiation lineage of CSCs, since miPS-LLCcm could not differentiate into vascular endothelial lineages after cultivation of only stem cell population.

Our results indicate that the differentiated progenies of CSCs containing vascular endothelium play important roles for regulating the CSC's properties. Therefore, miPS-LLCcm cells create their own in vitro niche to maintain themselves in the hierarchy of differentiating CSCs.

Citation Format: Akifumi Mizutani, Shuichi Matsuda, Ting Yan, Marta Prieto-Vila, Ling Chen, Ayano Satoh, Tomonari Kasai, Junko Masuda, Takyuki Kudoh, Hiroshi Murakami, Li Fu, David S. Salomon, Masaharu Seno. Cancer stem cells maintain a hierarchy of differentiation by creating their niche. [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 3026. doi:10.1158/1538-7445.AM2014-3026
Abstract 3032: The effect of a tankyrase inhibitor on the small intestine tissue homeostasis
Authors: Norum Jens Henrik , Skarpen Ellen , Brech Andreas , Kuiper Raoul , Waaler Jo , Krauss Stefan , Sørlie Therese
Published: May 2015

In the small intestine the Wnt/β-catenin pathway is involved in regulation of stem cells and tissue homeostasis. Aberrant Wnt signaling is known to cause intestinal cancers. Tankyrase enzymes play crucial roles in the regulation of the Wnt signaling pathway. Inhibition of tankyrase activity is an effective way of inhibiting Wnt/β-catenin signaling. A small molecule tankyrase inhibitor, G007-LK, was developed that inhibits tumor growth in a APC-mutant colorectal cancer xenograft model. In in vivo experiments at high doses (60 mg/kg), G007-LK treatment resulted in severe necrosis and inflammation in the small intestine, possibly affecting the stem cells. Different cell populations in the small intestine have been shown to have stem cell properties, including the Lgr5+ cell population located at the bottom of the crypts and the cells residing in +4 position counting from the crypt base. Both of these cell populations can give rise to all the various cell types of the small intestine.

In the current project, we have used in vivo models to address the effect of the tankyrase inhibitor, G007-LK, on small intestine tissue homeostasis. H&E staining of fixed tissue sections showed no significant differences between mice treated with 10 mg/kg G007-LK or vehicle. We performed lineage tracing from the Lgr5+ stem cells of the small intestine and observed that treatment with G007-LK reduced the number of cells traced from Lgr5+ stem cells. Immunohistochemistry (IHC) staining for the proliferation marker Ki67 showed reduced number of positive cells in the small intestinal crypts of the G007-LK- compared to vehicle-treated mice. As expected, IHC staining for β-catenin showed reduced number of positive nuclei in the G007-LK- compared to vehicle-treated mice, suggesting reduced signaling via the Wnt/β-catenin pathway.

Taken together, our data show that a daily dose of the tankyrase inhibitor G007-LK (10 mg/kg), is well tolerated by mice. Administration of the tankyrase inhibitor to the mice reduces the number of lineage traced cells from the Lgr5+ stem cell population in the small intestine, without altering the general histology of the tissue.

Citation Format: Jens Henrik Norum, Ellen Skarpen, Andreas Brech, Raoul Kuiper, Jo Waaler, Stefan Krauss, Therese Sørlie. The effect of a tankyrase inhibitor on the small intestine tissue homeostasis. [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 3032. doi:10.1158/1538-7445.AM2014-3032
Abstract 3034: Integrin αvβ3 drives Slug activation and stemness in the pregnant and neoplastic mammary gland
Authors: Desgrosellier Jay S. , Lesperance Jacqueline , Seguin Laetitia , Shattil Sanford J. , Cheresh David A.
Published: May 2015

While integrin αvβ3 expression is linked to breast cancer progression its role in epithelial development is unclear. Here, we show that αvβ3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. While αvβ3 is a luminal progenitor marker in the virgin gland, we noted increased αvβ3 expression in MaSCs at mid-pregnancy. Accordingly, mice lacking αvβ3 or expressing a signaling deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity and expression of Slug, a master regulator of MaSCs. Surprisingly, αvβ3 deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. TGFβ2, but not RANKL, induced αvβ3 leading to Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvβ3 was necessary and sufficient for Slug activation and tumorsphere formation. Thus, pregnancy-associated MaSCs require a TGFβ2/αvβ3/Slug pathway, which may also contribute to breast cancer progression.

