Tumor Cells Of Origin Research Articles

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3909.

Linfoma intravascular de células B grandes como causa inusual de ictus isquémico

Intravascular lymphoma is a type of extranodal lymphoma, usually composed of B-cells, resulting from a proliferation of atypical lymphocytes within the lumina of small to medium sized vessels. Since its initial description in 1959, it has had many names but currently the World Health Organization refers to this entity as intravascular large B-cell lymphoma. We present a case which presented with rapid progressive neurological deterioration and consecutive progressive multifocal and recurrent ischemic events of unknown origin. The postmortem study of the cranial cavity revealed atypical cell proliferation within the lumina of small to medium sized vessels. The lymphoid origin of intravascular tumor cells was confirmed by immunohistochemistry, establishing a diagnosis of intravascular large B-cell lymphoma.

Establishment and characterization of a novel osteosarcoma cell line: CHOS.

Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016.

PO-23 - Expression of heparanase in cancer as biomarker of malignancies: overexpression in an aggressive, poor survival gastric cancer “gastric signet ring cell carcinoma” compared with that of other gastric cancers

Gastric signet ring cell carcinoma (GSRC) is a distinct entity among of other gastric cancer. With unknown etiopathology, their incidence is increasing and it presents a low sensitivity to chemotherapy. Here, we studied the expression of the heparanase (HPA) in cancer tissues from GSRC patients and several cancer cell lines. HPA is an endo-β-D-glucuronidase, capable of cleaving the lateral chains of heparan sulfate on cell surfaces and the extracellular matrix at acidic pH. Apart from its well-characterized enzyme activity, HPA also has independent enzymatic functions, such as up-regulation of vascular endothelial growth factor (VEGF) -A and VEGF-C and activation of intracellular signaling pathways involved in, survival, migration and proliferation of tumor cells. It can also induce an hypercoagulability by a non-enzymatic manner. HPA was tested in several cancer cell lines from ovaries (OVCAR-3, SKOV-3), breast (MDAMB231, MCF7) colon (LS-174), lung (A549), uterus (HELA) and gastric (adenocarcinoma (AGS) and GSRC (KATO-III) using several techniques such as RT-PCR, Q-PCR, immunocytochemistry, flow cytometry and degradation of Fondaparinux at pH 5, evaluated by its anti Xa activity evaluated by Factor Xa amidolytic activity. The amount of HPA mRNA in the biopsy simples of gastric adenocarcinoma (n=10) and GSRC (n=11) in tumors and their environment were analyzed. HPA gene is expressed in all cancer cell lines, but its level varies depending on the tumor cell line. In biopsies of gastric cancer, the HPA gene is more expressed in the tumor regions (p=0.0002) and tumor environment (p=0.015) in GSRC than in gastric adenocarcinoma. B) The activity of HPA, evaluated by degradation of Fondaparinux at pH 5, 1) in the supernatants of 10(6) cancer cells: the residual activity of Fondaparinux after 2 hours incubation at 37°C with OVCAR-3 supernatant was of 70% of control value, and of 80% with KATO-III cell supernatants. 2) in patient's plasmas, this technique cannot be used because the site of degradation of fondaparinux by heparanase is masked by AT present in plasma. Heparanase was found in in many cancer cell lines and its level depends on origin of tumor cells and on its aggressivity. Taking into account the pro-metastatic functions, proangiogenic and procoagulant activity of HPA and its overexpression in gastric signet ring cell carcinoma of poor prognosis and its cell line, HPA can be considered as a biomarker of malignancy and as a therapeutic target in GSRC patients.

