bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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<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
Abstract 4816: Development of an integrated analysis platform of circulating melanoma cells for PD-L1 expression as a predictive biomarker
Authors: Gibbs Benjamin K. , Strotman Lindsay N. , Thiede Stephanie M. , Sperger Jamie M. , Casavant Benjamin P. , Berry Scott M. , Beebe David J. , Lang Joshua M.
Published: May 2015

Novel therapies that target the PD-1/PD-L1 axis have shown great potential to improve survival for patients with advanced melanoma. However, there are limited predictive biomarkers to identify patients who may respond to this class of agents. Previous studies suggest that a subset of responders to PD-1/PD-L1 targeting therapies have expression of PD-L1 in tumor biopsies. The development of these predictive biomarkers is complicated by intratumoral heterogeneity across primary and metastatic lesions for PD-L1 expression. Circulating tumor cells (CTCs) are one potential source of tumor samples for serial analysis. An integrated CTC capture and analysis technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform permits evaluation of CTCs at the protein, DNA and mRNA level for biomarkers of both therapeutic response and resistance. Using this platform, we have developed a method to isolate circulating tumor cells from patients with advanced melanoma. Using a cocktail of capture monoclonal antibodies including targets of CD146, GD2 and NG2, we have recovered cells across multiple cell lines with high efficiency. Using single antibody capture of SK-Mel-28 cells, capture was 52%, 54%, and 45% with CD146, GD2 and NG2 targets respectively. However, when using a cocktail of capture antibodies containing CD146, GD2 and NG2, capture increased to 92%. Similar results were obtained with other melanoma cell lines including, 624 and M21 cell lines. Cells were also stained for PD-L1 as a potential predictive biomarker of response to PD-L1 and PD-1 targeting immunotherapies. We are now collecting patient samples with this capture platform to identify CTCs expressing PD-L1 as a potential predictive biomarker to PD-1/PD-L1 targeting therapies.

Citation Format: Benjamin K. Gibbs, Lindsay N. Strotman, Stephanie M. Thiede, Jamie M. Sperger, Benjamin P. Casavant, Scott M. Berry, David J. Beebe, Joshua M. Lang. Development of an integrated analysis platform of circulating melanoma cells for PD-L1 expression as a predictive 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 4816. doi:10.1158/1538-7445.AM2014-4816
Abstract 4824: Evaluation of CTCs enriched with a flexible microspring array device as a prognostic biomarker in lung adenocarcinoma
Authors: Harouaka Ramdane , Liu Xin , Khan Waleed , Khan Tasleema , Asiaii Ayeh , Christ Christine , Miller Rebecca , Rice Shawn , Belani Chandra , Zheng Siyang
Published: May 2015

The dissemination of circulating tumor cells (CTCs) implicated in the metastatic spread of cancer accounts for the majority of cancer-related deaths. CTCs have been established as a prognostic biomarker and are associated with worse survival outcomes in various cancer types. However, the implication of CTCs as a prognostic marker in non-small cell lung cancer (NSCLC) has not yet been established. We have employed a flexible micro spring array (FMSA) device for label-free mechanical enrichment of CTCs from whole blood samples obtained from patients with advanced and metastatic lung adenocarcinoma. CTCs were enriched and analyzed qualitatively and quantitatively based on immunocytochemical determination of phenotype and morphological characteristics. Follow up samples were analyzed during the course of therapy and CTC results were correlated to patient survival and tumor burden. FMSA enriched CTCs obtained from a blood sample may prove to be a prognostic biomarker for advanced lung cancer.

Citation Format: Ramdane Harouaka, Xin Liu, Waleed Khan, Tasleema Khan, Ayeh Asiaii, Christine Christ, Rebecca Miller, Shawn Rice, Chandra Belani, Siyang Zheng. Evaluation of CTCs enriched with a flexible microspring array device as a prognostic biomarker in lung adenocarcinoma. [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 4824. doi:10.1158/1538-7445.AM2014-4824
Abstract 4826: Mobilization of tumor cells into the circulation during radiation therapy for lung cancer
Authors: MacManus Michael , Anderson Robin , Russell Prudence , Cox Ashley , Swierczak Agnieszka , Doherty Judy P. , Jacobs Daphne , Smith Jai , Siva Shankar , Daly Patricia E. , Ball David L. , Martin Roger F. , Martin Olga A.
Published: May 2015

Thousands of patients die annually with distant metastasis after curative-intent or “radical” radiation therapy (RT). Because non-small cell lung cancer (NSCLC), the most common cause of cancer-related mortality, exhibits an especially high rate of distant metastasis after radical RT or chemo-RT for locoregionally-advanced disease, it represents a suitable model to investigate the relationship between RT and metastasis. We hypothesized that disruption of tumour architecture during RT could result in the release of viable tumor cells into the peripheral circulation.