Citation Format: Jay S. Desgrosellier, Jacqueline Lesperance, Laetitia Seguin, Sanford J. Shattil, David A. Cheresh. Integrin αvβ3 drives Slug activation and stemness in the pregnant and neoplastic mammary gland. [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 3034. doi:10.1158/1538-7445.AM2014-3034
Abstract 304: Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues
Authors: Carrick Danielle Mercatante , Altekruse Sean , Camalier Corrine , Cozen Wendy , Hernandez Brenda , Lynch Charles , McGregor Paul , Mehaffey Michele G. , McShane Lisa , Rohan JoyAnn Phillips , Williams Mickey , Gillanders Elizabeth M. , Mechanic Leah E. , Schully Sheri
Published: May 2015

Next Generation Sequencing (NGS) technologies are being used for detection of somatic mutations in tumors and studies of germline variation. However, most NGS studies used DNA isolated either from whole blood or fresh frozen tissue specimens. Meanwhile, the tissue specimens available from most National Cancer Institute (NCI) funded cohorts and the Surveillance, Epidemiology and End Results (SEER) registries (http://seer.cancer.gov/biospecimen/) are primarily formalin fixed paraffin embedded (FFPE). There are limited data, on a small number of FFPE tissue samples, which suggest NGS is feasible. Much less is known about the feasibility of these technologies for large scale studies or using older FFPE specimens (e.g. 5-30 years old).

The main objective of this project was to conduct a pilot study to determine whether the DNA obtained from archival FFPE tissue is of sufficient quality and quantity to conduct NGS. Sixty high-grade serous ovarian adenocarcinomas from FFPE tissues which were between 7 and 31 years old were obtained from three SEER registries. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing. DNA extraction (yields and quality) and whole exome sequencing (depths of coverage and exome coverage obtained) results from this study will be presented. Ultimately, data derived from this analysis could serve as the basis for determining the utility of archival FFPE biospecimens for characterization and discovery projects utilizing NGS technologies instead of relying on frozen biospecimens.

Citation Format: Danielle Mercatante Carrick, Sean Altekruse, Corrine Camalier, Wendy Cozen, Brenda Hernandez, Charles Lynch, Paul McGregor, Michele G. Mehaffey, Lisa McShane, JoyAnn Phillips Rohan, Mickey Williams, Elizabeth M. Gillanders, Leah E. Mechanic, Sheri Schully. Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues. [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 304. doi:10.1158/1538-7445.AM2014-304
Abstract 3040: Forced astroglial differentiation depletes glioblastoma stem cells
Authors: Spina Raffaella , Voss Dillon M. , Sloan Andrew E. , Bar Eli E.
Published: May 2015

Glioblastoma are among the least curable cancers in man, at least in part because of stem-like cellular subpopulations (herein referred to as glioma stem cells, GSC) refractory to current therapies. The cancer stem cell hypothesis suggests that tumor cells are organized in a pyramidal unidirectional differentiation cascade with GSC at the top and functionally defined by the ability to self-renew and initiate tumors identical to the original tumors from which they are derived. GSCs are maintained by both cell intrinsic and microenvironmental factors and conditions. In this study we focused on identifying potential small molecules which may target tumor heterogeneity by promoting differentiation of GSC into less aggressive, more differentiated subpopulations. To this end, we have developed reporter models for astroglial and neuronal differentiation in HSR-GBM1, HSR040622 and HSR040821 tumor-derived, GSC-enriched, neurospheres lines, using lineage-specific reporter constructs. We employed these reporter lines to screen a library of over 600 bioactive small molecules for potential inducers of GSC differentiation. We identified several molecules which significantly induce astroglial differentiation in multiple GSC-enriched lines. We focus in this report on one of these inhibitors (AD1 = Astroglial Differentiation-1) which induces astroglial differentiation by over 20 to 30-fold as compared with DMSO control. In addition, AD1 treatment resulted in a 40-60% reduction in cell growth in HSR-GBM1, 60% in HSR040622, and 40% reduction in HSR040821, as determined by direct cell counting. As differentiation should result in reduced self-renewal capacity, we next determined the effect of AD1 on sphere formation using the extreme limited dilution assay (ELDA). We found that AD1 significantly reduced clonogenic capacity of HSR-GBM1 reporter line by over 90%. These data strongly suggest that AD1-induced astroglial differentiation may be the cause for the reduced clonogenic capacity. We are currently determining the plasticity of the system in term of reversal of the differentiation state in vitro and in vivo. Our results support the therapeutic value of a drug-induced differentiation and define how differentiation affects the GSC compartment in glioma.