Abstract B20: Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin

Abstract Overexpression of Myc and loss of Pten are very common oncogenic events in prostate cancer. However, why these specific oncogenic events, as opposed to others, are involved in prostate cancer develop remains unclear. While the impact of Myc and Pten on cell proliferation is well-characterized, their role in differentiation and the resulting impact on tumorigenesis is poorly understood. We recently developed an in vitro differentiation model in which basal prostate epithelial cells (PrECs) can be differentiated into secretory luminal cells. Our model offers several advantages, in that it is human-specific and readily manipulated genetically and biochemically; providing a rare opportunity to dissect the specific genetic and biochemical events associated with human PrEC differentiation and determining how it is impacted by specific oncogenic events. Using this model, we tested the hypothesis that overexpression of Myc and loss of Pten induce prostate cancer because their dysregulation impairs differentiation of an intermediate progenitor cell population that marks the tumor cell of origin. Transient induction of both Pten and Myc are required for normal PrEC differentiation. Constitutive Myc overexpression initially accelerates differentiation, but the differentiated cells ultimately die via a p53-independent mechanism. Overexpression of Myc in combination with loss of Pten (Myc+shPten), is required for tumorigenesis because it rescues cell survival and blocks terminal differentiation. We identified the chromatin binding protein, ING4, as a Myc target required for PrEC terminal differentiation. ING4 is lost in over 60% of human prostate tumors and in the Myc+shPten cells, ING4 expression is blocked. Loss of ING4 in Myc overexpressing cells is sufficient to replace loss of Pten; ING4 loss, like Pten loss, is required for tumorigenesis to block terminal differentiation and keep cells alive. We further identified Miz1, a component of the Myc repressor complex, as a direct target of ING4 required for terminal PrEC differentiation. Myc/Miz1 is required to suppress integrin α6 and β1 expression during terminal PrEC differentiation; in Myc+shPten cells, Miz1 fails to be induced and integrin α6β1 is aberrantly co-expressed with AR in an intermediate progenitor and tumorigenic cell population. Conclusions: Prostate cancer oncogenesis requires dysregulation of Myc and Pten, because they are required for the normal PrEC terminal differentiation pathway. Pten loss is required to remove the ING4/Miz1-mediated terminal differentiation program initiated by Myc overexpression, to maintain the intermediate progenitor cells in a partially differentiated, yet proliferative state, and to evade cell death. Citation Format: Penny Berger, McLane Watson, Mary Winn, Cindy K. Miranti. Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B20.

Abstract B047: Adequate Antigen Availability (AAA) in antitumor immunity: Definition and consequences for novel strategies of tumor prevention and antitumor treatment

Abstract Present immunopreventive and immunotherapeutic approaches against cancer suffer from the limitation of being not “sterilizing” procedures, as very poor protection against the tumor is obtained. Thus, newly conceived anti-tumor strategies of intervention are urgently needed. Here we introduce the concept of Adequate Antigen Availability (AAA) defined as the optimal tumor antigen dose and related antigen processing and MHC class II-restricted presentation necessary to trigger efficient tumor-specific TH cells (1). We will present results on a preventive anti-tumor vaccination system that consists in injecting tumor cells genetically modified to constitutively express MHC class II molecules by transfecting the MHC-II transcriptional activator CIITA. AAA is generated by the function of tumor's newly expressed MHC-II molecules to present tumor-associated antigens to tumor-specific CD4+ TH cells. We will show that tumor rejection against the CIITA-transfected neoplastic cells is brought about by the generation of persistent anti-tumor TH cell memory, responsible of protecting the animals from subsequent challenges with parental, untransfected tumor cells. This approach has been validated in tumor cells of different histotypic origin and of distinct MHC genotype (H-2d and H-2b). Importantly, the anti-tumor TH cell response modifies the tumor microenvironment, favouring the infiltration of CD4+ TH cells, CD8+ cytolytic T cells and granulocytes and dramatically reducing both intratumoral and lymphnode infiltration of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Thus the optimal initiation of the adaptive immune response, dictated by AAA, is sufficient to reorient the tumor microenvironment from a pro-tumor to an anti-tumor microenvironment. Our approach is, at present, applied to human clinical setting in hepatocellular carcinomas (2) whereby human tumor cells are rendered HLA class II-positive by CIITA genetic transfer with the aim to purify and sequence immunogenically relevant HLA class II-bound tumor–associated antigenic (TAA) peptides. In conjunction with similarly derived HLA class I-restricted TAA peptides, they will be the basis for the construction of a multi-peptide, multi-epitope vaccine that can target both CD4+ TH and CD8+ CTL anti-tumor responses.

