bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 479: A novel dynamic delivery system enabling high efficiency transfection of cells
Authors: Ludtke James , Lauer Anthony , Storck Austin , Rossi Nicholas , Neder Karen , Pinchuk Anatoly , Juckem Laura 
Published: May 2015

Abstract
Transfection is an enabling technique for cellular and molecular biologists to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity in some cell types. To address these shortcomings, Mirus Bio has developed a novel non-liposomal technology called the TransIT-X2™ Dynamic Delivery System. This unique polymeric delivery system is able to effectively condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm.
In this study, we demonstrate functional transfection of labeled plasmid DNA co-delivered with labeled siRNA illustrating the dual abilities of the TransIT-X2 Dynamic Delivery System. Additionally, extensive head-to-head comparisons with a popular liposomal reagent, Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT-X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2-fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT-PCR. miRNA function was assessed by the decrease in the miR-1 target mRNA PTK9. The TransIT-X2 Dynamic Delivery System shows effective knockdown via siRNA or miRNA.
The TransIT-X2 Dynamic Delivery System offers researchers exceptional broad-spectrum transfection of plasmid DNA and siRNA. This cutting edge, non-liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies.
Citation Format: James Ludtke, Anthony Lauer, Austin Storck, Nicholas Rossi, Karen Neder, Anatoly Pinchuk, Laura Juckem. A novel dynamic delivery system enabling high efficiency transfection of cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2014-479
Abstract 4796: Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells
Authors: Lu Lin , Tao Huimin , Xia Yang , Owen John , Moyer Jeffrey S. , Prince Mark E.P , Chang Alfred E. , Wicha Max S. , Li Qiao 
Published: May 2015

Abstract
The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DC). Using mouse models we demonstrate that DC pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (CSC-DC) significantly reduced development of pulmonary metastases and prolonged survival. The effect was associated with down regulation of CCR7 and CCR10 in tumor cells and decreased CCL27 and CCL28 expression in lung tissue. CSC-DC vaccine significantly reduced ALDHhigh CSCs in the primary tumors. Direct targeting of CSCs was demonstrated by specific binding of IgG produced by ALDHhigh CSC-DC vaccine-primed B cells to ALDHhigh CSCs, resulting in lysis of these target cells in the presence of complement. When administered in the adjuvant setting after localized radiation therapy of established tumors, CSC-DC vaccine reduced development of local tumor growth as well as systemic disease and prolonged survival demonstrating the efficacy of this approach. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after conventional treatment of cancers.
Citation Format: Lin Lu, Huimin Tao, Yang Xia, John Owen, Jeffrey S. Moyer, Mark E.P Prince, Alfred E. Chang, Max S. Wicha, Qiao Li. Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4796. doi:10.1158/1538-7445.AM2014-4796
Abstract 48: A novel method for live cell sorting based on RNA expression levels of cancer biomarkers maintains normal cellular function as confirmed by downstream functional testing
Authors: Weldon Don , Koong Victor , Williams Yuko 
Published: May 2015

Abstract
Enrichment of cells within a mixed population has been confined to fixed cells or in the case of live cell sorting to a limited set of cell surface markers which are typically not specific to the target population. Furthermore, cell surface antibodies may activate cell surface receptors altering the state of the cells such that they are no longer representative of the original population. Intracellular staining requires fixation and permeabilization and thus renders the cells unusable in downstream functional assays.
Through the use of a unique nanoparticle sequence based detection technology we have shown the ability to detect specific RNA within live cells which does not affect or alter the cells and allows further testing of the target population following the enrichment. Both microRNA and mRNA species have been used to enrich for specific target populations through Fluorescence Activated Cell Sorting (FACS). Sorting cells based on the expression level of any specific RNA allows researchers the ability to utilize targets of biological interest within isolated populations of cells. Since this method is non-destructive to live cells the enriched cells can then be used for downstream functional assays.
Here we present data showing the live cell enrichment of a target cell population within a heterogeneous mixture of cells based on their RNA expression levels. RNA probes to miR-221 and miR-222 were generated for the enrichment of invasive cells from the non-invasive. A mixture of breast cancer cells (MD-MB-231 and T47D) were sorted based on their expression levels of the RNA probes through FACS. The high and low expressing sort products were then each subjected to a collagen invasion assay. As expected the fractions which showed high expression of miR-221 and miR-222 showed a high level of invasion through the matrix while the low expressing fractions showed extremely low invasion.
Live cell sorting based on RNA levels of cancer biomarkers in a non-destructive manner enables researchers the ability to study the expression of a diverse group of targets beyond the traditional surface markers with the added benefit of utilizing the enriched cells for downstream functional assays.
Citation Format: Don Weldon, Victor Koong, Yuko Williams. A novel method for live cell sorting based on RNA expression levels of cancer biomarkers maintains normal cellular function as confirmed by downstream functional testing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 48. doi:10.1158/1538-7445.AM2014-48
Abstract 4802: Human breast carcinoma-associated mesenchymal stem cells from primary breast cancer metastasis promote proliferation, invasion and mammosphere formation in breast cancer
Authors: Gonzalez Maria E. , Toy Kathy A. , Kleer Celina G. 
Published: May 2015