Citation Format: Raffaella Spina, Dillon M. Voss, Andrew E. Sloan, Eli E. Bar. Forced astroglial differentiation depletes glioblastoma stem 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 3040. doi:10.1158/1538-7445.AM2014-3040
Abstract 3044: Cancer associated fibroblasts-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma
Authors: Lau Yuen Ting , Lo Jessica , Ng Irene Oi Lin , Lee Terence Kin Wah
Published: May 2015

Cancer stem cells (CSCs) play pivotal roles in tumor growth and therapeutic response; hence it is of fundamental importance to understand how CSCs is regulated inside the tumor mass. Similar to the regulation of normal stem cells by their ‘niche’, CSCs are also regulated by cells within the tumor microenvironment. Although most cases of HCC develop within a background of cirrhosis in which liver tissue is enriched in activated myofibroblasts, the role of cancer associated fibroblasts (CAFs) on liver CSCs within the tumor microenvironment has not yet studied. In order to achieve the goal of developing an effective therapy for HCC by specifically eliminating liver CSCs, we aim to dissect the cross-talks between HCC cells and CAFs. To study this, CAFs from fresh HCC tissues was successfully isolated, cultured and characterized by α-SMA immunoreactivity. Conditioned medium from CAFs enriched the populations of liver CSCs as reflected by an increased ability of self-renewal, chemoresistance, invasiveness and expression of stemness-associated genes and markers CD44 and CD47; and its CSC- stimulating effect was further enhanced when CAFs was stimulated with the conditioned medium of HCC cells. Using cytokine antibody array, hepatocyte growth factor (HGF) was found to be preferentially secreted by CAFs and increased upon stimulation by HCC cells. To verify whether the effect of CAFs on regulation of liver CSCs is due to HGF, the effect of recombinant HGF on regulation of liver CSCs was examined. Recombinant HGF at the physiological levels of CAFs (2ng/mL and 10ng/mL) promoted stem-like properties of HCC cells, and the effect of CAFs on CSC properties was alleviated with addition of HGF neutralizing antibody and c-met inhibitor (PHA665-752). In a cohort of HCC patient’s samples, HGF expression was significantly correlated with α-SMA (p=0.0003), suggesting the paracrine effect of CAFs on HCC cells. Further experiments showed that HCC cells induced proliferation and migration of CAFs to tumor-associated stroma; and CAFs in turn secretes HGF to regulate the CSC plasticity by activating NF-κB-mediated EMT transition. This study potentially dissects the signaling cross-talks between HCC cells and CAFs within the tumor microenvironment which may potentially open a new avenue to develop targeted therapy against this deadly disease.

Citation Format: Yuen Ting Lau, Jessica Lo, Irene Oi Lin Ng, Terence Kin Wah Lee. Cancer associated fibroblasts-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma. [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 3044. doi:10.1158/1538-7445.AM2014-3044
Abstract 3045: Defining an elusive multiple myeloma stem cell population in mice
Authors: Kellner Joshua , Hua Yunpeng , Liu Bei , Li Zihai
Published: May 2015

Multiple Myeloma (MM) is a debilitating disease of proliferating and malignant plasma cells that is currently incurable. The ability of monoclonal recurrence of disease suggests it might arise from a stem cell-like population capable of self-renewal. The difficulty to isolate the cancer stem-like cell in MM in both mice and men has introduced confusion toward this hypothesis. XBP1s is a key positive regulator for unfolded protein response and a master transcription factor for plasma cell development. It was previously reported that transgenic overexpression of XBP1s in the B cell lineage of mice (XBP1s-Tg), induces a multiple myeloma phenotype resembling human MM. We have now defined a post-germinal center, pre-plasma B cell population that was significantly increased in XBP1s-Tg mice as the hallmarks of MM was demonstrated. This non-memory B cell population resembled a stem/progenitor cell population, with increased expression of aldehyde dehydrogenase (ALDH), Notch1 and c-kit. Importantly, utilizing an in vitro culture for differentiating plasma cells, the increased population began to differentiate into plasma cells, increasing in size and gaining CD138 expression, the hallmark marker for plasma cells. In conclusion, we have defined a transitional pre-plasma cell population that could potentially be a stem/progenitor population of multiple myeloma. These results provide a novel insight into stem cell biology with regard to multiple myeloma and the use of murine models of myeloma to study the disease.