Myoepithelioma of Soft Palate: A Case Report and Review of Literature

ABSTRACTMyoepitheliomas are rare benign neoplasms of salivary glands representing less than 1% of all salivary gland tumors. This tumor is usually located in the parotid gland and less frequently in the submandibular gland and minor salivary glands that are distributed throughout the oral cavity. Diagnosis of tumor is based on evaluation of histological feature, although immunohistochemical analysis confirms the myoepithelial origin of tumor cells and the final diagnosis. Herein, a case of plasmacytoid myoepithelioma of soft palate in an adult male is presented along with review of the literature.How to cite this articleYogee AN, Choudhary R, Mathur N, Bhuie HS. Myoepithelioma of Soft Palate: A Case Report and Review of Literature. Int J Otorhinolaryngol Clin 2016;8(1):32-34.

Molecular Characterization of an Endometrial Endometrioid Adenocarcinoma Metastatic to a Thyroid Hürthle Cell Adenoma Showing Cancerization of Follicles

Tumor-to-tumor metastasis is rare. Herein, we present a unique case of endometrial endometrioid adenocarcinoma metastatic to a thyroid Hürthle cell adenoma 9years after initial diagnosis. On histologic examination of the thyroid, the malignant endometrioid glands and single cells (donor tumor) were dispersed within the Hürthle cell adenoma (recipient tumor). In several sections of the adenoma with still preserved microfollicular architecture, malignant endometrial adenocarcinoma cells were admixed within oncocytic adenomatous epithelium (so-called "cancerization of the follicles"). This unusual phenomenon, to our knowledge, is a novel finding in the thyroid gland. Immunohistochemistry, subsequently elicited clinical history, and morphologic comparison of the tumor in the thyroid to the primary endometrial tumor confirmed the origin of the donor tumor cells. Molecular analysis of both the metastatic and primary endometrial tumors demonstrated PIK3CA and PTEN mutations in both tumors, as is characteristic of well-differentiated endometrioid tumors of the endometrium. Amplification of chromosome 1q was detected in both sites; however, only the metastatic tumor showed loss of chromosomes 2, 9, and 22. The morphologic differential diagnosis of metastatic endometrioid adenocarcinoma in the thyroid includes columnar cell variant of papillary thyroid carcinoma (CCVPTC) arising in a preexisting adenoma, endocrine glandular atypia within an adenoma, and metastasis from other anatomic sites. Histomorphologic differences among these entities may be subtle; therefore, knowledge of and morphologic comparison with prior malignancies and immunohistochemistry can be helpful in rendering the correct diagnosis.

Abstract A13: Functional genomic analysis of central nervous system hemangioblastomas

Abstract Hemangioblastomas (HBs) are highly vascular tumors that develop in the Central Nervous System, mainly in the cerebellum, spinal cord and retina. These tumors are usually associated with Von-Hippel Lindau (VHL) disease but they can also appear sporadically. HBs often contribute to the morbidity and mortality of VHL patients due to the location of the tumor and the lack of efficient treatment. The genomic landscape of VHL-associated HBs is underexplored. The tumors develop upon bi-allelic loss of VHL but it is unclear if there are additional oncogenic drivers that influence tumor development. In addition, HBs are highly heterogeneous tumors comprised of diverse cell sub-populations; the origin of the HB tumor cell is controversial. The lack of cell lines and animal models that mimic the disease strongly impairs the research on HB biology and the search for new therapeutic strategies. In order to create an in vitro model to study HB biology, we established tumor-derived cell lines from VHL-associated HB tumors. The cell lines are formed by sub-populations that are morphologically different and express known HB markers such as Podoplanin, VEGFR2 and CXCR4. Moreover, these cells express Hypoxia Inducible Factor 1a (HIF1a), which is stabilized in VHL-deficient cells. We characterized the cell populations corresponding to surface markers expression and we analyzed the signaling pathways of isolated single HB cells derived from human tumors. In addition, we performed next-generation sequencing in HB tumor samples to identify potential oncogenic drivers that contribute to the development of HBs. We found a list of potential hits that we are now confirming with complementary techniques. These data can lead us to the identification of abnormal oncogenic pathways that could be therapeutically targeted in this disease. Finally, we are establishing a zebrafish model that mimics the HB disease. Vhl mutants develop hyper-vascularization in the brain and in the retina which is a common initiation factor in HB development. We are characterizing the vhl brain lesions using transgenic models with labeled vasculature and hematopoietic stem cell compartment. In conclusion, there are no current therapies for HBs and the lack of research models profoundly compromises the research advancement. In this work we established, for the first time, long-term cell lines that can be used to study the disease. The use of next-generation genomics allowed us to dissect the genomic landscape of this tumor type and will allow us to identify the HB cell of origin. All these new genomic data combined with novel resources, including the zebrafish model, will allow us to advance the current knowledge about HBs and may help to identify HB specific therapeutic targets. Citation Format: Ana Martins Metelo, Atanas Kamburov, James Kim, Elizabeth Lockerman, Dana Lee, Frederick Barker, Jeffrey Engelman, Anat Stemmer-Rachamimov, Gad Getz, Othon Iliopoulos. Functional genomic analysis of central nervous system hemangioblastomas. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A13.

