S544 I. J. Radiation Oncology d Biology d Physics Volume 75, Number 3, Supplement, 2009

Results: Gene array analysis comparing 6D12+ vs. 6D12- populations revealed significant changes in �600 genes. Gene arrayanalysis comparing spheres vs. matched non-tumorigenic in vitro epithelial cells revealed significant changes in �3100 genes.The tumorigenic 6D12- and sphere populations shared 160 gene changes, against a background of 1-4 shared gene changes fornon-congruent groups. To rank these genes for clinical and biological significance, we curated publicly available gene array da-tabases for advanced EOC with clinical outcome correlates. Parametric p values based on single gene Cox hazard ratios were gen-erated. The resultant genes were then filtered for surface expression to produce a list of candidate markers of the CSC population, tobe confirmed by single cell expression profile and xenograft modeling.

Conclusions: Merging traditional purification methods with gene array analysis, and publicly available outcomes data may speeddiscovery of CSC markers. Identification of surface markers permits generation of imaging reagents for target identification, as wellas radioimmunological reagents for direct therapy. Identification of the CSC population itself permits identification and inhibitionof active radioresistance pathways.

Author Disclosure: R.K. Chin, None; D. Sahoo, None; S. Willingham, None; T. Storm, None; S. Schmidt, None; J. Ross, None; K.Yang, None; L. Ailles, None; N. Teng, None; I. Weissman, None.

2807 Alpha5beta1 Integrin and Oncofetal Fibronectin are Upregulated in Malignant Breast Cells in Three-

dimensional Culture That can be Specifically Targeted for Radiation Therapy

J. Nam1 C. C. Park2

1Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, San Francisco, San Francisco, CA

Purpose/Objective(s): Integrin beta1 signaling has been implicated in breast cancer progression and has been shown to facilitateresistance to radiation that could be abrogated using beta1 integrin inhibitory antibody, AIIB2. In the present study, we investigatedwhether a specific alpha heterodimer of beta1 integrin was preferentially mediating a pro-survival signal in human breast cancercells, and if it could specifically be targeted for radiation therapy.

Materials/Methods: We used a three-dimensional laminin rich extracellular matrix (3D lrECM) culture to compare the relativelevels of alpha heterodimer expression in malignant HMT3522-T4-2 and nonmalignant cell line HMT3522-S-1. Total cell lysateswere probed by western blot and subjected to immunoprecipitation to detect several possible alpha subunits. In addition, to inves-tigate whether alpha5beta1 integrin mediated an important pro-survival signal, we treated 3D lrECM cultures with a small peptideinhibitor of alpha5beta1 integrin and fibronectin (FN) interaction, Ac-PHSCN-NH2 (ATN-161) and measured apoptotic responseusing TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling) assay.

Results: We found that expression of alpha5beta1 integrin and its ligand, FN were upregulated in T4-2 malignant breast cell linecompared to nonmalignant cell line S1. We further discovered that oncofetal FN, containing the EDA+ domain was specificallyover-expressed in malignant T4-2 cells in 3D lrECM culture. Apoptosis was increased in T4-2 cells cultured in 3D lrECM by ATN-161 treatment at 0.5 mg/ml (mean=8.3%, p\0.001) or 2.0 mg/ml (mean=23.4%, p\0.001). Moreover, treatment with 0.5 mg/mlATN-161 and 2 Gy radiation together increase apoptosis compared to single treatment in breast cancer cell lines, T4-2 cells, andMDA-MB-231 cells.

Conclusions: We have previously shown that inhibitory antibody to beta1 integrin, AIIB2, leads to selective apoptosis and de-creased proliferation in human breast cancer cells in 3DlrECM culture and in vivo. In addition, combining beta1 integrin inhibitionwith IR allowed for reduction of IR dose necessary to achieve growth inhibition in vivo. Here, we show that in HMT3522-T4-2breast cancer cell line, inhibition of alpha5beta1 integrin and FN interaction using ATN-161 significantly enhanced apoptosis in 3DlrECM. These data indicate that FN and its receptor, alpha5beta1 integrin play a pivotal role for survival signaling in breast cancerand may be an important target to enhance radiotherapy efficacy.

Author Disclosure: J. Nam, None; C.C. Park, None.