Citation Format: Joshua Kellner, Yunpeng Hua, Bei Liu, Zihai Li. Defining an elusive multiple myeloma stem cell population in mice. [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 3045. doi:10.1158/1538-7445.AM2014-3045
Abstract 3047: Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells
Authors: Marhold Maximilian , Tomasich Erwin , Pernicová Zuzana , Fedr Radek , Soucek Karel , Spittler Andreas , Krainer Michael , Horak Peter
Published: May 2015

Tumor initiating subpopulations of cancer cells, also known as cancer progenitor or cancer stem cells (CSCs) were recently identified and characterized in prostate cancer. Emerging evidence suggests that hypoxia-inducible factor 1 alpha (HIF1A) contributes to CSC maintenance and influences the PI3K/AKT/mTOR axis, a pathway which is frequently altered in prostate cancer. In our study, we assessed the effects of hypoxia on PI3K/AKT/mTOR signaling in a subset of basal prostate cancer stem cells in a murine model of prostate cancer. First, we successfully isolated Lin-/Sca-1+/CD49f+ basal stem cell subpopulations from primary prostate tumors of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. We observed a consistent decrease in S6 phosphorylation within the CSC subpopulation, in agreement with an mTOR pathway inhibition. However, AKT phosphorylation in the CSC subpopulation was simultaneously elevated, thus prompting further investigations. To analyze the molecular pathways leading to this deregulation, we used a cell line model and isolated Sca-1+/CD49f+ CSC-like subpopulations from the murine prostate cancer cell line TRAMP-C1 and CD44+/CD49f+ CSC-like subpopulations from the androgen independent human prostate cancer cell line DU145. We used sphere formation assays and expression of Notch 1 and Oct 3/4 to confirm the stem and progenitor cell properties of the sorted subpopulations. We could confirm the decrease in mTOR/S6 signaling and identify the upregulation of AKT through IRS-1 mediated feedback loop in prostate CSC-like cells under hypoxic conditions. This AKT/mTOR deregulation was promptly reversed in normoxia, suggestive of the involvement of hypoxia inducible factors. We found elevated HIF1A protein levels as well as increased target gene expression in CSC subpopulations in vitro and in vivo. Further, lentiviral knockdown of HIF1A restored AKT/mTOR activity in CSC-like cells. We could show that CSC-like prostate cancer cells with elevated expression of basal stem cell markers, such as CD44, Sca-1 or CD49f, show a hypoxic deactivation of feedback inhibition on AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway via HIF1A may be critical for cancer stem cell quiescence and maintenance, slowing down cancer stem cell metabolism and growth via mTOR and promoting their survival via AKT signaling.

Citation Format: Maximilian Marhold, Erwin Tomasich, Zuzana Pernicová, Radek Fedr, Karel Soucek, Andreas Spittler, Michael Krainer, Peter Horak. Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem 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 3047. doi:10.1158/1538-7445.AM2014-3047
Abstract 3048: OMP-59R5 (Anti-Notch2/3) inhibits tumor growth and reduces cancer stem cell frequency in patient derived SCLC xenografts
Authors: Fischer Marcus M. , Shah Jalpa , Cain Jennifer , Cancilla Belinda , Evans James W. , Murriel Christopher L. , Tang Tracy , Wei Jie , Yen Wan-Ching , Zhang Chun , Gurney Austin , Lewicki John , Kapoun Ann M. , Hoey Timothy
Published: May 2015