Abstract A89: Istiratumab (MM-141), a bispecific antibody co-targeting IGF-1R and ErbB3, potentiates the activity of immune checkpoint inhibitors

Abstract Purpose: Regulatory T cells (Tregs) help maintain immunological tolerance to cancer cells by suppressing T cell effector function. The depletion of Tregs with the CTLA-4 inhibitor antibody ipilimumab has been proven to be a productive therapeutic strategy in melanoma. Additionally, levels of insulin-like growth factor 1 (IGF-1) are often elevated following chemotherapy treatment and have been associated with poor prognosis in multiple malignances. We demonstrate that IGF-1 can stimulate Treg proliferation. Istiratumab, a tetravalent bispecific antibody that targets both the IGF-1 receptor (IGF-1R) and ErbB3, inhibits IGF-1 signaling by blocking ligand binding and inducing rapid receptor internalization. Based on its mechanism of action, we evaluated the activity of istiratumab on Treg proliferation and in combination with immune checkpoint targeting antibodies in murine models of cancer. Experimental Procedures: Mouse-derived splenocytes were harvested to evaluate the effect of IGF-1 and/or istiratumab treatment on Treg expansion and proliferation in vitro. Flow cytometry studies assessed surface receptor expression of T cell populations isolated from murine splenocytes. Efficacy studies using immunocompetent mice bearing syngeneic murine tumors determined the activity of istiratumab and immune checkpoint targeting antibodies, alone and in combination, on tumor growth alongside isotype-matched controls. Mice whose tumors were eradicated by treatment were maintained and subsequently used in tumor re-challenge studies to assess the development of tumor-specific, immunological, long-term memory. In addition, pharmacodynamic studies analyzed the expression of tumor cell signaling pathway markers, effects on relevant T cell sub-populations and effects on post-treatment cytokine profiles. Data Summary: Our in vitro T cell analyses indicated that sub-populations of murine CD4+ CD25+ FoxP3+ Tregs express IGF-1R, and that istiratumab can reverse IGF-1 driven Treg proliferation in vitro. In addition, istiratumab monotherapy treatment had significant anti-tumor activity in vivo in immunogenic, syngeneic murine tumor models. Moreover, istiratumab potentiated the activity of immune checkpoint targeting antibodies in efficacy studies leading to curative outcomes in a subset of treated animals. These animals developed long-term immunological memory directed against the original tumor cell line. A pharmacodynamic analysis of tumor cell signaling- and immunology-related markers demonstrated that istiratumab impacts tumor cell survival signaling and contributes to overall immunological changes that favor breaking immunological tolerance in relevant syngeneic tumor models, thus enabling the development of an effective and durable anti-tumor immune response. Conclusions: Our in vitro and in vivo pre-clinical studies demonstrate that istiratumab (MM-141) inhibits both tumor cell survival and Treg proliferation in host mice, thereby potentiating the anti-tumor activity of clinically-relevant immune checkpoint inhibitor antibodies. Citation Format: Sharlene Adams, Michael D. Curley, Adam J. Camblin, Sergio Iadevaia, Lin Nie, Gege Tan, Chrystal U. Louis, Alexey A. Lugovskoy. Istiratumab (MM-141), a bispecific antibody co-targeting IGF-1R and ErbB3, potentiates the activity of immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A89.

