bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5205: Selectively high Wnt pathway activity in subpopulations of colon cancer cells and its significance for tumor initiation
Authors: Horst David , Shivdasani Ramesh A.
Published: June 2012

Several cell surface markers that are reported to enrich for tumor-initiating colon cancer cells are presumed targets of the canonical Wnt/beta-catenin pathway. Furthermore, colon cancers typically express the Wnt-effector beta-catenin in a heterogeneous pattern, with strong nuclear expression detected only in a small fraction of tumor cells. We therefore hypothesized that high beta-catenin expression and Wnt pathway activity mark a tumorigenic subpopulation. To label viable tumor cells according to the level of Wnt pathway activity, we constructed lentiviral vectors that express GFP under control of an optimal, beta-catenin-responsive TCF/LEF1 promoter (TOP-GFP). We transduced primary colon cancer xenografts and two well characterized colon cancer cell lines with these vectors, used flow cytometry to isolate cell subpopulations with differential GFP expression, and examined these subpopulations for tumorigenicity and gene expression. High GFP expression accurately marked tumor cells with nuclear beta-catenin immunostaining in Caco2 but not SW1222 cells and in a fraction of primary colon cancer xenografts. Gene expression analysis in the concordant cases was consistent with increased Wnt-pathway activity within the GFP-high cell population, including high expression of the putative stem cell markers CD133, CD44 and LGR5. In SW1222 cells and some primary tumor xenografts, the GFP-high fraction correlated poorly with nuclear beta-catenin localization and GFP flow cytometry did not separate cells with a gene expression profile suggesting Wnt pathway activity. Among these various sources, only Caco2 cells showed increased tumorigenicity of GFP-high cells in immunodeficient mouse xenografts. The utility of TOP-GFP and related reporters to identify tumor-initiating colon cancer cells with high Wnt activity thus varies among primary human colorectal tumors and established cell lines. Furthermore, Wnt pathway activity might fluctuate substantially within individual tumor cells. We conclude that high Wnt activity among tumor cell subpopulations does not necessarily signify tumor-initiating potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2011-5205
Abstract 5208: Simultaneous color-coded imaging to distinguish colon cancer stem-like and non-stem cells in the same tumor
Authors: Suetsugu Atsushi , Hassanein Mohamed K. , Nagaki Mashito , Moriwaki Hisataka , Bouvet Michael , Moossa Abdool R. , Hoffman Robert M.
Published: June 2012

In this study, CD44+ HCT-116 cells were considered as colon cancer stem-like cells (CSCs) and CD44− HCT-116 cells were considered colon non-cancer stem cells (NCSCs). CD44+ cells were isolated by magnetic bead sorting. CD44+ cells outgrew CD44− cells in vitro. GFP-CD44+ cells preferentially migrated from the center of the spheroids after co-culture with RFP-CD44− cells. GFP-CD44+ cells grew to a greater extent subcutaneously in nude mice, when compared to the CD44− cells. Equal numbers of GFP CSCs and the RFP NCSCs were mixed and co-injected in the spleen of nude mice where the cells metastasized to the liver. GFP CSCs were found in greater numbers compared to RFP NCSCs in the liver of nude mouse. GFP CSCs and RFP NCSCs were co-injected in the wall of the cecum. GFP CSCs were observed in metastases formed in the small intestine and colon wall, to a much greater extent than RFP-NCSC. Simultaneous color-coded imaging of CSCs and NCSCs in the present study has demonstrated two very different subsets of cells within the HCT-116 cell line with respect to tumor growth and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5208. doi:10.1158/1538-7445.AM2011-5208
Abstract 5219: The LIM protein LIMD2 functions as an effector and biomarker for metastasis in multiple tumor types
Authors: Peng Hongzhuang , Hou Zhaoyuan , He Mei , Kebebew Electron , Orntoft Torben F. , Herlyn Meenhard , Caton Andrew J. , Fredericks William J. , Malkowicz Bruce , Rauscher Frank J.
Published: June 2012

The LIM domain is found in proteins from a wide variety of eukaryotic organisms. The LIM domain is organized as a tandem zinc-finger structure that functions as a modular protein-binding interface. LIM domain containing proteins have diverse cellular roles such as regulators of gene expression, cytoarchitecture, cell adhesion, cell motility, signal transduction, and biosensors. LIMD2 belongs to the LIM only family of LIM proteins and was identified as a biomarker for papillary thyroid carcinoma lymph node metastasis from molecular profiling of matched samples. However, the biological function of LIMD2 remains unknown. To identify whether LIMD2 is a biomarker for other cancers, highly specific LIMD2 polyclonal and monoclonal antibodies were developed and several cancer cell lines were tested for LIMD2 expression. These cancer cell lines were derived from breast cancer, melanoma, bladder cancer, and thyroid cancer. We found that the cancer cell lines derived from more aggressive tumors exhibited higher levels of LIMD2 protein expression. Furthermore, cells expressing higher levels of LIMD2 had increased migratory capabilities. Over-expression of LIMD2 protein in the less aggressive bladder cancer cell line (RT4) showed an increase in migration and colony formation ability. Conversely, knockdown of LIMD2 using siRNA decreased the migratory ability of the malignant melanoma cancer cell line and caused morphological changes in these cells. These data not only underscore the importance of LIMD2 as a key metastatic biomarker, but also suggest that LIMD2 may play a functional role in tumorgenicity. LIMD2 may also serve as a novel anti-metastatic target to improve the efficacy of conventional cancer therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5219. doi:10.1158/1538-7445.AM2011-5219
Abstract 522: CXCR4 inhibition sensitizes prostate cancer cells to conventional chemotherapy in an in vitro co-culture model
Authors: Domanska Urszula M. , Kruizinga Roeliene C. , Nagengast Wouter B. , Bosscha Hetty Timmer – , de Vries Elisabeth G.E. , Huls Gerwin A. , Walenkamp Annemiek M.E.
Published: June 2012