Abstract
Background: Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment; however controversy exists regarding their role in solid tumors. In this study, we identified the presence of carcinoma-associated MSCs (CA-MSCs) isolated from breast cancer metastasis to a human lymph node (LNM) and liver (LM).
Methods: Tissue harvesting and culture: CA-MSCs were isolated from fresh samples of lymph nodes and liver breast cancer metastasis obtained from the operating room. A portion of each specimen was mechanically dissected and digested with collagenase. Red cells were lysed with ACK buffer and cell suspensions were cultured in Human Epithelial Medium and isolated adherent CA-MSCs were subculture in MSC medium for up to 12 passages for each experiment. Another portion of the tumor samples were subjected to IHC for H&E staining.
Differentiation assays: For bone differentiation cells were plated in StemPro Osteogenesis differentiation or control medium and allowed to grow for 14 days, and then washed and stained with Alizarin Red S.
For cartilage differentiation cells were pelleted and grown in 15 ml tubes for 21 days, immobilized in histogel and stained with Alizarin Blue.
For adipose differentiation cells were plated in StemCell differentiation and control medium, after 21 days the cells were stained with 0.3% Oil Red O.
Proliferation, invasion, mammosphere assays, cytokine array and RT-PCR for EMT signature were performed seeding the same number of cells and comparing the single culture of CA-MSCs with co-culture of CA-MSCs and a panel of breast cancer cells lines
Results: CA-MSCs had spindle morphology, normal karyotype, were nontumorigenic, and possessed tri-lineage differentiation ability (osteoblast, adipocyte, and chondrocyte). When co-cultured with breast cancer cells, CA-MSCs promoted proliferation, invasion, and mammosphere formation, increased IL6 and IL8 secretion into the media, and induced a gene expression profile of EMT when compared to the single cultures.
Conclusion: We successfully isolated and characterized CA-MSCs, confirming their presence in human breast metastasis. We found that they provide a favorable microenvironment for tumor cell growth, enhance mammosphere formation, and promote an EMT signature and invasion in breast cancer cells.
Citation Format: Maria E. Gonzalez, Kathy A. Toy, Celina G. Kleer. Human breast carcinoma-associated mesenchymal stem cells from primary breast cancer metastasis promote proliferation, invasion and mammosphere formation in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4802. doi:10.1158/1538-7445.AM2014-4802
Abstract 4810: Identification of molecular markers of pathological vascular subtypes with differential sensitivity to therapies targeting the VEGF pathway
Authors: Falcon Beverly L. , O'Mahony Marguerita , Stewart Julie , Liu Jiangang , Nagy Janice A. , Xue Qi , Pan Yong , Jeffries Cynthia , Hatten Emma , Holzer Tim R. , Dvorak Harold F. , Nasir Aejaz , Pytowski Bronek , Benjamin Laura , Uhlik Mark T. 
Published: May 2015