Gene expression of cytokeratin 19 and its molecular detection in human breast cancer cell lines

Cytokeratins have been identified as useful tools in oncology diagnostics. In this study, cytokeratin19 (CK19) expression was studied in three human breast cancer cell lines, SKBR3, BT549, and BT474 using RT-PCR. CK19 was expressed in tumor cell of different origin, showing higher expression in invasive breast cancer with ER+ (BT474) than invasive breast cancer with ER− (BT549) and breast adenocarcinoma with ER− (SKBR3). Two primer sets were used to evaluate CK19 expression. Primer set I (hCK19/1) and primer set II (hCK19/2) were used to amplify the CK19 human gene at a 215bp and 384bp, respectively, whereas PBMC and RAW264.7 (mouse macrophage) no detectable PCR products were obtained. The sensitivity for detection was determined by two methods, i.e., cDNA dilution (the dilution of cDNA from RNA of breast cancer cells) and cell dilution (the dilution of breast cancer cells in PBMC). hCK19/2 was more sensitive than hCK19/1. In cDNA dilution, the lower limits of primer set II for detection were 400, 40 and 40 cells for SKBR3, BT549 and BT474 cells, respectively. While in cell dilution all of the 3 breast cancer cells could be detected at 1 cancer cell in 104, 106 and 105 PBMC, respectively. The data supported the possibility that CK19 could be detected and be the marker for breast cancer in patient blood.

T117: The plasticity of tumor cell migration mechanism is adaptive factor increasing metastasis efficiency

During cancer development, tumor cells gain the ability to invade and metastasize. Individual cells use alternative migration modes based on different cellular mechanisms. One of them is mesenchymal motility mode which is driven by leading edge protrusion in the form of filopodia or/and lamellipodia based on Arp2/3 dependent actin polymerization. Mesenchymal motility depends on formation of cell-substrate adhesions, activity of matrix metalloproteases (MMPs) and on activity of small GTPaseRac. Another mode is amoeboid motility, which involves formation of blebs – hollow membrane protrusions extruded from the cell surface by actin-myosin contraction. Amoeboid motility does not need both pronounced cell-substrate adhesions and MMPs activity and required increase of activity of small GTPase Rho. Fibroblasts and scattered epithelial cells migrate by mesenchymal mode, while blood cells – lymphocytes or macrophages mainly use amoeboid mode for migration. It was shown that some treatments cause transition from one motility mode to another. Switches from mesenchymal to amoeboid motility and opposite are called mesenchymal-amoeboid transition (MAT) and amoeboid -mesenchymal transition (AMT) respectively. The ability of cells for such transitions was named as plasticity of migration. We compared the plasticity of migration of normal and tumor cells. To study plasticity of mesenchymally migrated cells (MAT) we choose fibrosarcoma cells HT1080 as tumor and non-transformed subcutaneous fibroblasts 1036 as normal counterpart. To study AMT we choose a few lines of myeloid leukemia THP1, K562, KG1 in contrast to normal leukocytes obtained from healthy donors. We showed that fibrosarcoma cells in opposite to non-transformed fibroblasts could undergo MAT under treatments, which limited mesenchymal migration. Two approaches to limit mesenchymal motility of cells were used. One was decrease of substrate adhesiveness by treatment of coverslips with PolyHema solutions, which simulated the alteration of environmental conditions during cell migration. The other approach was influence on cellular pathways regulated cell motility. We used CK666, the inhibitor of Arp2/3 activity, which stopped actin polymerization and thus lamellipodia formation through Arp2/3 dependent mechanism. We showed that under both treatments the fraction of tumor cells switched from lamellipodia formation to blebbing and thus underwent MAT, while in non-transformed fibroblasts these treatments led to retraction of lamellipodia and significant failure of motility. Both leukemia cells and leucocytes of healthy donors showed blebs formation (amoeboid motility). We induced transition to mesenchymal motility by alteration of culture conditions. The first approach was the increase of substrate adhesiveness by treatment with fibronectin. Another way was to inhibit of small GTPase Rho activity. In result of both treatments, leukemia cells switched from amoeboid to mesenchymal motility (underwent AMT), but leucocytes of healthy donor could not do such transition. For the first time it was shown that AMT is features of leukemia cells but not leucocytes from healthy donors. Both MAT and AMT are reversible, meaning that cells exhibiting plasticity could change motility mode in dependence on environment. Our results demonstrate that tumor cells of different origin could transit from one mode of motility to another and normal cells could not undergo such transitions. We also investigate the effectiveness of amoeboid and mesenchymal motility during migration in different environments. It was shown that the mesenchymal motility is more effective for 2D migration, while the amoeboid motility is more effective in 3D conditions. During dissemination, cells go beyond the borders of original tissues and pass through environment with different properties. The plasticity of migration triggered by alteration of entire or internal conditions dramatically increases ability of cells to disseminate. The ability of tumor cells to plasticity permits them to choose optimal mode for migration, thus leading to metastasis development.