We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated sorting (FACS) or by immunofluorescence microscopy. Twenty seven RT-treated NSCLC patients had blood samples analyzed by one or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. CTCs were also identified, singly and in clumps in large numbers during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied), and first experiments revealed that these cells survive in bloodstream of NOD/SCID mice for several months. CTCs formed γ-H2AX foci in response to ex vivo irradiation providing further evidence of their viability.

Although of concern, these findings could represent an opportunity to monitor and target CTCs during RT. They provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by co-administering systemic therapies.

Citation Format: Michael MacManus, Robin Anderson, Prudence Russell, Ashley Cox, Agnieszka Swierczak, Judy P. Doherty, Daphne Jacobs, Jai Smith, Shankar Siva, Patricia E. Daly, David L. Ball, Roger F. Martin, Olga A. Martin. Mobilization of tumor cells into the circulation during radiation therapy for lung 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 4826. doi:10.1158/1538-7445.AM2014-4826
Abstract 4827: Investigation of the survival of circulating breast tumor cells using biomimetic microfluidic circulation system
Authors: Wang Yang
Published: May 2015

Distant metastases are the cause of about 90% of breast cancer patient death and the overall survival rate for metastatic breast cancer is only about 16%. Circulating tumor cells (CTCs) shed from the primary tumor, travel through the blood to distant anatomic sites and cause metastasis. The survival of CTCs in blood vascular system is a challenging pre-requisite of successful metastasis, as the biochemical and mechanical environment of circulation is vastly different from the primary tumor sites. Studies related to the fate of CTCs in circulation are mainly limited by lack of well-controlled and realistic experimental models.

We have developed a microfluidic device that mimics the human blood circulatory system in order to study the mechanism underlying the survival of cancer cells in the blood circulation. The circulation device integrates a network of channels with a self-contained micropump can generate pulsatile fluid flow pattern and corresponding fluidic forces, which can represent the nature of blood flow and haemodynamic forces in the vasculature.

The survival rate of breast cancer cell line MCF7 in circulation was measured via this microfluidic platform. MCF-7 cells in circulation device had a much lower survival rate compared with those in control device. The survival rate of MCF-7 cells after circulating 1, 2 and 4 hours are respectively 48.8%, 36.3% and 10.7%, while the survival rate in control device after 1, 2 and 4 hours are 93.7%, 77.6% and 94.30%. We further compared the survival rate of MCF-7 cells with SUM-159 cells. We hypothesize that by assessing the differential survival ability of breast cancer cells with varied EMT traits can reveal the key signatures of the metastatic tumor cells.

Citation Format: Yang Wang. Investigation of the survival of circulating breast tumor cells using biomimetic microfluidic circulation system. [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 4827. doi:10.1158/1538-7445.AM2014-4827
Abstract 4831: Breast cancer cells metastasize to bone through E-selectin+ vascular gateways
Authors: Price Trevor , Burness Monika , Sivan Ayelet , Cheng Renee , Magnani John L. , Sipkins Dorothy A.
Published: May 2015

