bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4966: Influence of the microenvironment on drug sensitivity in breast cancer using a three-dimensional cell culture model.
Authors: Lovitt Carrie J. , Avery Vicky M.
Published: August 2014

Three-dimensional (3D) cell culture is an important research tool and an invaluable alternative to two-dimensional (2D) cell culture as it is more suitable to represent in vivo conditions. The tumor microenvironment influences cellular activity and using 3D cell culture allows these interactions to be studied in vitro. We have evaluated the efficacy of a novel 3D cell culture model for breast cancer and determined cellular responses caused by drug activity. We have recently developed and optimized a miniaturized 3D breast cancer culture model. Our model uses reconstituted basement membrane from Growth Factor Reduced Matrigel™ as a scaffold and is suitable for semi-automated early stage drug discovery. The newly developed 3D culture model has been extensively characterized for reproducibility and the activity of known anti-breast cancer therapies. Notably, anthracyclines and taxanes showed significantly altered drug sensitivity in 3D when compared to monolayer cell cultures of breast cancer cell lines. To determine the mechanisms underlying the altered sensitivity in 3D cell culture, we sought to identify the differences between untreated and drug treated spheroids, as well as the differences between 2D and 3D cell culture responses to anthracyclines and taxanes. We evaluated proliferation, penetration, cellular architecture and signaling of major cellular pathways in our 3D model. We will discuss the proliferation rate comparing 2D and 3D cell culture models and drug penetration (exemplified by doxorubicin) in 3D cell culture. The effect of anthracyclines and taxanes on the 3D cellular architecture, without the influence of extracellular matrix proteins in the microenvironment, will also be presented. In addition, we have characterized the signaling pathways in 2D and 3D cell culture commonly associated with enhanced drug resistance and other relevant proteins including those associated with apoptosis. Deconstructing the cellular processes in 3D breast cancer cell culture in response to breast cancer therapies has the potential to elucidate drug resistance mechanisms. The results obtained from this research illustrate the characteristics and mechanisms of both monolayer and 3D cell culture with respect to anthracycline and taxane drug activity. These results demonstrate the benefits of 3D cell culture in early stage novel compound evaluation for breast cancer.

Citation Format: Carrie J. Lovitt, Vicky M. Avery. Influence of the microenvironment on drug sensitivity in breast cancer using a three-dimensional cell culture model. [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 4966. doi:10.1158/1538-7445.AM2013-4966
Abstract 4970: Folate metabolism defect induce invasion and Gefinitib resistance in lung cancer.
Authors: Ho Chun-Te , Liu Tsan-Zon , Chou Yu-Ting , Hsiao Shih-Hsin , Liu Jun-Jen
Published: August 2014

Folate, act as an one-carbon units donator or acceptor, participate in numerous biochemical reactions. Because mammals can't synthesize folic acid de novo, obtain from diet or from microbial breakdown in the gut is essential. Pathological study revealed that tumor cell expressing high level of folate receptors (FR) comparing to normal tissue. Therefore, exploring the application of folate-related physiological properties as an anticancer strategy arose worldwide interesting. Previous studies in lung cancer revealed a positive correlation in folate receptors overexpression and efficacy of antifolate chemotherapy. Here we describe prolong Alimta treatment would prompt lung cancer cell's malignancy. Cultivation of lung cancer CL1-0, CL1-5, and A549 in low dose Alimta (15-30 ng/mL) for 2-3 weeks would dramatically cause cell morphology change . Compare to control cell with round and flat epithelial like morphology, low dose Alimta treated cells showed mesenchymal to amoeboid phenotype. Further examination of epithelial to mesenchymal transition (EMT) markers showed the expression level of N-cadherin, matrix metalloproteinase 1 (MMP-1) and matrix metalloproteinase 2 (MMP-2) increased. Associated with morphology and EMT marker changed, cell migration and invasion ability of those Alimta treated cells were 3 folds higher than control cells. Besides, evaluated drug sensitivity by sulforhodamine B (SRB) method showed that those alimta treated cells were more resistant to gefitinib. These prominent events provoked our interesting to investigate the mechanism involving in the oncogenic effect induced by low dose Alimta treatment. While examing EMT-related transcription factors, both Slug and SOX-9 expression increased significantly. These evidences indicated that the folate metabolism defect induced metastasis could through the SOX-9 and Slug mechanism.