PIK3CA(H1047R) induces multipotency and multi-lineage mammary tumours.

The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

Abstract 3677: Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system

Abstract We report a development of a novel localized nanodelivery system featuring an enhanced targeted and controlled paclitaxel (PTX) delivery to epithelial and neuroectodermal origin tumor cells. The presented drug delivery system consists of a chitosan hydrogel embedded with PTX loaded non-ionic surfactant vesicles (PTX-niosomes) and chlorotoxin (CTX). We found a higher accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to overexpress MUC1 antigen as compared to normal ovarian epithelial cells. We therefore hypothesized that besides serving as a localized drug delivery platform, chitosan hydrogel can actively target MUC1 overexpressing tumor cells. To further improve the tumor-specific delivery of PTX, we have incorporated CTX, a 36-amino acid peptide, which binds to tumor cells of neuroectodermal origin, but not to non-transformed cells. Using a High Performance Liquid Chromatography (HPLC) assay, we have measured the in-vitro release profiles of PTX from chitosan hydrogel embedded with PTX-niosomes and CTX. We have assessed the morphology of CTX and PTX-nisomes as well as chitosan hydrogel embedded with PTX-niosomes and CTX using Transmission Electron Microscopy (TEM). We have used Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to investigate the possible molecular interactions between chitosan hydrogel and various tumor cells. To better understand the mechanism involved in interactions between chitosan and specific tumor cells, we have developed a new Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) allowing us to quantify the expression level of MUC1 in epithelial and neuroectodermal origin tumor cells. Our Cell ELISA results revealed higher expression level of MUC1 (around 1.5 fold) in ovarian epithelial carcinoma cell line (OV2008) when compared to normal ovarian epithelial cell line (MCC3). The ATR-FTIR results showed a specific interaction of chitosan with OV2008 cells implying the mucoadhesive property of chitosan and its potential in targeting of MUC1 overexpressing tumor cells. We observed a sustained PTX release from chitosan hydrogel embedded with PTX-niosomes (< 40% of cumulative release occurred within two weeks). Release studies in cell-free system also indicated that embedding CTX along with the niosomes does not have any adverse effect on the controlled release of PTX from the chitosan network. In fact, attachment of CTX to the surface of PTX-niosomes shown by TEM imaging improves the stability of PTX-niosomes resulting in extended release rates. Together these findings indicate that the proposed drug delivery system with the distinctive tumor targeting features and extended release profiles would improve the specific delivery of PTX to epithelial and neuroectodermal origin tumor cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2015-3677

Abstract 1935: Gene expression analysis reveals distinct pathways of resistance to antiangiogenic therapy in ovarian cancer