The bone marrow (BM) has been suggested to serve as a protective environment for disseminated breast cancer cells (BCCs). The precise molecular signals that circulating BCCs use to identify and adhere to BM vasculature are unknown, yet these interactions are essential for the development of bone metastases. To address this question, we used in vivo fluorescence microscopy to track the interactions of estrogen receptor (ER)+ and ER- BCC lines with bone in real time in an intracardiac injection xenograft mouse model.  Our data show that circulating tumor cells enter the bone through unique vascular niches where endothelial cell expression of the adhesive molecule E-selectin and of the chemokine SDF-1 are both upregulated. These vascular niches are the same sinusoidal capillary beds that serve as the site of entry for peripherally circulating hematopoietic stem and progenitor cells (HSPCs) returning to the BM. Similar to HSPCs, BCCs aberrantly express the major cell surface molecules known to regulate hematopoietic cell entry and exit through the BM, including CXCR4 (SDF-1 receptor) and multiple E-selectin ligands. While neither CXCR4 blockade nor general chemokine signaling inhibition in BCCs prevented BM dissemination, E-selectin blockade by the specific glycomimetic antagonist GMI-1271 decreased BCC entry in the BM by approximately three fold at 2 and 24 hours after treatment. Moreover, in studies comparing FACS isolated stem and non-stem MCF-7 BCCs, GMI-1271 inhibition of E-selectin specifically targeted CD44+CD24- breast cancer stem cell homing. Non-stem CD44+CD24+ BCCs demonstrated significantly less BM homing compared to stem BCCs, and their BM trafficking was less E-selectin dependent. These data reveal for the first time a molecular mediator of bone metastases at the point-of-entry for BCCs in the BM. In addition, the ability of BCCs to hijack HSPC trafficking pathways to metastasize to bone correlates with E-selectin ligand expression and may be a specific characteristic of the BCC stem cell subpopulation.

Citation Format: Trevor Price, Monika Burness, Ayelet Sivan, Renee Cheng, John L. Magnani, Dorothy A. Sipkins. Breast cancer cells metastasize to bone through E-selectin+ vascular gateways. [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 4831. doi:10.1158/1538-7445.AM2014-4831
Abstract 4840: DNA methyltransferase 3B as a mediator in cancer associated inflammation and progression
Authors: Skrypek Nicolas G. , Achyut B R. , Hollander Christine , Yang Li
Published: May 2015

For a long time ignored, the tumor microenvironment is now known as an important contributor in tumor progression and metastasis. However, the molecular mechanisms as how cancer-associated inflammation promotes metastasis remain to be investigated. Previous work in our lab showed an inflammatory and immune suppressive microenvironment in the pre-metastatic lung prior to tumor cell arrival. Recently, we found that inflammation is responsible for down-regulation of cyclin-dependent kinase inhibitors p15, p16, and p21 in a mouse model of invasive squamous cell carcinoma, in which gene encoding TGFb receptor 2 (Tgfbr2) was deleted in the stromal fibroblasts (Tgfbr2fspKO). There was an induction of inflammation and significant DNA damage in the neighboring epithelia of the forestomach due to the stromal deletion of Tgfbr2. We found that an up-regulation of DNA methyltransferase 3B (DNMT3B) was responsible for these epigenetic alterations. Based on these studies, we hypothesize that the cancer-associated inflammation could induce DNMTs activity leading to the repression of genes important for inhibition of tumor progression. We are currently investigating the impact of inflammation on DNMTs expression using 4T1 mammary tumor models. We hope that our studies will provide insight for molecular mechanisms underlying tumor-host interaction in the metastatic process and identify the novel therapeutic targets.

Citation Format: Nicolas G. Skrypek, B R. Achyut, Christine Hollander, Li Yang. DNA methyltransferase 3B as a mediator in cancer associated inflammation and progression. [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 4840. doi:10.1158/1538-7445.AM2014-4840
Abstract 4856: Survivin packaging into exosomes is lipid raft-dependent
Authors: Valenzuela Malyn May Asuncion , Bennit Heather R. Ferguson , Osterman Carlos Joel Diaz , Khan Salma , Casiano Carlos , Wall Nathan R.
Published: May 2015