Citation Format: Chun-Te Ho, Tsan-Zon Liu, Yu-Ting Chou, Shih-Hsin Hsiao, Jun-Jen Liu. Folate metabolism defect induce invasion and Gefinitib resistance in lung cancer. [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 4970. doi:10.1158/1538-7445.AM2013-4970

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 4976: Characterization of the neuroinflammatory response in a mouse model of brain metastatic inflammatory breast cancer.
Authors: Reed L. Tiffany , Palmieri Diane , Mason Ginny , Steeg Patricia
Published: August 2014

Inflammatory Breast Cancer (IBC) is a rare form of breast cancer, accounting for approximately 2% of all breast cancer cases worldwide. While rare, it is extremely progressive and has a high propensity for metastatic spread and decreased overall survival compared to non-IBC breast cancers. IBC has a five-year overall survival rate of 30%; the median age at diagnosis is 57 years of age. Approximately 15% of IBC patients develop brain metastasis, and most of the brain metastasizing IBC tumors are triple negative (ER-, PR- and HER2-) or Her-2 overexpressing. The goal of this study was to investigate the neuroinflammatory response of SUM190 cells, a Her-2 overexpressing IBC cell line, in a mouse model of brain metastasis. The SUM190 brain metastatic mouse model of IBC was developed following three-sequential rounds of injection of cells into the left cardiac ventricle, isolation of metastatic tumor cells from the brains of the mice, and growth in tissue culture to maintain brain metastatic potential of the cell line. In this model, brain metastases were widely disseminated throughout the neuroparenchyma. The lesions were round, and measured 0.5-1 mm in maximal diameter, with a central area of necrosis characterized by shrunken hypereosinophilic epithelial cells with karryorhectic or absent nuclei. There was no significant difference in vascular proliferation in the peritumoral microenvironment compared to the unaffected neuroparenchyma (p=0.57) following quantitative immunofluorescent analysis. However, there was a 4-fold increase in GFAP-positive astrocytes (p=<0.0001) and CD11b-positive microglia (p=0.001) and a 3-fold increase in F4/80-positive macrophages (p=0.001) in the tumor microenvironment compared to the unaffected neuroparenchyma. CD3+ T cells were widely disseminated throughout the tumor microenvironment. The influx of inflammatory cells in brain metastases associated with IBC is greater than that of other brain-tropic breast cancer cell lines including MDA-MB-231-BR, and may explain the increased incidence of metastasis in IBC patients. This research was supported by the NIH Intramural Research Program and a grant from the Inflammatory Breast Cancer Research Foundation.

Citation Format: L. Tiffany Reed, Diane Palmieri, Ginny Mason, Patricia Steeg. Characterization of the neuroinflammatory response in a mouse model of brain metastatic inflammatory breast cancer. [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 4976. doi:10.1158/1538-7445.AM2013-4976
Abstract 4978: Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival.
Authors: Kadel Edward (Ward) E. , Walter Kimberly , Desai Rupal , Carbon Juliet , Boe Marigold , Shames David , Kowanetz Marcin
Published: August 2014

The immune system has multiple mechanisms by which it can eliminate cancer and yet, tumors are able to adapt resistance to host immune surveillance and continue to survive and grow. One critical resistance mechanism involves Programmed cell death 1 ligand 1 (PD-L1, CD274, B7-H1), the predominant ligand for PD-1, an inhibitory receptor expressed on T cells following activation. PD-L1 also binds to B7.1 (CD80), inhibiting its ability to provide an immune stimulatory signal. PD-L1 is expressed broadly on multiple peripheral blood mononuclear cell subtypes, placenta, and numerous cancers, including NSCLC. Tumor-specific T cells infiltrate tumors and recognize tumor cells, releasing Interferon-gamma (IFNγ), initiating signaling of the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in the tumor cells. IFNγ induces and/or greatly enhances the expression of PD-L1 in the tumor, among other cell types, allowing the tumor to become resistant to the host T cell response. Blockade of PD-L1 binding to PD-1 and B7.1 can reinvigorate the host immune response against the tumor and overcome tumor adaptive resistance. The regulation of PD-L1 expression is complex and likely involves multiple types of pre- and post-translational events. We show that basal PD-L1 expression levels can vary greatly in cancer cells, as can PD-L1 induction by IFNγ. Here we describe the different categories of PD-L1 basal expression and IFNγ–dependent regulation across multiple lung cell lines and human tumor samples. Reverse Phase Protein Array and RNA microarray data show that the JAK/STAT canonical pathways are still intact among all of these distinct categories, so alternative mechanisms of expression regulation must be active in these cell lines and tumor samples. We provide evidence that a combination of mechanisms regulate both the basal and stimulated expression levels of PD-L1 across these distinct categories. This data regarding PD-L1 expression regulation provides valuable information to better understand the PD-1/PD-L1 pathway as a therapeutic target.

Citation Format: Edward (Ward) E. Kadel, Kimberly Walter, Rupal Desai, Juliet Carbon, Marigold Boe, David Shames, Marcin Kowanetz. Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival. [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 4978. doi:10.1158/1538-7445.AM2013-4978
Abstract 4985: Expression of FasL on the tumor endothelium establishes a selective immune barrier in ovarian cancer.
Authors: Motz Greg , Coukos George
Published: August 2014