Background: Chemokine receptor 4 (CXCR4) mediates homing of hematopoietic stem cells to niches, where stromal cells express CXCL12, the CXCR4 ligand. Indeed, in clinical practice the CXCR4 inhibitor AMD3100 is used to mobilize hematopoietic stem cells. Also in several solid cancer types, including prostate cancer, tumor cells show CXCR4 overexpression. In solid cancers this receptor is involved in tumor metastasis, invasiveness and primary tumor growth. It is postulated that in these tumors, the stromal microenvironment expressing CXCL12, protects tumor cells from chemotherapy. We therefore aimed to study whether inhibition of the CXCR4/CXCL12 axis, with the specific CXCR4 inhibitor AMD3100, sensitizes prostate cancer cells for conventional chemotherapy and improves treatment outcome.

Methods: A co – culture model of murine stroma (MS5) and human prostate cancer cells (PC3-luc) has been developed. To assess the effect of stromal cells on sensitivity to chemotherapy, PC3-luc cells labeled with fluorescent DiI dye were cultured with or without MS5 cells and treated with 1 μM docetaxel for 40 hours. To determine whether observed effect can be reversed by CXCR4 inhibition, experiments were repeated with addition of 25μg/ml AMD3100. After 40 hours treatment, the percentage of viable PC3-luc cells in the field of view was assessed by fluorescent microscopy with DAPI nuclear staining. For each condition, six fields of view were counted and the average percentage was calculated. Experiments were performed in three repetitions.

Results: In the absence of MS5 stromal cells, docetaxel treatment of PC3-luc cells resulted in only 14.1 % survival. Co-culturing with stromal cells demonstrated a protective effect on chemotherapy induced cell death, with up to 61,8 % survival of PC3-luc cells after docetaxel treatment. AMD3100 eversed this protective effect completely, as combined treatment with AMD3100 and docetaxel in the co-culture setting resulted in a drop in the percentage of viable cells from 61.8 % to 19.9 % (P<0.0042).

Conclusion: Murine stromal MS5 cells protect PC3-luc tumor cells against docetaxel – induced cell death in an in vitro co-culture system. Treatment with AMD3100 almost completely abolished the effect of the presence of stromal cells when the prostate cancer cells were treated with docetaxel. These data underscore the relevance to focus for further research on the role of CXCR4 inhibitors as a potentiating adjunct to conventional chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 522. doi:10.1158/1538-7445.AM2011-522
Abstract 5228: Multi-modal in vivo imaging of breast cancer cells expressing dual reporters
Authors: Urban Konnie , Ang Angel , Kuo Chaincy , Francis Kevin , Singh Raj , Roskey Mark , Kim Jae-Beom
Published: June 2012

We previously engineered tumor cell lines with bioluminescent markers and fluorescent proteins for whole animal imaging. In addition to conventional non-invasive tumor growth detection, these cells when harvested from animals can also be used to analyze cell-cell interactions by immunohistochemistry. Moreover, fluorescent cells can be isolated using fluorescent activated cell sorting (FACS) for gene expression profiling. In this study, we used a mouse mammary gland tumor cell line 4T1 that was labeled with firefly luciferase and tdTomato fluorescent protein (4T1-luc2-tdTomato). The generated cell line was tested for in vitro signal stability with respect to bioluminescence and fluorescence prior to injection into the animals. Tumor cells were implanted subcutaneously and monitored for tumor development by acquisition of bioluminescent and fluorescent images. In a separate study, 4T1-luc2-tdTomato cells were implanted orthotopically into mammary fat pads. Tumor growth was monitored and signals from the implantation sites were reconstructed into three dimensional images. In order to non-invasively detect bone metastases by 4T1-luc2-tdTomato cells, we applied both microCT and optical imaging. For this, an interchangeable imaging platform between an optical imager and microCT machine was developed. 4T1-luc2-tdTomato cells were injected into nu/nu mice by intracardiac injection. Bioluminescent imaging was performed immediately to verify the injection of cells into the left ventricle. Fluorescent signals were not detected, likely due to the small number of cells in the secondary tumor sites. Animals demonstrating substantial whole body bioluminescence were selected for further monitoring until post injection day 9. We found that bioluminescent signals were located in the knee joint regions. For these animals, bioluminescent and X-ray images were also taken without changing the positions of the animals using the interchangeable platform. Bioluminescent images in the knee joints were reconstructed using a diffused luminescence imaging tomography algorithm. These images were then co-registered with microCT images. The results showed that bioluminescent signals were colocalized within the joint area obtained by microCT. Moreover, high resolution images of the joints from these mice revealed bone erosion in the tibia induced by 4T1-luc2-tdTomato cells. These findings demonstrate that multimodal imaging can pinpoint the tumor lesions non-invasively. In addition, low dose microCT imaging can be used to acquire high resolution skeletal images longitudinally without inducing adverse effects on the animals.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5228. doi:10.1158/1538-7445.AM2011-5228
Abstract 523: Modeling the crosstalk between head and neck carcinomas and fibroblasts that underlies malignant invasion and drug resistance
Authors: Räsänen Kati A. , Basu Devraj , Montone Kathleen , Zhang Gao , Rustgi Anil , Herlyn Meenhard
Published: June 2012