Small cell lung cancer (SCLC) is a devastating disease with very poor prognosis and few treatment options. We have established patient derived xenografts from SCLC tumors and determined that Notch pathway target genes are up-regulated in this disease relative to normal lung tissues and relative to other tumor types. We examined the in vivo efficacy of OMP-59R5, an antibody that was initially identified by binding to Notch2 and was subsequently shown to block signaling of both human and mouse Notch2 and Notch3. We performed in vivo growth inhibition and tumorigenicity studies with this Notch2/3 antibody alone or in combination with chemotherapeutic agents in a panel of six patient-derived SCLC xenograft models. We found that OMP-59R5 treatment significantly reduced tumor recurrence in combination with chemotherapy in five of the six models. Through a serial transplantation study from a sensitive tumor, we found that OMP-59R5 in combination with chemotherapy (cisplatin plus irinotecan) profoundly reduced the cancer stem cell (CSC) frequency. In contrast, treatment with chemotherapeutic agents alone had the opposite effect and increased CSC frequency. In minimal residual disease models, we analyzed the ability of OMP-59R5 to delay tumor recurrence following chemotherapy-induced tumor regression. While chemotherapeutic agent-treated tumors re-grew rapidly following treatment termination, the growth of recurrent tumors was delayed by OMP-59R5. We also evaluated changes in gene expression post treatment and found that OMP-59R5 modulated the expression of Notch pathway, epithelial-to-mesenchymal transition, stromal and vasculature genes. Furthermore, we observed an increase in expression of neuroendocrine differentiation markers after OMP-59R5 treatment in sensitive tumors. Following up on these preclinical findings, OMP-59R5 is currently being evaluated for safety and efficacy in combination with cisplatin and etoposide for treatment of SCLC in an ongoing Phase 1b/2 clinical trial (PINNACLE).

Citation Format: Marcus M. Fischer, Jalpa Shah, Jennifer Cain, Belinda Cancilla, James W. Evans, Christopher L. Murriel, Tracy Tang, Jie Wei, Wan-Ching Yen, Chun Zhang, Austin Gurney, John Lewicki, Ann M. Kapoun, Timothy Hoey. OMP-59R5 (Anti-Notch2/3) inhibits tumor growth and reduces cancer stem cell frequency in patient derived SCLC xenografts. [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 3048. doi:10.1158/1538-7445.AM2014-3048
Abstract 3049: Regulation of cancer cell stemness by delta-like 1 homolog (Drosophila) and prohibitins
Authors: Lin Qun , Begum Asma , Yu Chenye , Hu Ming-Yeah , Kim Yuri , Yun Zhong
Published: May 2015

Cancer stem cell characteristics, especially their self-renewal and clonogenic potential, play an essential role in malignant progression and response to anti-cancer therapies. Currently, it remains largely unknown which pathways are involved in the regulation of cancer cell stemness and differentiation. Previously, we found that delta-like 1 homolog (Drosophila) or DLK1, a developmentally regulated gene, plays a critical role in regulation of differentiation, self-renewal, and tumorigenic growth of neuroblastoma cells. In order to gain mechanistic understanding of the involvement of DLK1 in intracellular signal transduction, we identified DLK1-interacting proteins using an affinity purification approach. As reported herein, we have found that DLK1 specifically interacts with the prohibitin (PHB) complex via the DLK1 cytoplasmic domain. PHB1 and the closely related PHB2 are encoded by evolutionarily conserved genes and possess diverse functions from mitochondrial structural integrity and oxidative phosphorylation to gene transcription in the nucleus. We have found that DLK1 regulates mitochondrial membrane potential and production of reactive oxygen species (ROS). Our data further reveal a role of PHBs and especially PHB2 in the regulation of cancer cell self-renewal as well as their clonogenic potential. Hence, the DLK1-PHB interaction constitutes a new signaling mechanism that promotes the maintenance of cancer cell stemness.

This work was supported by a grant from the National Institutes of Health to ZY (R01CA125021). YK was supported in part by an institutional postdoctoral training grant (T32) from the National Institutes of Health and an Anna Fuller Fund Fellowship from Yale School of Medicine.