There is an intimate relationship between angiogenesis and immune suppression in cancer, and anti-angiogenesis strategies can complement cancer immunotherapy. Previous research has implicated the tumor endothelium as a critical regulator of anti-tumor immunity, and our current work establishes a new role for the tumor endothelium as a critical barrier to T cell infiltration mediated through FasL. FasL is an important regulator of immunity and apoptosis, and we have found that, in contrast to the endothelium of normal tissues, the endothelium of multiple tumor types expressed FasL. FasL expression was restricted to tumor islets, and hypoxic tumor cell-conditioned media induced FasL on endothelial cells in vitro. Combination treatment with proangiogenic growth factors VEGF and PGE2 induced endothelial FasL expression in vitro, and COX1 and VEGF expression were associated with endothelial expression of FasL in ovarian cancer patients. Combinatorial blockade of VEGF and COX enzymes reduced endothelial FasL expression in mice, decreased tumor volume, and increased the CD8/FoxP3 ratio. In patients, low endothelial FasL expression was associated with a high CD8/Foxp3 ratio and increased survival. We found that endothelial FasL contributes to an unfavorable CD8/FoxP3 ratio through preferential killing of tumor-reactive CD8 T cells relative to Tregs. Increased intratumoral CD8/FoxP3 ratio was also observed in tumors grown on gld and lpr mice, but tumor growth was not robustly inhibited in these mice. Tumor control was dependent on the induction of an anti-tumor immune response, and CD8 depletion reversed the effects of VEGF and COX blockade. Additionally, combinatorial VEGF and COX blockade, tumors grown on gld mice, or the use of anti-FasL antibody all enhanced infiltration of adoptively transferred anti-tumor T cells and reduced apoptosis of transferred cells. Together, this work lends mechanistic insights into existing observations regarding T cells and tumor angiogenesis, and provides additional evidence for the paradigm that angiogenesis and immune suppression are intricately linked. Additionally, this work provides new strategies for cancer immunotherapy that may result in superior tumor control in patients.

Citation Format: Greg Motz, George Coukos. Expression of FasL on the tumor endothelium establishes a selective immune barrier in ovarian cancer. [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 4985. doi:10.1158/1538-7445.AM2013-4985

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 4988: FOXP3, a critical mediator of regulatory T-cell function, is expressed in subsets of myeloid cells.
Authors: Gara Sudheer Kumar , Pang Yanli , Hollander Christine , Yang Li
Published: August 2014

Forkhead box P3 (FOXP3) is a well-known molecular marker for regulatory T cells (Tregs) and is considered as a master regulatory transcription factor for the development, maturation and function of Tregs. Mutations in FOXP3 lead to severe immune deficiencies and cancer in human and mice. Administration of Foxp3-expressing Tregs drastically reduced the disease severity in animal models of diabetes, asthma and multiple sclerosis giving rise to hope for therapeutic options. It is not clear whether cell types other than Treg express FOXP3. While investigating TGFβ-mediated signaling in the myeloid lineage, we discovered a subset of myeloid derived suppressor cells (Gr-1+CD11b+ cells or MDSCs) that express FOXP3. Indeed these cells were CD3 negative suggesting that they are not of T cell lineage. Upon further investigation, we observed that these FOXP3 expressing myeloid cells are present in different tissues of mouse. Moreover, we found that they are elevated under pathological conditions. We are currently identifying the molecular function and mechanisms of Foxp3 expression in the myeloid subsets. Our work will provide new insight into the FOXP3 research area beyond regulatory T cells, and might reveal its role in regulating the immunosuppressive activity of MDSCs.

Citation Format: Sudheer Kumar Gara, Yanli Pang, Christine Hollander, Li Yang. FOXP3, a critical mediator of regulatory T-cell function, is expressed in subsets of myeloid cells. [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 4988. doi:10.1158/1538-7445.AM2013-4988
Abstract 4994: Hypoxia-induced autophagy and TNF-alpha resistance in breast cancer cells leads to tumor evasion from NK-mediated immunosurveillance by downregulation of ICAM1.
Authors: Janji Bassam , Baginska Joanna , Medves Sandrine , Van Moer Kris , Chouaib Salem , Berchem Guy
Published: August 2014

The major obstacle for defining efficient immunotherapeutic approaches for the treatment of solid tumor is the capability of tumor cells to evade host immune response under hypoxic microenvironment. We have investigated how breast cancer cells manage to evade effective immunosurveillance. Since the host's immune evasion of many breast cancer cells is correlated with the acquisition of resistance to TNF-α, we used TNF-sensitive (MCF-7) and TNF-resistant (1001) breast cancer cells. We showed that the acquisition of TNF resistance in 1001 cells is correlated with the induction of hypoxia inducible factor (HIF)-1α and a constitutive activation of autophagy. When cultured under hypoxia, TNF-sensitive tumor cells activate autophagy and resist to the cytotoxic effect of TNF-α. This data strongly argue for a role of hypoxia in the development of TNF-resistant tumor cell population. Furthermore, we showed that TNF-resistant cells evade natural killer (NK) cells-mediated lysis by a mechanism involving a decrease in the conjugate formation between TNF-resistant and NK cells. The decrease in the ability of NK cells to recognize TNF-resistant tumor cells is related to a dramatic down-regulation of Intercellular Cell Adhesion Molecule (ICAM-1) at the surface of tumor cells. The mechanism responsible for the decrease of ICAM-1 expression in TNF-resistant cells and its functional link with autophagy is currently under investigation. Together, this study discloses the major role of hypoxic microenvironment in the activation of the autophagic process in tumor cells which ultimately leads to tumor evasion from immunosurveillance.