There is an ongoing need to advance in vitro models of carcinoma invasion and drug resistance, which are both processes likely to be shaped by the complex crosstalk between normal and malignant cells at stromal interfaces. Epithelial to mesenchymal transition (EMT) generates a mesenchymal-like malignant subpopulation within carcinomas with enhanced invasive potential. Here this subpopulation is hypothesized to play a critical role in a tumor-fibroblast crosstalk that enhances invasion and resistance to chemotherapy. To assess the biologic and therapeutic significance of this subpopulation, we have defined head and neck squamous cell carcinoma (HNSCC) cell lines where ongoing EMT sustains an “Ecad-lo” subset; these cells are distinguished by a mesenchymal-like overall gene expression profile and also display key markers typically associated with activated fibroblasts. The mesenchymal-like (Ecad-lo) subset is separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. As an in vivo correlate of Ecad-lo cells, we identified a subpopulation of vimentin-positive malignant cells in primary advanced stage human HNSCCs. In vitro, the Ecad-lo subset showed resistance to cytotoxic and EGFR-targeted therapy; comparable findings were seen in vivo, where treatment led to enrichment of vimentin positive cells in direct xenografts of human HNSCCs. Interactions of stroma with Ecad-hi versus Ecad-lo cells were compared using collagen embedded tumor spheroids and organotypic 3D reconstructs. Here Ecad-lo cells were shown necessary to create an invasive front into collagen matrix. Ecad-lo cells were also needed to drive fibroblast migration and formed a direct interface with fibroblasts in the collagen layer of organotypic 3D cultures. Activated stromal fibroblasts subsequently participated in a feedback loop by promoting EMT in the epithelial-like Ecad-hi subpopulation of cancer cells, increasing the size of the invasive, treatment-resistant Ecad-lo subset. These results define systems for modeling the coordinated acquisition of mesenchymal-like features, invasiveness, and drug resistance by HNSCC cells via active crosstalk with stromal fibroblasts. In this capacity, the models will serve as a platform for identifying targetable mechanisms underlying these central features of malignant behavior.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 523. doi:10.1158/1538-7445.AM2011-523
Abstract 5233: Abnormal lymphatic drainage and function from vascular endothelial growth factor (VEGF)-C overexpressing B16F10 melanoma
Authors: Kwon Sunkuk , Robinson Holly , Wu Grace , Lachance Pier-Anne , Sevick-Muraca Eva
Published: June 2012

The lymphatic system provides a major transport route for initial cancer cell dissemination for many cancers. Tumor lymphangiogenesis (i.e., the new formation of tumor-associated lymphatic vessels) is associated with metastatic potential and is an emerging therapeutic target to arrest metastasis. Recent studies show that the major lymphangiogenic growth factor, vascular endothelial growth factor (VEGF)-C induces tumor lymphangiogenesis and enhances tumor spread to the regional lymph nodes (LNs) and distant sites in mouse models of breast cancer and melanoma. Yet despite the important role of lymphatics as a critical pathway for cancer metastasis, relatively little is known about lymphatic function during tumor progression and metastasis, due mainly to the lack of lymphatic imaging approaches. Recently, we have developed in vivo dynamic near-infrared (NIR) fluorescence imaging methods for quantifying propulsive lymph flow. Herein, we employed a dynamic, functional lymphatic imaging technique and longitudinally imaged spatial and temporal changes in lymphatic function and architecture in mice bearing vascular endothelial growth factor (VEGF)-C overexpressing B16F10 (VEGF-C-B16F10) or mock-transduced B16F10 (mock-B16F10) melanoma tumors. C57BL6 mice were injected with 10ul of indocyanine green (ICG) or FITC-Dextran in the base of the tail and were dynamically imaged for baseline information before implantation of VEGF-C-B16F10 or mock-B16F10 in the hindlimb every three to four days for up to 27 days after inoculation. Our dynamic NIR fluorescence imaging data show that ICG-laden lymph accumulates into a VEGF-C-B16F10 tumor at 3 days post implantation as confirmed with FITC-Dextran leakage from lymphatic vessels; thereafter lymphatic leakage is visualized only in peritumoral lymphatic vessels. Our longitudinal imaging data also show dilated and tortuous lymphatic vessels and reduced or loss of propulsive lymphatic flow in the process of VEGF-C-B16F10 growth. However, mock-B16F10 tumors do not show lymphatic vessel leakage during tumor progression. NIR fluorescence imaging technology can be used to non-invasively and quantitatively detect functional lymphatic changes associated with cancer, which may enable accurate nodal staging of cancer patients and provide new approaches to diagnose and treat cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2011-5233
Abstract 5237: Development of organ-selective neuroblastoma cell lines to identify genes mediating bone marrow and liver colonization
Authors: Neiron Zillan , Jankowski Kacper , Murray Jayne , Champion Sophia , Norris Murray D. , Haber Michelle , Fletcher Jamie I.
Published: June 2012