Abstract Purpose: The rapid development of resistance after an initial response phase is a major challenge of antiangiogenic therapies. The molecular mechanisms underlying clinical resistance to antiangiogenic therapies are not yet well understood. We aimed to identify potential mediators of resistance to antiangiogenic therapy, with a view toward eventually developing therapeutic approaches to improve antiangiogenic therapies. Methods: We developed preclinical in vivo phenotypic resistance models of antiangiogenic therapy using human ovarian carcinoma cells. We used two representative anti-VEGF agents to block VEGF (bevacizumab, 10 mg/kg BIW, IP) or its receptor (sorafenib, 30 mg/kg daily, PO). The treatment was given until tumors showed a long-term trend toward continued progression after an initial response (resistant tumor). Whereas treatment-sensitive tumors were defined by a long-term trend toward stable disease or regression. We conducted next generation sequencing to compare changes in the gene expression in resistant and sensitive tumors, distinguishing their origin (tumor vs stroma cells). To get a more detailed perspective of the overall changes in gene expression, Gene Set Enrichment Analysis (GSEA) was performed by pair-wise comparison according to treatment outcome (resistant vs sensitive) with respect to bevacizumab and sorafenib, of which the entire MSigDB collections were compared to. Further, significantly enriched pathways were validated with clinical gene expression profiling studies of various tumor types deposited in GEO and ArrayExpress. Results: Our GSEA analysis revealed significant differences (FDR<0.25) in enrichment of various pathways between sensitive and resistant tumors. We observed ERK, P38 MAPK, ATP dependent helicase activity, and phosphatidylinositide 3-kinases pathways are differentially enriched between sensitive and resistant phenotypes of bevacizumab-treated group. In sorafenib-resistant group, six pathways were enriched including E2F1, KRAS and PDGF regulated pathways. Conclusions: Here, we report distinct pathways of response to bevacizumab and sorafenib in preclinical ovarian cancer models, which may provide critical insights into ovarian tumor's adaption during anti-VEGF blockade. These pathways may also provide important clues for identifying synergistic therapeutic strategies to overcome or delay resistance to antiangiogenic agents. Citation Format: Bharat Kumar Devapatla, Pharavee Jaiprasart, Sukyung Woo. Gene expression analysis reveals distinct pathways of resistance to antiangiogenic therapy in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1935. doi:10.1158/1538-7445.AM2015-1935

Abstract 484: Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project

Abstract Patient-derived xenografts and cell lines have been the underpinning of functional characterization and drug discovery efforts in cancer. The use of these models is often under the assumption that these systems are renewable, faithful representations of original progenitor tumor cell populations. To test this assumption, we performed whole exome (92X median coverage) and genome sequencing (36X median coverage) analysis of cell line and patient-derived mouse xenografts (PDXs) originating from 7 neuroblastoma patients. Data are from1 primary tumor, 3 PDXs, and 15 neuroblastoma cell lines cultured from tumor, bone marrow, or blood. The cell lines consisted of 4 pre-/post-therapy pairs and 3 pairs established and maintained in either hyperoxia (room air i.e. “standard” cell culture) or physiologic (bone marrow hypoxia = 5%) oxygen. 7 lymphoblastoid or fibroblast cell lines were used as matched normals to identify somatic mutations. Subclonal population structures were inferred from somatic mutation calls calibrated for copy number state and tumor purity. In all cell lines and xenografts, we observed 1-2 additional subclonal populations, primarily supported by deep coverage from exome sequencing. In nearly every case, we observed shifts in the proportional representation of genetic subclones and many subclones showed additional mutations not evident in the progenitor tissue or cancer line derived in parallel. Comparison of three cell line pairs established in bone marrow level hypoxia versus room air found only ∼40% of coding mutations in each line were shared (average 82 mutations per line), suggesting significant genetic impact of growing tumor cells in the two different culture conditions. Matched PDXs from these cases had only ∼17% of coding mutations shared across all three models. The greatest genetic similarity was seen between paired cell lines established from tissue obtained pre-/post-therapy from the same patient (36 coding mutations shared, 14 private to diagnosis and 13 private to progression). However, a second pre/post-therapy cell line pair did not share any coding mutations, although they did have 585 non-coding mutations in common (of 4,033 and 2,480 in each line), assuring that the relapse was derived from a diagnostic tumor clone. These results highlight a need for comprehensive subclonal analysis of human cancer laboratory models to better inform design and interpretation of biological and preclinical therapeutic studies. Citation Format: Maya Schonbach, Arnavaz Danesh, Jeff Bruce, Tito Woodburn, Tanja Davidsen, Leandro Hermida, Patee Gesuwan, Jaime Guidry Auvil, Oliver Hampton, David Wheeler, Julie Gastier-Foster, Malcolm Smith, Daniela Gerhard, John M. Maris, Patrick Reynolds, Trevor J. Pugh. Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2015-484

Are we ready for the use of intraoperative salvaged blood in metastatic spine tumour surgery?