Citation Format: Bassam Janji, Joanna Baginska, Sandrine Medves, Kris Van Moer, Salem Chouaib, Guy Berchem. Hypoxia-induced autophagy and TNF-alpha resistance in breast cancer cells leads to tumor evasion from NK-mediated immunosurveillance by downregulation of ICAM1. [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 4994. doi:10.1158/1538-7445.AM2013-4994

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 4995: Tumor-dervied gammadelta regulatory T cells suppress innate and adaptive immunity through the induction of immunosenescence.
Authors: Ye Jian , Ma Chunling , Hsueh Eddy C. , Zhang Yanping , Varvares Mark A. , Hoft Daniel F. , Peng Guangyong
Published: August 2014

Fundamentally understanding the suppressive mechanisms utilized by different subsets of tumor-infiltrating regulatory T (Treg) cells is critical for the development of effective strategies for anti-tumor immunotherapy. Gammadelta Treg cells have recently been identified in human diseases including in cancer patients. However, the suppressive mechanisms and functional regulations of this new subset of unconventional Treg cells are largely unknown. In the current studies, we explored the suppressive mechanism(s) utilized by human breast tumor-derived gammadelta Treg cells on innate and adaptive immunity. We found that gammadelta Treg cells induced immunosenescence in targeted naïve and effector T cells, as well as dendritic cells (DCs). Furthermore, senescent T cells and DCs induced by gammadelta Treg cells had altered phenotypes and developed potent suppressive activities, further amplifying the immunosuppression mediated by gammadelta Treg cells. In addition, we demonstrated that manipulation of TLR8 signaling in gammadelta Treg cells can block gammadelta Treg-induced conversion of T cells and DCs into senescent cells in vitro and in vivo. Our studies identify the novel suppressive mechanism mediated by human tumor-derived gammadelta Treg cells on innate and adaptive immunity, which should be critical for development of strong and innovative approaches to reverse the tumor suppressive microenvironment and improve effects of immunotherapy. This work was supported by the grant from the American Cancer Society (RSG-10-160-01-LIB).

Key words: gammadelta T cells; Regulatory T cells; Dendritic cells; Senescence; Immune suppression

Citation Format: Jian Ye, Chunling Ma, Eddy C. Hsueh, Yanping Zhang, Mark A. Varvares, Daniel F. Hoft, Guangyong Peng. Tumor-dervied gammadelta regulatory T cells suppress innate and adaptive immunity through the induction of immunosenescence. [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 4995. doi:10.1158/1538-7445.AM2013-4995
Abstract 5018: Evidence for a dynamic interaction between bone marrow and the cutaneous epithelium during skin carcinogenesis by exposure to solar ultraviolet radiation.
Authors: Morris Rebecca J. , Readio Nyssa AK , Singh Ashok , Singh Anupama
Published: August 2014

Non-melanoma skin cancer induced by solar ultraviolet radiation (UV) is the most common of epithelial cancers and occurs more frequently in the human population than any other type of malignancy. Therefore, determining its etiology is a significant problem. The objective of this investigation was to determine whether there might be an interaction between epidermal keratinocytes and bone marrow during carcinogenesis induced by solar UV. First, we determined that bone marrow cells were recruited to the cutaneous epithelium where a subpopulation assumed epithelial characteristics during chronic exposure of mice to solar UV radiation. Second, we documented the presence of keratins in blood and bone marrow prior to and during exposure of mice to solar UV radiation by quantitative real time polymerase chain reaction, fluorescence activated cell sorting of freshly harvested cells, and by immunofluorescence microscopy of bone marrow smears. We observed low, but reproducible levels of keratins 8, 14 and 15 in both blood and bone marrow of un-irradiated mice, with a relatively higher expression of keratin 8 in blood, and a relatively higher expression of keratins 14 and 15 in bone marrow. Third, during chronic exposure to solar UV, we detected a dose dependent increase in the percentage of cells expressing keratin 14 in bone marrow: increasing from approximately 1% in un-irradiated control bone marrow, to approximately 10% after 10 weeks of irradiation, with greater increases at later time points. Surprisingly, this increase in keratin expression in bone marrow began many weeks prior to development of benign or malignant cutaneous neoplasms. Fourth, because these results could suggest a possible egress of keratinocytes from the cutaneous epithelium and homing to the bone marrow, we performed in vitro keratinocyte migration assays with bone marrow as bait. We observed a significant and reproducible migration of epidermal keratinocytes towards bone marrow over controls such as 3T3 cells, fetal bovine serum, or other keratinocyte cultures. We conclude from these studies that there is a dynamic interaction between the cells of the cutaneous epithelium and the bone marrow. The cells and the mechanisms involved in this interaction are currently under investigation.