Neuroblastoma is a pediatric tumor with a high propensity for metastasis to bone marrow, cortical bone and liver, and almost 50% of patients have metastatic disease at diagnosis. This study aims to identify genes contributing to neuroblastoma colonization of the bone marrow and liver through a combination of in vivo cell selection techniques and gene expression analyses. Ultimately, it aims to identify potential therapeutic targets for this disease.

When the human neuroblastoma cell line SK-N-BE(2) is xenografted into immunodeficient mice, metastases arise in the adrenal medulla, liver and bone marrow. In contrast, an established subclone forms metastases limited to the bone marrow, suggesting that the original line contains populations with different abilities to colonise each site. We xenografted luciferase positive SK-N-BE(2) cells into immunodeficient mice and isolated pure tumour cell populations from the bone marrow and liver. Increased selectivity for the organ of origin was confirmed for some of these populations using bioluminescent imaging following engraftment into secondary recipients.

We conclude that neuroblastoma cell populations with selectivity for particular organs can be generated using in vivo selection methods. Whole genome expression analyses will now be used to identify candidate genes underlying this selectivity. These genes will subsequently be validated in xenograft models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5237. doi:10.1158/1538-7445.AM2011-5237
Abstract 524: Stromal fibroblasts diminish the growth-inhibitory effect of the epidermal growth factor receptor antagonist cetuximab in head and neck squamous cell carcinoma cell cultures
Authors: Johansson Ann-Charlotte , Ansell Anna , Östman Arne , Roberg Karin
Published: June 2012

There is a growing body of evidence that components of the tumor microenvironment, including stromal fibroblasts, may modulate the treatment sensitivity of tumor cells. Thus, we aimed to investigate the possible influence of stromal fibroblasts on the cetuximab sensitivity of head and neck squamous cell carcinoma (HNSCC) cells.

Stromal fibroblasts isolated from 7 HNSCC patients were co-cultured with HNSCC cells in a transwell system. Mono- and co-cultures were exposed to the epidermal growth factor receptor (EGFR) antagonist cetuximab (30 nM) and cell proliferation was determined by crystal violet staining. The level of EGFR, Akt, Erk (p42/p44 MAPK), and Met activation was assessed by western blot analysis.

Cetuximab elicited an anti-proliferative response in tumor cell monocultures while the growth rate of stromal fibroblasts remained unchanged. When tumor cells were co-cultured with fibroblasts or grown in media conditioned from fibroblast cultures the anti-proliferative effect of cetuximab was diminished. Furthermore, stromal fibroblasts desensitized tumor cells to treatment with gefitinib, an EGFR tyrosine kinase inhibitor, excluding the possibility that fibroblast-derived factors confer resistance by interfering with the EGFR-cetuximab interaction. Results from experiments using size-fractionated conditioned media suggest that at least two soluble factors, 50-100 kDa and greater than 100 kDa in size, confer treatment resistance. In cetuximab-treated cultures, phosphorylation of EGFR and Erk was reduced, while the level of Akt phosphorylation remained unchanged. The cetuximab resistance observed in co-cultures could not be explained by changes in either EGFR or Erk phosphorylation. However, there was a higher level of activation of the hepatocyte growth factor (HGF) receptor Met in co-cultures, and when recombinant HGF was added to tumor cell monocultures the response to cetuximab was reduced.

These results identify a previously unrecognized fibroblast-dependent modulation of cetuximab sensitivity, and also suggest fibroblast-derived HGF as one of the mediators of this paracrine cross-talk. The results also suggest targeting of fibroblast-derived factors, possibly including HGF, as a novel strategy to improve the therapeutic effects of cetuximab.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2011-524
Abstract 5244: Genistein's anti-invasion and anti-metastatic effects are linked to decreases in the expression of several different gene in Human prostate cancer that regulate metastatic behavior
Authors: Huang Xiao Ke , Bergan Raymond
Published: June 2012

Genistein has been shown by our group to inhibit Human prostate cancer (PCa) cell invasion in vitro and metastasis in vivo. Related studies demonstrate that endoglin (ENG), a TGF b superfamily receptor, suppresses invasion via activation of Smad 1, and that Genistein therapeutically compensated for endoglin loss by activating Smad 1 signaling. The mechanisms underlying these effects by Genistein are not clear. We hypothesized that they resulted from effects by genistein upon multiple genes that together regulate metastatic behavior.