We have shown that Survivin is exported out of the cancer cell via exosomes despite its lack of a secretory signaling peptide. However, the mechanism of Survivin exosomal loading is not yet known. Exosomal protein sorting can be endosomal sorting complex required for transport (ESCRT)-dependent or ESCRT-independent. ESCRT dependent protein sorting requires monoubiquitination of the target protein, which serves as an exosome sorting signal. The ESCRT independent mechanism is through preferential aggregation of the proteins which is based on intrinsic physical properties to lipid-rafts present in exosomes. Our lab has recently shown that exosomal Survivin is associated with both Hsp70 and Hsp90, as well as PRDX1 which have also been shown associated with detergent-resistant lipid rafts. Here, we determine whether Survivin is lipid-raft dependent and whether Hsp70 and Hsp90 and/or PRDX1 are required for exosomal Survivin loading. Exosomes were collected from conditioned media taken from HeLaS cells and isolated by centrifugation. Colocalizations of Survivin with Hsp70, Hsp90 and PRDX1 in the cell and exosomes were detected by immnocytochemistry visualized by confocal microscopy and immunoprecipitation. HeLaS cells were treated with increasing concentrations of methyl-β-cyclodextrin to disrupt lipid rafts as a way to determine protein lipid raft association. Hsp70, Hsp90 and PRDX1 knock-down using siRNAs will be accomplished to verify the requirement of these proteins for Survivin exosome loading and lipid-raft dependents. Here we show that Survivin colocalizes with Hsp70, Hsp90 and PRDX1 in HeLaS cells as well as their exosomes. Increasing concentrations of methyl-β-cyclodextrin disrupts lipid rafts in the HeLaS cells and releases exosomes which also subsequently contain decreasing PRDX1 and Survivin protein amounts. The release of Survivin into the tumor microenvironment is an important finding as this IAP may contribute to the aggressiveness of the cancer as well as the development of chemoresistance. Despite lacking a secretory signaling peptide, Survivin is being released into the extracellular space via exosomes using chaperones. Knowing how Survivin is exosomally packaged may prove useful in selecting patient specific treatment regimens.

Citation Format: Malyn May Asuncion Valenzuela, Heather R. Ferguson Bennit, Carlos Joel Diaz Osterman, Salma Khan, Carlos Casiano, Nathan R. Wall. Survivin packaging into exosomes is lipid raft-dependent. [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 4856. doi:10.1158/1538-7445.AM2014-4856
Abstract 4857: Clonal expansion of Lkb1-deficient stromal cells underlies polyp development in mouse models of Peutz-Jeghers syndrome
Authors: Ollila Saara , Laajanen Kaisa , Wong Iris , Vaahtomeri Kari , Mäkelä Tomi P.
Published: May 2015

While the role of LKB1 mutations in the Peutz-Jeghers polyposis syndrome (PJS) is uncontroversial, the originating cell type remains unclear as Lkb1 mutations in both epithelial cells and stromal smooth muscle cells (SMCs) have been proposed as tumor drivers. Since SMCs do not represent a major fraction of stromal cells in polyps, altered signaling from Lkb1-deficient SMCs to epithelium has been suggested as a possible mechanism for polyposis. Here we investigate the cell type origin and tumor development mechanism of PJS type polyps in mice by targeting Lkb1 in either early mesenchymal progenitors (Twist2-Cre) or in stromal fibroblasts (Fsp1-Cre). Remarkably, both Twist2 (Twist2-Cre;Lkb1flox/+) and Fsp1-driven (Fsp1-Cre;Lkb1flox/+ and Fsp1-Cre;Lkb1flox/flox) Lkb1 deletion led to formation of PJS-type gastrointestinal polyps predominantly in the stomach, as noted before for Lkb1+/- mice. Furthermore, lineage-tracing experiments demonstrated that Lkb1 deletion results in early local expansion of Lkb1-deficient myofibroblast-like alpha smooth muscle actin-expressing cells between gastric glands, and subsequent clonal expansion filling the stroma of the forming polyps. The simultaneous expansion of the adjacent epithelium seems to be secondary to the stromal growth, based on lack of noticeable genetic alterations in the epithelial cells. Immunohistochemical and mRNA analyses demonstrate that the polyps arising from the stromal deletion models are indistinguishable from Lkb1+/- mice and PJS patient polyps. These results indicate that polyps in the Twist2-Cre and Fsp1-Cre mice represent stromal tumors, and suggest that the identified tumorigenic mechanism is shared in PJS syndrome.