Citation Format: Rebecca J. Morris, Nyssa AK Readio, Ashok Singh, Anupama Singh. Evidence for a dynamic interaction between bone marrow and the cutaneous epithelium during skin carcinogenesis by exposure to solar ultraviolet radiation. [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 5018. doi:10.1158/1538-7445.AM2013-5018
Abstract 5019: Stem cell and neuroendocrine cell phenotypes of normal and malignant colonic cells.
Authors: Modarai Shirin R. , Opdenaker Lynn M. , Boman Bruce M.
Published: August 2014

In the development of colorectal cancer (CRC), stem cell (SC) overpopulation underlies tumor initiation and progression, but the underlying mechanisms are poorly understood. We hypothesized that neuroendocrine cells (NE) cells play a key role in maintaining the SC population in the normal colon and, aberrant signaling from NE cells contributes to tumor initiation and increased stemness. Indeed, NE cells and SCs reside in close proximity to each other in the SC niche at the bottom of the normal human colonic crypt. Therefore, it seems feasible that the communication between NE cells and SCs is crucial to normal crypt homeostasis and that dysregulation of the interactions between the two cell types could lead to SC overpopulation during cancer progression. Our studies on human colonic tissues show that in normal crypts, most cells (>80%) expressing the colonic SC marker ALDH1 co-express NE markers such as chromogranin-A (CGA). Neither ALDH1+ nor CGA+ cells co-stained for markers for proliferating cells. Thus, in normal crypts, many ALDH1+ cells have NE characteristics (likely representing progenitor NE cells). In APC mutant crypts, the size of the undifferentiated SC population (ALDH+/CGA-) progressively increased relative to the size of the NEC population (both ALDH1+/CGA+ and ALDH1-/CGA+) during the stepwise progression to cancer. The distributions of these populations also extended father up the crypt (outside the normal SC niche). To evaluate how NE factors might regulate SCs, we did further studies to assess how NE factors might be regulating SCs. Because we found, by flow cytometric analysis, that various CRC cell lines (COL0320, DiFi, LoVo, SW480, HCT116 & HT29) each maintains a unique proportion of SCs and NE cells over time, we surmised these proportions are maintained constant through feedback mechanisms involving SC and NEC subpopulations. When these cell lines were induced to differentiate along the NE lineage upon retinoid treatment, they showed a decrease in the proportion of ALDEFLUOR+ cells and an increase in NE cell differentiation and enhanced collagen IV production. Retinoid treatment to induce NE differentiation also decreased the sphere-forming ability in terms of the number and sizes of spheres that were formed from each cell line. Taken together, our findings indicate that: (1) NE factors contribute to colonic SC regulation via an autocrine or paracrine mechanism in normal colon, and (2) dysregulation of these mechanisms contributes to colon cancer development.

Citation Format: Shirin R. Modarai, Lynn M. Opdenaker, Bruce M. Boman. Stem cell and neuroendocrine cell phenotypes of normal and malignant colonic cells. [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 5019. doi:10.1158/1538-7445.AM2013-5019
Abstract 5020: Characterization of epidermal stem cells in SKH1 hairless mice, a widely used mouse model to investigate ultraviolet radiation carcinogenesis.
Authors: Singh Ashok , Singh Anupama , Verma Ajit K.
Published: August 2014

Human cutaneous squamous cell carcinoma (SCC) is the second most common type of non-melanoma skin cancer (NMSC) after basal cell carcinoma (BCC) in US. Evidence indicates that the precursor cells are the precursor cells for the origin of SCC. The SCCs probably arise from bulge stem cell niche of hair follicle but not from the transit amplifying cells of epidermis. The SKH1 hairless mice is a widely used mouse model for ultraviolet radiation (UV)-induced carcinogenesis. However, SKH1 mice have never been evaluated for status of skin stem cell probably due to absence of hair and other associated hairless phenotype. It has recently become possible to isolate living hair follicle stem and progenitor cells from mouse skin because of the discovery of cell surface marker (CD34) that facilitate enrichment. The cell surface protein CD34, more widely known as a hematopoietic stem and progenitor cell marker, was found to be uniquely expressed in the mouse hair follicle bulge region using immunohistochemistry staining techniques. In addition, CD34 expression was restricted to the hair follicle regardless of hair follicle stage, making this a potentially important tool for selected enrichment of hair follicle bulge region keratinocytes. In combination with alpha 6-integrin staining and fluorescence activated cell sorting (FACS), CD34 is used to successfully isolate a small population of CD34+/alpha 6-integrin+ cells from a single cell preparation of mouse keratinocytes. CD34-expressing keratinocytes were confirmed to have properties consistent with stem and progenitor cells in that they were shown to be slowly cycling and have a high proliferative capacity in culture, growing larger colonies relative to those from CD34− keratinocytes. In the present experiments, the SKH1 mouse skin stem cell population was identified using stem cell markers (CD34+/α6-integrin+, Keratin-15, Gli1, and Sox9). The results of FACS analysis of live cells, isolated from untreated SKH1 mouse skin, indicate that there are 0.23% CD34+ and 1.05% CD34+/α6-integrin+ cells. The FACS sorted double positive (CD34+/α6-integrin+) stem cells of SKH1 are also observed to be positive for K15, Gli1, and Sox9 markers of stem cells in cytospin slides. In summary, the quantitative FACS analysis and qualitative immunofluorescence data in skin tissues confirms the presence of the stem cell populations in SKH1 mice. Support: (RO1CA102431).