2808 Identification of Conserved Gene Expression Programs in Epithelial Cancer Stem Cells

M. Diehn1, R. W. Cho1, L. Ailles2, J. S. Lam1, M. J. Kaplan1, G. Somlo3, I. L. Weissman1, M. F. Clarke1

1Stanford University School of Medicine, Stanford, CA, 2Ontario Cancer Institute, Toronto, ON, Canada, 3Hope NationalMedical Center, Duarte, CA

Purpose/Objective(s): The cancer stem cell (CSC) hypothesis proposes that tumors are maintained by a subset of cancer cellswhich has the ability to proliferate indefinitely and which can give rise to non-tumorigenic cancer cells (NTCs) with limited pro-liferative potential. Although normal stem cells are not necessarily the target cell of transformation, a growing body of evidenceindicates that there are significant similarities between normal stem cells and CSCs. We hypothesized that CSCs from differenttumor types display conserved gene expression programs and that these are related to normal stem cell gene expression profiles.

Materials/Methods: We performed gene expression profiling of human CSCs from primary breast and head & neck cancers toexplore relationships between CSCs and normal stem cells. We employed unsupervised and supervised analyses to evaluate thedata. Specifically, we searched for conserved CSC gene expression patterns between the two tumor types and compared CSCgene expression profiles to those of normal stem cells. Finally, we used clonogenic survival assays to assess CSC sensitivity tocandidate self-renewal/survival pathway inhibition.

Results: Unsupervised analyses of gene expression profiles of primary human breast and head & neck CSCs revealed that CSCsfrom the two types of cancers were more similar to each other than to their corresponding NTCs. Supervised analyses confirmed theexistence of conserved gene expression programs in CSCs from different types of carcinomas. The CSC gene expression signaturewas strongly enriched for genes expressed by normal adult tissue stem cells, including mouse mammary stem cells and humanhematopoietic stem cells. Furthermore, genes implicated in stem cell self-renewal were highly overrepresented in the CSC signa-ture. Inhibition of self-renewal pathways significantly reduced clonogenicity of breast CSCs.

Conclusions: Our findings indicate that CSCs from different types of carcinomas express a conserved gene expression programwhich includes genes involved in stem cell self-renewal. Targeting of self-renewal-associated pathways in CSCs represents

Proceedings of the 51st Annual ASTRO Meeting S545

a potential therapeutic approach for eliminating CSCs. The development of multimodality treatment regimens consisting of radio-therapy and CSC-directed therapies has the potential to improve clinical outcomes.

Author Disclosure: M. Diehn, None; R.W. Cho, None; L. Ailles, None; J.S. Lam, None; M.J. Kaplan, None; G. Somlo, None; I.L.Weissman, None; M.F. Clarke, Oncomed Pharmaceuticals, F. Consultant/Advisory Board.

2809 Brain Tumor Stem Cell Marker - Distinct Populations of Tumor-initiating Cells Expressing CD44/CD24

Derived from Cancer Stem Cells Mediates Chemo and Radio Resistance

K. Palanichamy, A. Chakravarti

The Ohio State University Medical School, Columbus, OH

Purpose/Objective(s): Glioblastoma multiforme (GBM) is the most frequently diagnosed neoplasm of the central nervous system.The standard of care consists of surgery (when feasible), followed by radiation and chemotherapy. Resistance to treatment repre-sents a major obstacle to recovery. Although tumors may respond initially, recurrence is highly likely because of a minor sub-pop-ulation of cells within the tumor known as tumor initiating cells or cancer stem cells (CSCs). The molecular and cellularmechanisms underlying this resistance in CSCs have been under active investigation, and represent an opportunity for therapeuticexploitation.

Materials/Methods: We exposed our panel of CSCs to varying doses of radiation and comprehensive FACS analysis shows thatthe Chemo-/radio- resistance subpopulation of CSCs encodes antigens comprising CD44 and CD24. Not all CSCs exhibit treat-ment resistance, it is only a sub-population of CSCs exhibit Chemo-/Radio- resistance. For the first time here we are reportingthat CD44/CD24 expression encodes chemo-/radio-resistant subpopulation in Glioma CSCs.