Citation Format: Qun Lin, Asma Begum, Chenye Yu, Ming-Yeah Hu, Yuri Kim, Zhong Yun. Regulation of cancer cell stemness by delta-like 1 homolog (Drosophila) and prohibitins. [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 3049. doi:10.1158/1538-7445.AM2014-3049
Abstract 3054: Snail-induced epithelial-mesenchymal transition generates the properties of cancer stem cells in head and neck cancer cells
Authors: Ota Ichiro , Masui Takashi , Mikami Shinji , Yamanaka Toshiaki , Hosoi Hiroshi
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) is known to its rapid clinical progression and poor prognosis. Notably, regional and distant metastases in HNSCC correspond to an extremely poor prognosis with limited treatment options. The treatment resistance and tumor recurrence are important clinical problems in the management of HNSCC. Epithelial-mesenchymal transition (EMT) is a key process in successful execution of many steps such as invasion and metastasis for cancer cells. Snail is one of master regulators that promote EMT in many types of malignancies including HNSCC. Recently, it has been reported that Snail-induced EMT could induce cancer stem cell (CSC)-like phenotype in a number of tumor types. Herein, we investigated the role of Snail in inducing EMT properties and CSC-like phenotype in HNSCC. We constructed HNSCC cell lines transfected with Snail. E-cadherin was analyzed using western blotting and immunofluorescence staining. Cell migration and invasion were assessed using wound healing assay and modified Boyden chamber assay, respectively. CSC markers of HNSCC, CD44 and aldehyde dehydrogenase 1 (ALDH1), were also evaluated with western blotting, and chemosensitivity was assessed with WST-8 assay. Introduction of Snail induced EMT properties in HNSCC cells and enhanced cell migration and invasion. Moreover, Snail-induced EMT gained CSC-like phenotype and was associated with increased chemoresistance. These results suggest that Snail can be one of the attractive targets for the development of therapeutic strategies in HNSCC.

Citation Format: Ichiro Ota, Takashi Masui, Shinji Mikami, Toshiaki Yamanaka, Hiroshi Hosoi. Snail-induced epithelial-mesenchymal transition generates the properties of cancer stem cells in head and neck cancer 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 3054. doi:10.1158/1538-7445.AM2014-3054
Abstract 3062: Isolation and characterization of circulating tumor cells (CTCs) from peripheral blood specimens of patients with advanced solid tumor malignancies (using ApoStream™ instrumentation)
Authors: Balasubramanian Priya , Wang Lihua , Lawrence Scott M. , Navas Tony , Kummar Shivaani , Hollingshead Melinda , Owusu Francis , Parchment Ralph E. , Tomaszewski Joseph E. , Doroshow James H. , Kinders Robert J.
Published: May 2015

Circulating tumor cells (CTCs) isolated from the peripheral blood of cancer patients provide prognostic information and may inform treatment decisions. Current strategies for CTC isolation and characterization commonly employ epithelial marker-specific antibody capture of fixed cells from blood and detection based on cytokeratin (CK) expression. Our laboratory is currently evaluating an antibody-independent CTC enrichment technology, ApoStream™, which isolates live CTCs from blood, enabled by critical dissimilarities in morphology and dielectric properties of CTCs and blood cells rather than surface marker expression. CTCs isolated with the ApoStream™ instrument are amenable to further high resolution phenotypic characterization, enabling unambiguous identification of enriched cells as malignant cells. We previously reported on the utility of ApoStream™ technology for the isolation of CTCs from patients with advanced alveolar soft part sarcoma (ASPS) with unambiguous confirmation of CTCs by break-apart fluorescent in situ hybridization (FISH) for ASPL-TFE3 gene translocation. Here, we show the isolation of viable CTCs from patients with advanced solid tumors from ongoing clinical studies at the NCI. Since during progression, cancer cells undergo epithelial to mesenchymal transitions (EMT) and mesenchymal to epithelial transitions (MET), we sought to characterize ApoStream™-isolated CTCs using a multiplex phenotyping assay for CD45 (hematopoietic marker), EMT markers (CK, EpCAM, β-catenin and Vimentin), and tumor specific markers (MUC1 and CEA). A user-defined processing algorithm and CTC scoring criteria developed using Definiens® software was used for rare cell detection and enumeration. Our current efforts are focused on evaluating the utility of ApoStream™-isolated CTCs for assessing pharmacodynamic effects of anticancer agents on DNA damage response in patients with refractory solid tumors. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: Priya Balasubramanian, Lihua Wang, Scott M. Lawrence, Tony Navas, Shivaani Kummar, Melinda Hollingshead, Francis Owusu, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Isolation and characterization of circulating tumor cells (CTCs) from peripheral blood specimens of patients with advanced solid tumor malignancies (using ApoStream™ instrumentation). [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 3062. doi:10.1158/1538-7445.AM2014-3062