Citation Format: Ashok Singh, Anupama Singh, Ajit K. Verma. Characterization of epidermal stem cells in SKH1 hairless mice, a widely used mouse model to investigate ultraviolet radiation carcinogenesis. [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 5020. doi:10.1158/1538-7445.AM2013-5020
Abstract 5024: Epigenetic regulation of cancer stem cell states in a p53/PTEN inactivated mammary epithelial cells.
Authors: Ouzounova Maria , Davis April , Kim Gwangil , Malik Fayaz , Quraishi Ahmed A. , Clouthier Shawn G. , Wicha Max S. , Korkaya Hasan
Published: August 2014

Inactivation of the tumor suppressors, TP53 and PTEN in pre-neoplastic lesions has been implicated in tumor progression as well as in regulation of normal and malignant stem cell self-renewal. Down regulation of both p53 and PTEN in non-transformed MCF10A cells synergized to expand the cancer stem cell compartment transforming these cells and generating an EMT phenotype in vitro which generated highly metastatic tumors when injected in NOD/SCID mice.

These tumors could be fractionated based on their expression of CD49f and EPCAM. Gene expression analyses of these sub-fractions demonstrate an EMT gene signature in CD49f+/EPCAM- and CD49f-/EPCAM- cell populations. These findings are also supported by DNA methylation analyses. Although CD49f+EPCAM+ cell population express epithelial (MET) gene signature, these cells also generate tumors in mice and give rise to cells with EMT phenotype. Interestingly CD49f+EPCAM+ subpopulation expresses higher ALDH than other two subpopulations with EMT phenotype. Therefore we propose that breast CSCs may exist in two states; EMT state may be represented by CD49f or CD44 and MET state by ALDH expression. Consistent with our hypothesis, ALDH-positive cells compared to ALDH-negative cells within the CD49f+EPCAM+ phenotype have greater capacity to generate CD49f+/EPCAM- phenotype upon IL6 or TGF-b stimulation. Interestingly blocking IL6 and TGF-b in CD49f+/EPCAM- cell population results in MET-CSC phenotype characterized by ALDH expression. It is well known that EMT is characterized by reversible epigenetic changes and therefore we hypothesized that these epigenetic changes might be involved in acquisition of different CSC states.

Our results suggest that interplay between genetic and epigenetic changes during malignant transformation of mammary epithelial cells may activate multiple signaling cascades resulting in expansion of stem cells which display an EMT phenotype. Furthermore, these studies provide a strong rationale for the development of alternative therapeutics which are able to target these different states of cancer stem cells.

Citation Format: Maria Ouzounova, April Davis, Gwangil Kim, Fayaz Malik, Ahmed A. Quraishi, Shawn G. Clouthier, Max S. Wicha, Hasan Korkaya. Epigenetic regulation of cancer stem cell states in a p53/PTEN inactivated mammary epithelial cells. [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 5024. doi:10.1158/1538-7445.AM2013-5024
Abstract 5025: Presence of meningioma stroma mesenchymal stem-like cells.
Authors: Kim Eui Hyun , Kang Seok Ku
Published: August 2014

Presence of cells resembling bone marrow-mesenchymal stem cells (BM-MSCs) from glioma was proved, however it has been little known about existence of MSCs from meningioma. We try to investigate whether we are able to isolate cells resembling BM-MSCs. Thus, we hypothesized that cells similar to BM-MSCs exist in meningioma specimen.

We cultured fresh meningioma specimen using the same protocols for culturing BM-MSCs. The cultured cells were analyzed by fluorescence-activated cell sorting (FACS) for surface markers associated with BM-MSCs. These cells were exposed to mesenchymal differentiation conditions. To evaluate the tumorigenecity of cells resembling MSCs from meningioma, we injected these cells into the brain of athymic nude mice. To find possible locations, sections of meningioma specimens were analyzed by immunofluorescent labeling.

In 5 of 17 meningioma specimen, we isolated cells similar to BM-MSCs which were called meningioma stroma mesenchymal stem-like cells (MS-MSCs). MS-MSCs were spindle shaped and adherent cells. FACS analysis showed that the MS-MSCs had similar surface marker to BM-MSCs. (Cells that were positive for two of the markers among CD105, CD90, CD73 and negative for two of the markers among CD45, CD31, NG2 were chosen to see their differentiation capability.) Chosen cells were observed for their ability to differentiate into adipocytes, osteocytes and chondrocytes. MS-MSCs immnohistological analysis indicated that were closely associated with the endothelial cells and pericytes cells. No tumor was discovered within the brains of mice that received the MS-MSCs orthotopic inoculation. Our results recognized that cells similar to BM-MSCs exist in meningioma specimen for the first time. MS-MSCs could be a source of meningioma microenvironment.