Results: CD44 is a transmembrane glycoprotein which participates in many cellular processes, including growth, survival,differentiation, and motility. It is a unique adhesion molecule and plays a role in cancer cell migration and matrix adhesionin response to a cellular microenvironment, thus enhancing cellular aggregation and tumor cell growth. Several reports haveshown that CD44+ cells possessed higher tumorigenicity and metastatic potential and has been used as a marker to isolateCSCs. We demonstrate with this work that CD44 is a potential marker to identify the treatment resistance sub-populationof CSCs in association with CD24. The treatment resistance subpopulation of CSCs expresses higher levels of ABC familygenes in addition to stem cell genes such as Nanog, Sox2, Oct4, etc. They also have a higher expression of CXCR4 and IAPfamily proteins. We have generated brain tumor spheroid cultures of primary GBM and this sub-population of CSCs are con-sistently capable of inducing tumors upon xenotransplantation that resemble the original malignancies, both in morphologyand marker expression.

Conclusions: To fully ascertain that this is also true for directly isolated tumor cells, we used a third primary GBM and initiatedsingle cell cultures with purified CD44+/CD24+ cells directly ex-patient. Also in this setting the CD44+/CD24+ cell fraction gen-erated single cell derived spheroids, whereas the negative cells were incapable of doing so. This clearly confirmed that the clono-genic and treatment resistance potential of our spheroid cultures resides in the CD44+/CD24+ cells.

Author Disclosure: K. Palanichamy, None; A. Chakravarti, None.

2810 Identification of Novel Radiation Induced Immune Signaling Changes in Antigen Presenting Cells

J. J. Parker, J. C. Jones, S. Strober, S. Knox

Stanford University School of Medicine, Stanford, CA

Background: The success of transplantation and tumor vaccine protocols depends on effective immune modulation, but theeffects of radiation (RT) on the immune system in such protocols are not well characterized. When RT is used as a conditioningregimen prior to hematopoietic stem cell transplantation, host antigen presenting cells (APC) persist in the patient immediately aftertransplantation.

Purpose/Objective(s): Characterization of RT-induced changes in APC will facilitate the optimization of protocols that use RT forthe treatment of malignant and hereditary diseases with transplantation. The goal of our research is to identify RT-induced changesin APC intracellular signaling profiles. In the past, changes at the molecular level have been difficult to elucidate due to the het-erogeneity and small population of immune cells present after RT treatment. Phospho-flow cytometry (pFCS), a recently developedmethod (Krutzik PO, et al., Clin Immunol 2006), provides a new method for studying cell signaling in rare cell subsets in complex,heterogeneous populations of cells. We hypothesized that RT-induced changes in the phosphorylation state of intracellular signal-ing proteins in immunologically important signaling pathways can be detected by pFCS, and we investigated whether RT-inducedphosphorylation of NFkB family members was modified by p53 function.

Materials/Methods: We compared the effects of RT (0, 2, and 20 Gy) with the effects of cytokines (IL-4 and IFN-g) and che-motherapeutic agents (Cisplatin and Doxorubicin) at various timepoints on a human APC line (U937) using standard pFCS stainingmethods and cytometric analysis (FACS Calibur cytometer and FlowJo software).

Results: With this pFCS-based method, we identified novel RT-induced phospho-specific changes in U937 cells. RT enhances theexpression of NFkB family members: IKKg (NEMO), IKKa/b, IkBa, and NFkB within one to four hours after RT with 2 Gy and20 Gy, while RT does not induce STAT1 and STAT6 expression in these cells. These specific RT-induced phosphorylation profilesare not associated with other stress conditions (chemotherapeutic agents and proinflammatory cytokines). Cisplatin, doxorubicin,IL4, and IFN-g do not induce the RT-specific phosphorylation of NFkB family members. Although p53 is functionally inactive inU937 cells, restoration of p53 by transfection did not modify the phosphorylation profiles seen after RT.

Conclusions: With pFCS we identified RT-induced changes in the phosphorylation state of NFkB family members in APC, and theexpression of this profile is not modified by p53 activity. Our work is the first to use pFCS to examine the effects of RT on immunecell subsets. Future experiments will help to determine the physiologic/functional significance of these changes in human periph-eral blood mononuclear cells.

Author Disclosure: J.J. Parker, None; J.C. Jones, None; S. Strober, None; S. Knox, None.