Methods: PC3M cells were treated or not with 10uM Genistein × 3 days, RNA was isolated, treated with DNAse, and effects upon gene expression were screened for using SAbiosciences metastasis specific and extracellular matrix and adhesion molecule specific gene expression array platforms. Array experiments were performed 2-3 separate times. Positive array findings were then evaluated by real time qRT/PCR, using gene-specific primer/probe sets. These experiments were performed 3 separate times. A student's T-test P-value</= 0.05 was required.

Results: Genistein significantly decreased MMP2, MMP10 and MMP 13 by 2.81, 3.98 and 4.87 fold, respectively, on the metastasis array, and decreased MMP1, MMP10, MMP13 and SPARC (osteonectin) by 2.29, 3.25, 5.36 and 3.57 fold, respectively, on the matrix /adhesion array. With qRT/PCR, Genistein decreased MMP1, MMP2, MMP10, MMP13 and SPARC by 2.13, 2.17, 4.43, 6.82 and 2.57 fold, respectively. We then treated previously characterized stably engineered ENG human PCa cell lines. Hi-ENG, vector control (VC) and NO-ENG, with Genistein or not, followed by qRT/PCR, ENG expression did not alter gene expression in a consistent fashion. Importantly, ENG status had no effect upon Genistein's ability to decrease the expression of any of the five genes.

Conclusions: We demonstrate for the first time that Genistein inhibit MMP1, MMP2, MMP10, MMP13 and SPARC gene expression. Also, Genistein's effect upon these genes is not dependent upon the endoglin pathway. Further, endoglin does not appear to regulate these genes. Finally these data support the notion that Gensitein's anti-invasion and anti-metastatic actions result from effects upon several genes that together affect invasive and metastatic behavior.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2011-5244
Abstract 5248: Quantum dot multiplexing of prognostic marker and stem cell marker expression in colorectal cancer circulating tumor cells
Authors: Das Avisnata , Allen Joshua E. , Dicker David T. , Peters Kristi L. , Joudeh Jamal , El-Deiry Wafik S.
Published: June 2012

Circulating tumor cells (CTCs) reflect disease burden in metastatic colorectal cancer (mCRC) and show reduced numbers in patients who are responding to chemotherapy. Current analysis is limited to counting CTCs and little additional marker analysis. We sought to develop methodology to allow multiplexing of marker detection in individual CTCs to assess tumor cell heterogeneity and to gain insight into relationships of prognostic and therapy response markers. This ongoing study is evaluating expression of up to 8 markers in a single CTC by a multispectral multiplex imaging technique using nano-particle type quantum dot (Q-Dot) dyes. We hypothesize that CTCs that are extremely rare (∼ 1 in 10 million RBCs) in the blood of cancer patients play an important role in metastasis. In view of the small number of such cells in the blood of mCRC patients (prognostication based on <3 versus >3 CTCs/7.5 ml of blood) there is a need to derive more molecular correlations from single CTCs. We designed a clinical protocol to collect blood from mCRC patients receiving chemotherapy with the intent to evaluate multiplexed molecular marker expression in CTCs in responders versus non-responders using the FDA-approved Veridex CellSearch technology. We are evaluating CD26, CD133, EGFR, Ki 67, p27, c-Myc, ALDH1, Bcl-Xl, Mcl-1, phosho-Akt, phospho-ERK, TRAIL receptors, IAPs, and p53 using antibodies conjugated to different Q-Dots. Ongoing studies include prognostic marker, stem cell marker, and cell proliferation/death response marker analysis in human tumor xenografts of HCT116, p53-null HCT116, DLD1, SW620, and SW480. Initial profiling of tumor cell lines revealed that mutant p53 and KRAS-expressing SW620 derived from a patient with mCRC has CD26 and CD133 expression including a sub-population of tumor cells that co-express these two stem cell markers. HCT116 showed positive EGFR, CD26, but little or no CD133 expression. We are evaluating correlations between the primary tumors in mice as compared to the CTCs in mice and similar experiments are part of the clinical protocol. The studies should unravel the complexity of CTC heterogeneity, and define informative subsets of markers that can be routinely included in studies to assess prognosis and response to therapy. The ease with which CTCs can be isolated makes these studies of comparisons with primary tumors critical in order to establish and validate new methods for assessing tumor behavior and response to therapy in the clinic through this CTC “liquid biopsy” technique.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2011-5248
Abstract 5280: PET imaging of proteolysis: Evaluation of 68Ga-DOTA and 68Ga-NODAGA chelates of an uPAR-specific peptide in a human glioblastoma xenograft model
Authors: Persson Morten , Madsen Jacob , Østergaard Søren , Ploug Michael , Kjaer Andreas
Published: June 2012

Background & Aim: The urokinase-type plasminogen activator receptor (uPAR) has been shown to facilitate cancer cell invasion and metastasis. uPAR is over-expressed in various cancers, including human breast, prostate and colorectal cancer, and over-expression has been shown to correlate with poor prognosis. The established correlation between uPAR expression and disease progression provides an opportunity to develop an in vivo imaging agent, which could identify cancer patients at risk. In the present study, we produced and compared two new PET tracers based on 68Ga, the chelators DOTA and NODAGA and the uPAR antagonist peptide AE105 in a human glioblastoma xenograft model using microPET/CT.