Citation Format: Eui Hyun Kim, Seok Ku Kang. Presence of meningioma stroma mesenchymal stem-like cells. [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 5025. doi:10.1158/1538-7445.AM2013-5025

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 503: Mammary fibroblasts induce hormone-independent growth in estrogen receptor-positive breast cancer cells via an epithelial-to-mesenchymal transition in a 3D cell culture model.
Authors: Holton Sarah E. , Bergamaschi Anna , Katzenellenbogen Benita S. , Bhargava Rohit
Published: August 2014

The majority of breast cancers diagnosed in the United States are estrogen receptor positive (ER+). These patients may respond to targeted endocrine therapies, but up to 30% of patients experience a cancer recurrence. We hypothesized that the molecular interaction of ER+ breast cancer cells with mammary fibroblasts might lead to endocrine resistance and, eventually, breast cancer relapse. We developed a three-dimensional (3D) co-culture model representing cancer cell-fibroblast interactions during early tumor invasion. We show that the proliferation of MCF7 ER+ cells switches from hormone-dependent to growth factor-dependent in the presence of human mammary fibroblasts. Further, we determine that this transition is associated with gene and protein hallmarks of the epithelial-mesenchymal transition (EMT) in tumor cells, in particular upregulation of SNAIL and SLUG mRNA and loss of E-Cadherin protein. We identified the secreted proteins that arise from epithelial-fibroblast interactions in this system using an antibody-based array. The proteins secreted into the conditioned medium are sufficient to induce EMT markers in primary normal human mammary epithelial cells (HMECs). Our report demonstrates that estrogen receptor activity is dynamic and therefore is not best assessed with immunohistochemical techniques alone. For the facile and rapid determination of ER activity in a clinical setting, we propose the use of an emerging imaging method, highly sensitive label-free Fourier Transform infrared (FT-IR) spectroscopic imaging. We validate this technology in predicting endocrine sensitivity in breast cancer here, by detecting significant changes in spectroscopic peaks correlated with proliferation and metabolism of MCF7 cells grown in 3D culture.

(Supported by grants from The Breast Cancer Research Foundation to BSK, DOD postdoctoral fellowship W81XWH-09-1-0398 to AB, NIH NCI Alliance for Nanotechnology in Cancer ‘Midwest Cancer Nanotechnology Training Center R25 CA154015A to SEH, and National Institutes of Health RO1CA138882 to RB.)

Citation Format: Sarah E. Holton, Anna Bergamaschi, Benita S. Katzenellenbogen, Rohit Bhargava. Mammary fibroblasts induce hormone-independent growth in estrogen receptor-positive breast cancer cells via an epithelial-to-mesenchymal transition in a 3D cell culture model. [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 503. doi:10.1158/1538-7445.AM2013-503
Abstract 5041: The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma.
Authors: Knelson Erik H. , Gaviglio Angela L. , Tewari Alok K. , Armstrong Michael B. , Nixon Andrew B. , Starr Mark D. , Mythreye Karthikeyan , Blobe Gerard C.
Published: August 2014

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Here we report that expression of type III TGF-beta receptor (TβRIII) mRNA and protein decreases with advancing stage of neuroblastoma and positively correlates with prognosis. TβRIII expression is epigenetically suppressed by MYCN oncogene amplification and TβRIII expression can be used as a prognostic marker in neuroblastoma patients with MYCN amplification. TβRIII expression in neuroblastoma cells promotes neuronal differentiation and enhances the differentiating effects of FGF2 treatment. Mechanistically, glycosaminoglycan modifications on TβRIII bind FGF2 and FGFR1 to promote neuronal differentiation via Erk MAPK and the transcription factor ID1. TβRIII-mediated differentiation suppresses tumor cell proliferation in vitro and in vivo. These studies characterize a novel co-receptor function for TβRIII in FGF2-mediated neuronal differentiation of neuroblastoma cells, while identifying potential therapeutic targets and clinical biomarkers for advanced-stage disease. More generally, our results suggest that the targeting of growth factor receptors and downstream signaling pathways may prove useful in promoting neuronal differentiation to suppress neuroblastoma tumor growth.

Citation Format: Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Andrew B. Nixon, Mark D. Starr, Karthikeyan Mythreye, Gerard C. Blobe. The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma. [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 5041. doi:10.1158/1538-7445.AM2013-5041
Abstract 505: Hypoxia inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis.
Authors: Chaturvedi Pallavi , Gilkes Daniele , Semenza Gregg
Published: August 2014

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We have demonstrated that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) that facilitate metastasis. In an orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to lymph nodes and lungs in a HIF-dependent manner. In vitro gene analysis of co-culture of MSCs with BCCs showed induced expression of the chemokine CXCL10 in MSCs and its cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. Further we showed that CXCR3 expression was blocked in co-cultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs co-cultured with BCCs that did not express CXCR3 or HIFs. MSC co-culture did not enhance the metastasis of CXCR3-deficient or HIF-deficient BCCs. Co-culture of MSCs with BCCs augmented HIF activity in BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGF1, respectively, in a HIF-dependent manner. The expression of CXCL10 by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis.