To evaluate the feasibility of using intraoperative cell salvage (IOCS) in combination with leucocyte depletion filter (LDF) in eliminating tumour cells from blood salvaged during metastatic spine tumour surgery (MSTS). This is with the view to pave the path for use of IOCS-LDF in MSTS and musculoskeletal oncological surgery. Sixty consecutive patients with known primary epithelial tumour, who were offered surgery for metastatic spine disease at our university hospital, were recruited. Blood samples were collected at three different stages during surgery: from operative field prior to IOCS processing, after IOCS processing and after IOCS-LDF processing. Three separate samples (5ml each) were taken at each stage. Samples were examined by cell block technique using immunohistochemical monoclonal antibodies to identify tumour cells of epithelial origin in the samples. Of 60 patients, ten were excluded for not fulfilling the inclusion criteria leaving 50 patients. Malignant tumour cells were detected in the samples from operative field prior to IOCS processing in 24 patients and in the samples from the transfusion bag post-IOCS processing in 4 patients. No viable malignant cells were detectable in any of the blood samples after passage through both IOCS and LDF. The findings support the notion that IOCS-LDF combination works effectively in eliminating tumour cells from salvaged blood so this technique can possibly be applied in MSTS and even musculoskeletal oncological surgery. This concept can then be extended to other oncological surgeries in general with further appropriate clinical studies.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Use of preoperative FLAIR MRI and ependymal proximity of tumor enhancement as surrogate markers of brain tumor origin

Neural stem cells proliferate in the subventricular zone and give rise to progeny that differentiate and migrate throughout the brain. We aimed to test the hypothesis that glioma behavior and grade may correlate with the identity of the tumor cell of origin. We evaluated three preoperative radiographic features (fluid attenuated inversion recovery [FLAIR] MRI characteristics, tumor proximity to ventricular ependyma, and subependymal representation) as surrogate markers of tumor origin using a retrospective cohort design. The medical records of 228 patients who underwent surgical resection of a glioma from January 2004 to August 2008 were reviewed. Average patient age was 54.5years (standard deviation [SD] 15.3) with a male predominance (62.9%). World Health Organization glioma grades amongst the cohort were Grade IV (71.6%), Grade III (21.3%) and Grade II (7.1%). Mean survival was 11.2months (SD 10.5) with a mean follow up of 12.8months (SD 11.3). Glioma tumor grade was significantly correlated to FLAIR signal proximity to the ependymal surface (p<0.01) and inversely with proximity of tumor mass to the ependyma (p<0.01). The mean distance of tumor-associated FLAIR signal from the ependymal surface for glioblastoma multiforme (GBM) was 1.2mm (SD 3.3) compared to 4.8 (SD 6.5) for anaplastic astrocytomas and 6.6mm (SD 6.7; p<0.01) for low grade gliomas. Conversely, the mean distance of the enhancing tumor mass from the ependyma for GBM was 7.3mm (SD 9.4), Grade III glioma 2.3mm (SD 4.9), and Grade II glioma 3.8mm (SD 6.8; p<0.05). These findings suggest that higher grade gliomas might arise from less differentiated neuroepithelial cells in the subventricular zone that possess greater migratory potential.

Differentiation of Glioma Stem Cells and Progenitor Cells into Local Host Cell-Like Cells: A Study Based on Choroidcarcinoma Differentiation of Choroid Plexus of GFP Transgenic Nude Mouse.

The idea of multiple differentiation capacity of glioma stem cells and progentior cells (GSCPs) has been accepted by most of the researchers, but the effect of local environment on the differentiation of GSCPs is unclear. GSCPs SU2 and CM-Dil-stained C6 cells (C6-Dil) were injected into the brain of GFP transgenic nude mice. The xenografts were sectioned. Morphological changes of tumor cells that resided in the choroid plexus, molecular markers expression, and the relationship between the original tumor cells and host cells were studied carefully. The tumorigenicity rate was 40/40 (100%) in all of the inoculated nude mice. Cell morphology and molecular expression of neoplasm settled in the choroid plexus showed that choroidcarcinoma derived from GSCPs was developed. These results showed that GSCPs may have the multiple differentiation capacity, which can be induced by the local environment of host brain as NSCs, and cell fusion may play an important role in the transformation.