Method: Both compounds were synthesized using standard Fmoc solid-phase peptide chemistry and subsequently N-terminal conjugated with either DOTA or NODAGA. The conjugated peptides were radiolabeled with 68Ga using a 68Ge/68Ga generator system, which render the production of the tracers independent of a onsite cyclotron. The compounds were evaluated in vitro by surface plasmon resonance against human uPAR and in vivo using micro-positron emission tomography in a mouse model bearing human glioblastoma U87MG xenografts. All findings were validated using uPAR ELISA on tumor tissue and uPAR immnohistochemistry on tumor slides.

Results: Both compounds were labeled under mild conditions with high yield and radiochemical purity (>95%). Both compounds showed excellent uPAR binding (IC50 values of 10-15 nM) in vitro, which was identical to the un-conjugated peptide AE105. In vivo, they displayed different tumor uptake, with 68Ga-DOTA-AE105 and 68Ga-NODAGA-AE105 having an uptake of 0.48±0.3 and 2.58±0.02 %ID/g 0.5 hr post injection, respectively. Besides tumor uptake, high uptake in the kidneys was also observed for both compounds. Dynamic PET scans illustrated higher stability in vivo for 68Ga-NODAGA-AE105 compared to 68Ga-DOTA-AE105, thus generating the best tumor-to-background contrast.

Conclusion: A new 68Ga-based PET tracer for tumor imaging of human uPAR has been developed. Based on head-to-head comparison, NODAGA seems to be the optimal chelator, giving the highest uptake in tumor tissue. The use of generator-produced 68Ga could further support translation into the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2011-5280
Abstract 529: WISP-1/CCN4 is responsible for mediating the inhibitory effect of Notch-engineered stromal fibroblasts on melanoma growth
Authors: Shao Hongwei , Cai Long , Livingstone Alan S. , Velazquez Omaida C. , Liu Zhao-Jun
Published: June 2012

Purpose. The tumor microenvironment plays a pivotal role in influencing melanoma progression. Fibroblasts are major components of tumor stroma and are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix, and direct cell-cell interaction. We aimed to alter the biological activity of stromal fibroblasts by modulating a specific signaling pathway to convert them from promoters to inhibitors of melanoma progression.

Methods. Enforced activation of the Notch pathway was achieved by transducing human dermal fibroblasts with lentiviral vector encoding activated Notch1 (NIC). The effect of NIC-engineered stromal fibroblasts on melanoma growth was tested by the in vivo tumor-fibroblast co-xenografting approach. The soluble factor produced by NIC-engineered fibroblasts was identified by RT2-PCRArray and validated by Western blot analysis. The biological effect of WISP-1 on melanoma cell growth was tested in vitro by MTT. The function of WISP-1/CCN4 in mediating the inhibitory effect of NIC-engineered stromal fibroblasts on melanoma growth was examined through an siRNA-mediated blockade approach and tested by in vitro cell co-culture and animal models of tumor-fibroblast co-xenografting.

Results. NIC-engineered fibroblasts significantly inhibited melanoma growth in vivo by suppressing melanoma cell proliferation and inducing melanoma cell apoptosis. Constitutive activation of Notch1 in fibroblasts up-regulated WISP-1 production. The inhibitory effect of NIC-engineered fibroblasts on melanoma growth was mediated, at least in part, by up-regulating WISP-1 expression, since recombinant WISP-1 was able to inhibit melanoma cell growth in vitro while blockade of WISP-1 production by NIC-engineered fibroblasts liberated their inhibitory effect on melanoma growth in vivo.

Conclusions. Activation of Notch pathway in stromal fibroblasts can suppress melanoma growth, partially through WISP-1. Our study provides a novel approach to target the tumor microenvironment through modulating stromal fibroblasts by manipulating Notch signaling, and highlights WISP-1 as a potential therapeutic target in melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2011-529
Abstract 5291: A preclinical model for investigation of nuclear factor-kappa B inhibitors in ovarian cancer
Authors: Wilson Andrew J. , Lee Hye-Jeong , Oshinko Halina , Yull Fiona , Khabele Dineo
Published: June 2012