Citation Format: Pallavi Chaturvedi, Daniele Gilkes, Gregg Semenza. Hypoxia inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. [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 505. doi:10.1158/1538-7445.AM2013-505
Abstract 5051: MYC-driven models of primitive neuroectodermal tumor are sensitive to inhibitors of glutamine metabolism.
Authors: Ahsan Sama F. , Hanaford Allison , Weingart Melanie , Taylor Isabella , Eberhart Charles , Raabe Eric
Published: August 2014

Brain tumors are a diverse group of cancers in which alterations in growth regulation pathways drive tumorigenicity. Histologically similar tumors may have significant differences in underlying molecular pathways and this in turn may have significant implications for the development of targeted treatment. The MYC family of proteins promotes proliferation and an aggressive phenotype in diverse cancers. Primitive Neuroectodermal Tumors (PNETs) are high grade tumors that can arise throughout the neuro-axis. One subset of PNETs is MYC amplified, and some PNETs are known to express LIN28A, a key regulator of MYC. MYC expression in tumors leads to increased reliance on glutamine metabolism (i.e. the Warburg effect). We hypothesized that MYC-driven PNET tumors would up regulate glutamine metabolism and that glutaminase inhibitors would selectively target MYC-driven tumors. We tested this hypothesis using the MYC-positive PNET cell line PFSK as well as MYC-transformed human neural stem cells. The glutamine methabolic inhibitors DON (6-Diazo-5-oxo-L-norleucine) and acivicin were used to disrupt glutamine metabolism in these cells. Using Tetrazolium dye reduction (MTT assay) and Bromodeoxyuridine assays (BrdU) as indicators for cell growth and replication, we found that PFSK growth is inhibited by acivicin at 10 micromolar concentration (MTT activity reduced by 88%, p<0.05, t-test acivicin treated compared to untreated) MYC-negative UW228 cells were resistant to DON and acivicin treatment. Similarly, DON or acivicin inhibited the proliferation of MYC-transformed human neural stem cells. We observed a 75% reduction in BrdU incorporation in MYC transformed cortex neural stem cells using acivicin, (p<0.001, t-test acivicin treated compared to vehicle treated), 60% reduction in cortex MYC cells using DON (p<0.05), and a 48% reduction in hindbrain MYC cells using DON (p<0.01). The growth of control MYC-negative cells was not significantly inhibited by either drug. This data shows that glutaminase inhibition limits cell replication in tissue culture models of MYC-driven PNET. We are currently investigating the mechanism of growth inhibition (e.g. apoptosis versus cell cycle arrest) caused by glutaminase inhibitors. Murine orthotopic xenograft experiments investigating the efficacy of DON and acivicin treatment in MYC-driven PNET are currently underway in our laboratory.

Citation Format: Sama F. Ahsan, Allison Hanaford, Melanie Weingart, Isabella Taylor, Charles Eberhart, Eric Raabe. MYC-driven models of primitive neuroectodermal tumor are sensitive to inhibitors of glutamine metabolism. [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 5051. doi:10.1158/1538-7445.AM2013-5051
Abstract 5054: The role of neurofibromin in stem cell differentiation.
Authors: Farbaniec Michelle , Chakraborty Adri , Coleman Natalia
Published: August 2014

Neurofibromin is a product of the tumor suppressor gene Nf1 which is responsible for neurofibromatosis type 1 (NF1). When NF1 is mutated, Ras is constitutively active, enabling affected cells to proliferate extensively. This proliferation can result in tumor formation, particularly in neural crest-derived cells in the peripheral nervous system. NF1 patients can also exhibit cognitive deficits and develop other symptoms unrelated to cancer. While it is common knowledge that neurofibromin is involved in tumor formation, little is known about its function in neural stem cell proliferation and differentiation in normal and pathological settings. In addition to Ras-GAP function, recent studies have demonstrated that neurofibromin has a novel role on neuronal differentiation via its GAP-related domain. Thus we have speculated that abnormal regulation of NF1-GAP in neuronal cells may be implicated in NF1-related learning and memory disorders. The aim of the present work is to evaluate the role of neurofibromin in neuronal cell differentiation using stem cells (SCs) expressing different levels of neurofibromin. We have developed a new system to differentiate mouse SCs via formation of an embryoid body, a floating cell cluster which, by markers and function, mimicks adult neural stem cells. We have shown that the mouse embryonic ESs of all NF1 genotypes in our possession retain their pluripotency and can be differentiated into neuronal cells. This systematic study of complex molecular events in signaling pathways that occur in mouse embryonic SCs that have one (Nf1+/-), both (Nf1+/+) or neither (Nf1-/-) functional allele during in vitro differentiation is critical for a better understanding of the biology of neurofibromatosis and identification of potential targets.

Citation Format: Michelle Farbaniec, Adri Chakraborty, Natalia Coleman. The role of neurofibromin in stem cell differentiation. [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 5054. doi:10.1158/1538-7445.AM2013-5054