Citation Format: Saara Ollila, Kaisa Laajanen, Iris Wong, Kari Vaahtomeri, Tomi P. Mäkelä. Clonal expansion of Lkb1-deficient stromal cells underlies polyp development in mouse models of Peutz-Jeghers syndrome. [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 4857. doi:10.1158/1538-7445.AM2014-4857
Abstract 4873: IKKα bridges central tolerance to innate immunity and inflammation
Authors: Zhu Feng , Chen Zhisong , Willette-Brown Jami , Lomada Dakshayani , Davis Sean R. , Back Timothy , Yoshimura Teizo , Sun Zhonghe , Wu Xiaolin , Wiltrout Robert , Richie Ellen , Siebenlist Ulrich , Trichieri Giorgio , Hu Yinling
Published: May 2015

IKKα has emerged as a tumor suppressor in squamous cell carcinomas (SCCs) of skin, head and neck, and lung. Our recent studies indicate that inflammation is involved in lung SCCs in kinase-dead knock-in IkkαKA/KA mice. However, how IKKα prevents inflammation has not been fully understood yet. In the present studies, we showed that IkkαKA/KA mice developed reduced thymic medullary regions, systemic inflammation, and severe skin damage, indicating that IKKα plays an essential role linking central tolerance to innate immunity and inflammation. In line with previous studies of the role of NF-κB pathway in thymic medulla formation and the establishment of central tolerance, we found that NF-κB pathway was inactivated in the presence of kinase inactive form of IKKα in thymic epithelial cells. Depleting the lymphocytes, thymus, T cells, macrophages, and/or reintroducing transgenic IKKα into the skin or thymus rescued the severe skin phenotypes. Transferring IkkαKA/KA T cells, but not wild-type T cells, reconstituted the severe skin phenotypes and SCCs in the lungs and forestomach of IkkαKA/KA/Rag-/- mice. We found that the self-reactive T cells provided TGFβ signaling, which enhanced the expression of monocyte chemoattractant protein-1 (MCP-1) in keratinocytes. As a result, increased level of MCP-1 led to robust recruitment of macrophages to the skin, resulting in skin inflammation and hyperproliferation. This study reveals an important IKKα/TGFβ/MCP-1 signaling axis that orchestrates central tolerance and inflammation in the maintenance of tissue homeostasis.

Citation Format: Feng Zhu, Zhisong Chen, Jami Willette-Brown, Dakshayani Lomada, Sean R. Davis, Timothy Back, Teizo Yoshimura, Zhonghe Sun, Xiaolin Wu, Robert Wiltrout, Ellen Richie, Ulrich Siebenlist, Giorgio Trichieri, Yinling Hu. IKKα bridges central tolerance to innate immunity and inflammation. [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 4873. doi:10.1158/1538-7445.AM2014-4873
Abstract 4876: Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models
Authors: Nygaard Vigdis , Prasmickaite Lina , Vasiliauskaite Kotryna , Clancy Trevor , Hovig Eivind
Published: May 2015

The brain offers a unique microenvironment to which malignant melanoma frequently spreads leading to dismal prognosis for the patients. The molecular determinants that promote melanoma brain metastases however, remain largely unknown. Here, we show an experimental strategy utilizing two species of immune-compromised animals in a metastasis model to generate in vivo-cultivated metastatic tissues along with their corresponding host tissues in order to identify molecular events associated with brain metastases. The study was based on the melanoma cell lines, Melmet 1 and Melmet 5 with opposing phenotypes (invasive/proliferative, respectively) in the in vitro setting and distinct metastatic patterns in vivo, where Melmet 1 showed a preference to the brain. Transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, revealed the opportunistic nature of melanoma in this context. We detected transitioning from the invasive phenotype to the proliferative phenotype, as the brain metastases passed from early to late growth phase. Of significance, we identified a brain-adaptive phenotype with synaptic characteristics and where glutamate signaling and Ca2+-dependent effectors played a central role. The brain-adaptive phenotype was more prominent in the earlier metastatic growth phases compared to a later phase. Analysis of host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells. Altogether, this suggests that acquisition of the brain-adaptive phenotype in a permissive environment might be critical for the establishment of metastatic growth in the brain microenvironment and further, targeting glutamate signaling may represent a therapeutic approach in brain metastasis. Global network modeling and functional annotation analysis revealed that several of the central molecular factors and pathways mediating melanoma brain metastasis in this study, are also central to other pathological diseases, in particular neurodegenerative diseases. The identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

Citation Format: Vigdis Nygaard, Lina Prasmickaite, Kotryna Vasiliauskaite, Trevor Clancy, Eivind Hovig. Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models. [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 4876. doi:10.1158/1538-7445.AM2014-4876