Most women diagnosed with ovarian cancer initially present with metastatic disease characterized by peritoneal implants and ascites. Critical to the distinct clinical pattern of spread of ovarian tumors is activation of a pro-inflammatory process mediated by nuclear factor-kappa B (NF-κB). However, NF-κB inhibitors such as bortezomib have been disappointing in the clinic. Alternative NF-κB inhibitors such as thymoquinone (TQ) and curcumin, both derived from natural products, exhibit promise as potential treatments for ovarian cancer and are well tolerated. Therefore, our goals were to validate a newly developed preclinical model system for NF-κB activity in ovarian cancer cells and test the efficacy of TQ and curcumin in combination with cisplatin, standard therapy for ovarian cancer. Mouse ovarian epithelial cancer cells (ID8) derived from C57BL/6 mice were stably transfected with a fusion reporter that expresses a green fluorescent protein (GFP)/luciferase fusion product under the control of a synthetic NF-κB dependent promoter (ID8-NGL). Tumor necrosis factor-α (TNF-α) stimulated NGL reporter activity approximately 2.5-fold in luciferase assays in ID8-NGL cells, an effect confirmed by bioluminescence imaging of cell monolayers. The specificity of this effect was demonstrated by the ability of the NF-κB inhibitor, TQ, to abrogate TNF-α-stimulation of NF-κB reporter activity. These effects in ID8-NGL cells were confirmed by parallel Western Blot analysis of IκBα phosphorylation (p-IκBα), with TQ markedly inhibiting TNF-α-induction of p-IκBα. Treatment of ID8-NGL cells with TQ and curcumin inhibited NGL reporter activity and inhibited growth in MTT assays in a dose-dependent manner. Furthermore, the combination of TQ or curcumin with cisplatin induced greater inhibitory effects on growth of ID8-NGL cells than either drug alone, indicative of synergistic effects. Finally, we have confirmed ID8-NGL cells transplanted intraperitoneally into C57BL/6 mice form peritoneal tumors and ascites in a similar manner to parental ID8 cells in vivo. This syngeneic model of ovarian cancer progression will allow us to examine in more detail the effects of promising NF-κB inhibitors in combination with platinum therapy in ovarian cancer cells in vitro and in immunocompetent mice in vivo, providing a powerful platform for future preclinical investigation of novel therapeutic agents that target NF-κB in ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5291. doi:10.1158/1538-7445.AM2011-5291
Abstract 53: Exploiting heterogeneity to develop better treatment strategies using an evolutionary multiscale mathematical model of tumor-vessel interactions
Authors: Robertson-Tessi Mark , Estrella Veronica C. , Chen Tingan , Lloyd Mark C. , Gillies Robert J. , Gatenby Robert A. , Anderson Alexander R A
Published: June 2012

Tumor heterogeneity in cancer is an observed fact, both genetically and phenotypically. Cell-cell variation is seen in almost all aspects of cancer from early development all the way through to invasion and subsequent metastasis. The tumor microenvironment is also very heterogeneous and dynamic with multiple zones of hypoxia, normoxia, acidosis and necrosis. Central to our understanding of this heterogeneity is how the tumor cells interact with each other and with their microenvironment. Since the microenvironment is modulated by the tumor itself, as well as the surrounding stroma and the vasculature supply, these interactions rapidly become complex. In order to better understand these complex interactions, we have developed an integrated approach utilizing a hybrid multiscale mathematical model in parallel with an experimental murine window chamber model. This study aims to understand the development of the vasculature in response to a growing tumor, the effects of a heterogeneous microenvironment, and how interactions between the tumor and its vasculature modulate tumor progression and treatment resistance. By incorporating phenotypic heterogeneity into the model, a range of tumor phenotypes emerges due to the heterogeneous microenvironment. The response of the tumor to a treatment will depend on the distribution of phenotypes as well as the local conditions. At the same time, treatment will alter the phenotypic distribution and the microenvironment, so that subsequent tumor progression will be profoundly different than it was prior to treatment. By using the spatial and temporal projections of the mathematical model, optimal treatment strategies are derived and classified based on tumor and microenvironmental parameters. One particularly intriguing result is that tumor control, as opposed to eradication, may be more readily achieved by modulation of the tumor heterogeneity via treatment-mediated microenvironmental selection.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2011-53
Abstract 5306: 18F-EF5 microPET imaging of treatment response from a novel, hypoxia-selective cytotoxin SN30000 in a human lung cancer xenograft model
Authors: Chitneni Satish K. , Bida Gerald T. , Hay Michael P. , Zalutsky Michael R. , Melcher Thorsten , Wilson William R. , Dewhirst Mark W.
Published: June 2012

Introduction: SN30000 (CEN-209) is a structural analogue of the clinically tested hypoxic cell cytotoxin tirapazamine, with improved tumor penetration and increased hypoxic cell kill activity in vitro and in vivo. In this study, we investigated the use of the hypoxia PET imaging agent 18F-EF5 to measure changes in tumor hypoxia before and after treatment with SN30000 in a rat model of human non-small cell lung cancer (H460).

Methods: Xenografts were established by subcutaneous injection of about 5 million H460 cells into the lower hind limb of NIH-RNu nude rats, and after 12-13 days the tumors were used for imaging experiments. Using two groups of animals we studied the changes in hypoxia at early time point (5-6 h) and at about 24 h after intraperitoneal administration of a single dose SN30000 (90 mg/kg body weight) or saline. Each group consisted of two control tumors (saline) and three SN30000 treated tumors. In both pre- and post-treatment scans, animals were scanned for 15-20 min at 2.5 h after i.v. injection 0.7-1.4 mCi of 18F-EF5. After reconstruction, PET images were analyzed by drawing regions of interest (ROI) covering whole tumor tissue and on contralateral leg muscle as background ROI. Standard uptake values (SUV) were calculated from the radioactivity concentration in both tumor and muscle tissue after normalization to the injection dose and body weight of the animal.