Abstract
Therapeutic targeting of the VEGF pathway has not matched the efficacy suggested by preclinical tumor models. To date, the limitations of this form of therapy stem either from inherent or acquired resistance of the tumor vasculature to the reduction of VEGF signaling. Previous studies have described morphological phenotypes of different blood vessel types in human and mouse tumors. Recapitulation of these vascular phenotypes with a surrogate Ad-VEGFA164 model has shown differential sensitivity of these vascular subclasses to inhibitors of the VEGF pathway. The goal of the current study was to identify molecular markers for the various subtypes of pathological vessels and to assess whether similar molecular signatures are found in blood vessels in mouse tumor models and human disease. Using the Ad-VEGF-A164 flank model, microarray analysis was performed to identify molecular markers at each time-point, representing distinct stages of vessel development and maturation. We identified unique gene signatures at the various time-points and confirmed differential expression of the genes by qRT-PCR and/or immunohistochemistry. We found that vascular markers such as CD31, Ang2, and Tie1 were pan-endothelial markers at all time-points. However, other markers such as CD34, MECA-32, vWF, SMA, Tie2, CD105, and AQP1 were expressed on subclasses of endothelial cells. Expression of the molecular markers in tumors models revealed different subclasses of tumor vessels in patient-derived xenografts which were sensitive or resistant to anti-VEGF-A or anti-VEGFR-2 treatment. These vascular subtypes were also found in tissues from human breast and gastric cancers. Together these results indicate that multiple molecular markers can be used to identify unique pathological subclasses of tumor vessels. Some of these vascular subtypes may be insensitive to inhibitors targeting the VEGF pathways and their signatures may be useful biomarkers to predict vascular sensitivity to anti-angiogenic therapy.
Citation Format: Beverly L. Falcon, Marguerita O'Mahony, Julie Stewart, Jiangang Liu, Janice A. Nagy, Qi Xue, Yong Pan, Cynthia Jeffries, Emma Hatten, Tim R. Holzer, Harold F. Dvorak, Aejaz Nasir, Bronek Pytowski, Laura Benjamin, Mark T. Uhlik. Identification of molecular markers of pathological vascular subtypes with differential sensitivity to therapies targeting the VEGF pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2014-4810
Abstract 4813: DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients
Authors: Chimonidou Maria , Strati Areti , Malamos Nikos , Georgoulias Vassilis , Lianidou Evi 
Published: May 2015

Abstract
Introduction: Blood based specimens such as Circulating Tumor Cells (CTCs) and cell free DNA (cfDNA) provide a very promising and non-invasive liquid biopsy approach. In our study, we first compared the promoter methylation status of BRMS1 and SOX17 genes in DNA samples isolated from primary tumors, matched CTCs and cfDNA isolated from the same breast cancer patients.
Methods: We first developed and validated real time Methylation Specific PCR (MSP) assays for studying BRMS1, CST6 and SOX17 promoter methylation. We further analysed 165 paired DNA samples isolated from: a) the EpCAM positive CTC fraction from 92 operable, 61 metastatic breast cancer patients and 12 healthy individuals, b) the corresponding plasma from the same individuals. All CTC were also checked for CK-19 mRNA expression by RT-qPCR. Additionally, we investigated the promoter methylation status of BRMS1 and SOX17 in corresponding primary tumors (n=75).
Results: In operable breast cancer, comparison of SOX17 promoter methylation status in CTCs and corresponding cfDNA revealed a direct association (P = 0.001), while it was also highly correlated with CK-19 mRNA expression (P=0.006). In patients with verified metastasis, there was a direct connection between SOX17 promoter methylation in CTCs and cfDNA (P=0.046), while SOX17 promoter methylation in cfDNA was highly correlated with CK-19 mRNA expression (P=0.04). Our results are shown below
Conclusion: Our findings indicate a direct connection between SOX17 promoter methylation in CTCs and cfDNA both in patients with operable breast cancer, and verified metastasis.
resultsBreast CancerGenesCTC fractioncfDNAFFPEsOperable (N=92)CST624/92(26.1%)33/92(35.9%)not analyzedSOX1719/92(20.7%)24/92(26.1%)26/43(60.5%)BRMS120/92(21.7%)22/92(23.9%)19/43(44.1%)Verified metastasis (N=61)CST621/61(34.4%)26/61(42.6%)not analyzedSOX1726/61(42.6%)22/61(36.1%)21/32(65.6%)BRMS126/61(42.6%)4/61(6.6%)5/32(15.6%)
Citation Format: Maria Chimonidou, Areti Strati, Nikos Malamos, Vassilis Georgoulias, Evi Lianidou. DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4813. doi:10.1158/1538-7445.AM2014-4813
Abstract 4814: Molecular interrogation of the androgen receptor in circulating tumor cells to identify therapeutic resistance to androgen targeting therapies
Authors: Strotman Lindsay N. , Sperger Jamie , Casavant Benjamin , Theide Stephanie , Beebe David , Lang Joshua 
Published: May 2015