Results: All tumors (n=10) showed tumor-to-muscle ratios (2.2 to 3.1) high enough to delineate the tumor from background activity. The SUV values in pre-treatment scans ranged from 0.43 to 0.79. No change in 18F-EF5 uptake was observed when animals were imaged at 5-6 h post-SN30000 therapy (mean SUV 0.59 ± 0.18 vs 0.59 ± 0.08 at baseline). However, tumors when imaged at 24 h after the treatment showed a decrease in tumor accumulation of 18F-EF5 by 19.7% to 54.6% when compared to pre-treatment scans (SUV 0.39 ± 0.12 vs. 0.63 ± 0.09 at baseline, p=0.06). Three out of four control tumors showed a slight increase in SUV in post-treatment scan compared to pre-treatment scan.

Conclusions: The results from this study suggest that SN30000 may reduce hypoxia as measured by 18F-EF5 PET imaging at a later time point (1 day) rather than a few hours after treatment, and suggest potential use of 2-nitroimidazole-based PET imaging as an early response biomarker during clinical development of this compound.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5306. doi:10.1158/1538-7445.AM2011-5306
Abstract 5309: Antivascular activities of novel tubulin binding agent BPR0L075
Authors: Liu Xinli , Liu Li , Beck Haley , Hsieh Hsing-Pang , Boreddy Srinivas , Srivastava Sanjay , Mason Ralph P.
Published: June 2012

BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole, is a novel tubulin binding agent that inhibits tubulin polymerization through binding to the colchicine binding site. We evaluated the antiangiogenic and antivascular properties of BPR0L075 in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were treated with BPR0L075, and effects on endothelial cell morphology, migration, and inhibition of tube formation on extracellular matrix were determined in vitro. The BPR0L075 induced inhibition of neovasculature formation was evaluated by rat aortic ring ex vivo assay. Using a validated dynamic bioluminescence imaging method, we assessed acute vascular disrupting effects of BPR0L075 on luciferase-expressing MCF7 human breast mammary fat pad xenografts. The disruption of tumor vascular perfusion after BPR0L075 treatment was confirmed using the perfusion marker Hoechst 33342 and CD31 staining by fluorescence microscopy. Exposure of BPR0L075 (10 nM) inhibits the endothelial cell migration (within 24 hr) and tube formation on Matrigel (3-6 hr). BPR0L075 treatment also blocked angiogenesis in rat aortic ring assay. A single dose of BPR0L075 induced rapid temporary tumor vascular shutdown evidenced by rapid and reproducible decrease of light emission from luciferase-expressing breast tumors. Histological staining using Hoechst dye and anti-CD31 antibody revealed a time-dependent reduction of tumor perfusion after 2, 4, and 24h of BPR0L075 treatment. Taken together, our results show that the novel tubulin-binding agent BPR0L075 alters endothelial cell tubule formation, prevents cell migration, and inhibits the formation of neovessels. These properties of BPR0L075 may ultimately contribute to the loss of tumor blood vessel integrity causing rapid tumor vascular shutdown in experimental breast tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5309. doi:10.1158/1538-7445.AM2011-5309
Abstract 5313: Real-time video imaging of protease expression in vivo
Authors: Lee Seulki , Zhu Lei , Mansfield James R. , Chen Xiaoyuan
Published: June 2012

Most new drug candidates generated during in vitro screening turn out to be invalid after time-consuming and costly testing in animal models. Therefore, there is an urgent need for development of noninvasive, real-time, sensitive, and cost-effective tools with high throughput for monitoring and early detection of drug efficacy in vivo.

Proteases are known as exceptionally critical signaling proteins that are involved in numerous processes such as cancers. Considerable efforts have been made to identify the role of certain proteases in given biological processes and to screen specific molecules that can regulate protease expression. Most experimental methods are based on the use of protease reporters or molecular beacons. Moreover, they are limited to in vitro applications.

To date, protease-activatable probes have already emerged as powerful tools for in vivo imaging because of their potential to offer high specificity and lower background. However, current activatable probes strive to image protease activity and effects in real-time. Although major improvements have been made, such as the conjugation of macromolecules to improve in vivo stability, these probes still suffer in sensitivity and specificity. In addition, conventional protease activatable probes typically take about 24 hours to activate fully, impeding real-time imaging and high throughput in in vivo applications. Here, we report a simple methodology to improve real-time imaging of protease activity by in vivo activatable probes by balancing competing needs for stability and sensitivity in applications where there are high target-to-background signal ratios.

We demonstrate the first true real-time in vivo video imaging of extracellular protease expression using an ultrafast-acting and extended-use activatable probe. This simple, one-step technique is capable of boosting fluorescent signals upon target protease cleavage as early as 30 minutes from injection in a small animal model and is able to sustain the strong fluorescent signal up to 24 hours. Using this method, we video imaged the expression and inhibition of matrix metalloproteinases (MMPs) in a MMPs-positive SCC-7 tumor-bearing mouse model. The current platform can be universally applied to any target protease of interest with a known peptide substrate and is adaptable to a wide range of real-time imaging applications with high throughputs such as for in vivo drug screening, examinations of the therapeutic efficacy of drugs, and monitoring of disease onset and development in animal models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5313. doi:10.1158/1538-7445.AM2011-5313