Abstract
There is a critical need to evaluate and target mechanisms of resistance to Androgen Receptor (AR) targeting agents, such as Enzalutamide and Abiraterone Acetate. Preclinical models have identified resistance mechanisms that include AR gene mutations and rearrangements, expression of AR splice variants and AR overexpression. Circulating tumor cells (CTCs) represent a potential source of tumor samples for further molecular interrogation into resistance mechanisms of AR directed therapy. Here we present, an integrated CTC capture and analysis technology known as VERSA (Vertical Exclusion-based Rare Sample Analysis) to allow evaluation of the AR at the protein, DNA and mRNA level. Using the VERSA platform we have captured EpCAM+ CTCs from castrate-resistance prostate cancer patients and quantified nuclear localization of the AR in CTCs as a potential predictive therapeutic protein biomarker. Using quantitative immunocytochemistry, results have shown patients that are responding to various AR-targeting and chemotherapy treatments have lower percentages of AR nuclear localization (38.3±7.4%) as compared to patients that have progressed on AR-targeting therapies (60.4±4.9%). We have also identified significant heterogeneity in both AR localization and total AR protein expression that correlated with treatment history. Furthermore, CTCs have been identified in having 100 % AR nuclear localization in patients undergoing AR targeted therapies, indicating a possible subpopulation of CTCs that exhibit resistance phenotypes. mRNA was also extracted from CTCs of men with CRPC for analysis of AR splice variant expression. Results showed various levels of splice variant expression from men previously on AR targeting therapies. These studies indicate that a comprehensive, real-time analysis of the AR can be serially assessed for early signs of tumor resistance to AR targeting agents.
Citation Format: Lindsay N. Strotman, Jamie Sperger, Benjamin Casavant, Stephanie Theide, David Beebe, Joshua Lang. Molecular interrogation of the androgen receptor in circulating tumor cells to identify therapeutic resistance to androgen targeting therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2014-4814
Abstract 4815: A novel AR splice variant identified in circulating tumor cells from castration-resistant prostate cancer patient blood as a potentially prognostic biomarker
Authors: Kang Zhigang , Yu Yunkai , Cao Liang , Meltzer Paul S. , Zhu Yuelin , Shah Avan , Gulley James , Duht William , Madan Ravi , Bhagat Ali , Zhao Kyra 
Published: May 2015

Abstract
Circulating tumor cells (CTCs) are rare cancer cells circulating in the bloodstream that are thought to have a key role in cancer metastasis. They have been associated with worse prognosis in several major cancer types. There is an intense interest in the molecular analysis of CTC to gain information that would assist treatment decisions and potentially improve the disease outcome. There are enormous interests in the analysis of CTCs for castration-resistant prostate cancer (CRPC) due to the nature of the tumor that tends to metastasize to bone, making them generally inaccessible to obtain biopsies. In this study, we applied a surface marker independent, label-free cell trap microfluidics device for CTCs isolation and retrieval based on cell size and deformability. A total of 47 blood samples drawn from 36 patients with CRPC were preceded for CTC isolation followed by molecular characterization using epithelial tumor markers and prostate cancer specific markers. We identified a novel androgen receptor (AR) splice variant from four patients' CTCs, and confirmed by DNA sequencing that this variant lacks a 554bp region in the ligand binding domain of the receptor in comparison with wild type human AR. Patients with this splice variant were found to be associated with poor survival (p&lt;0.0001). Our study suggests that the presence of the AR splice variant in CTC may serve as a novel prognostic biomarker for CRPC patients.
Note: This abstract was not presented at the meeting.
Citation Format: Zhigang Kang, Yunkai Yu, Liang Cao, Paul S. Meltzer, Yuelin Zhu, Avan Shah, James Gulley, William Duht, Ravi Madan, Ali Bhagat, Kyra Zhao. A novel AR splice variant identified in circulating tumor cells from castration-resistant prostate cancer patient blood as a potentially prognostic biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4815. doi:10.1158/1538-7445.AM2014-4815