Cancer Cells Of Epithelial Origin Research Articles

Synthetic circuits based on split Cas9 to detect cellular events

Synthetic biology involves the engineering of logic circuit gates that process different inputs to produce specific outputs, enabling the creation or control of biological functions. While CRISPR has become the tool of choice in molecular biology due to its RNA-guided targetability to other nucleic acids, it has not been frequently applied to logic gates beyond those controlling the guide RNA (gRNA). In this study, we present an adaptation of split Cas9 to generate logic gates capable of sensing biological events, leveraging a Cas9 reporter (EGxxFP) to detect occurrences such as cancer cell origin, epithelial to mesenchymal transition (EMT), and cell–cell fusion. First, we positioned the complementing halves of split Cas9 under different promoters—one specific to cancer cells of epithelial origin (phCEA) and the other a universal promoter. The use of self-assembling inteins facilitated the reconstitution of the Cas9 halves. Consequently, only cancer cells with an epithelial origin activated the reporter, exhibiting green fluorescence. Subsequently, we explored whether this system could detect biological processes such as epithelial to mesenchymal transition (EMT). To achieve this, we designed a logic gate where one half of Cas9 is expressed under the phCEA, while the other is activated by TWIST1. The results showed that cells undergoing EMT effectively activated the reporter. Next, we combined the two inputs (epithelial origin and EMT) to create a new logic gate, where only cancer epithelial cells undergoing EMT activated the reporter. Lastly, we applied the split-Cas9 logic gate as a sensor of cell–cell fusion, both in induced and naturally occurring scenarios. Each cell type expressed one half of split Cas9, and the induction of fusion resulted in the appearance of multinucleated syncytia and the fluorescent reporter. The simplicity of the split Cas9 system presented here allows for its integration into various cellular processes, not only as a sensor but also as an actuator.

Analysis of Differential Gene Expression and Core Canonical Pathways Involved in the Epithelial to Mesenchymal Transition of Triple Negative Breast Cancer Cells by Ingenuity Pathway Analysis.

Triple Negative Breast Cancer (TNBC) is a malignant form of cancer with very high mortality and morbidity. Epithelial to Mesenchymal Transition (EMT) is the most common pathophysiological change observed in cancer cells of epithelial origin that promotes metastasis, drug resistance and cancer stem cell formation. Since the information regarding differential gene expression in TNBC cells and cell signaling events leading to EMT is limited, this investigation was done by comparing transcriptomic data generated by RNA isolation and sequencing of a EMT model TNBC cell line in comparison to regular TNBC cells. RNA sequencing and Ingenuity Pathway Software Analysis (IPA) of the transcriptomic data revealed several upregulated and downregulated gene expressions along with novel core canonical pathways including Sirtuin signaling, Oxidative Phosphorylation and Mitochondrial dysfunction events involved in EMT changes of the TNBC cells.

Tumour-associated Mucin1 correlates with the procoagulant properties of cancer cells of epithelial origin

BackgroundCancer-associated thrombosis (CAT) is caused, at least in part, by procoagulant factors produced by the tumour itself. Although MUC1 is an established biomarker for the diagnosis, immunotherapy, and prognosis of cancer, it is unclear whether it contributes to the procoagulant phenotype of cancer cells. MethodsMUC1 knockdown breast cancer MCF-7 cells were used to investigate the influence of overexpression of MUC1 on procoagulant parameters. In addition, the effect of treating normal human epithelial cells with extracellular vesicles from several human breast and pancreatic cancer cell lines, which overexpress MUC1, was determined. The impact of a pharmacological anti-MUC1 antibody on cancer cells was also analysed. ResultsThe level of a range of procoagulant proteins was observed to correlate with the MUC1 level of human breast and pancreatic cancer cell lines. MUC1 downregulation in MCF-7 cells led to a reduction in the procoagulant parameters particularly thrombin activity. The levels of selected tumorigenic markers, procoagulant proteins and miRNAs associated with tumorigenicity and thromboembolism were also modulated by treatment of normal cells with tumour cell derived extracellular vesicles in correlation with that of the extracellular vesicles donor cells. Moreover, the procoagulant properties were also reduced by an anti-MUC1 antibody in these cancer cells. ConclusionsA range of procoagulant proteins found in human breast and pancreatic cancer cells were shown to exhibit a positive correlation with the level of MUC1 and thereby potentially contribute to the pathogenesis of CAT. The reduction of the procoagulant activity by MUC1 antibody could be an additional beneficial effect of its therapeutic efficacy. These findings also suggest that the level of tumour associated MUC1 could be of use as a risk factor for CAT.

Intersections of endocrine pathways and the epithelial mesenchymal transition in endometrial cancer.

The epithelial mesenchymal transition (EMT) is the process by which cancer cells of epithelial origin, including endometrial cancer, acquire a mesenchymal phenotype with enhanced migratory and invasive capacity, to facilitate metastasis. The regulation of EMT is tissue-specific, and in endometrial cancer, endocrine signaling pathways serve as critical regulators of EMT. The intersections of endocrine signaling and EMT highlight potential avenues for therapeutic intervention to target cancer metastasis with the aim of reduced mortality.

Prolactin and its significance in the placenta.

Prolactin, a pituitary hormone that was discovered about 80years ago and is primarily known for its functions in mammary gland development and lactation, is now known to participate in numerous functions across different phylogenetic groups. Fundamentally known for its secretion from lactotroph cells in adenohypophysis region of pituitary gland, newer studies have demonstrated a number of extrapituitary sites which secrete prolactin, where it acts in an autocrine, paracrine, and endocrine manner to regulate essential physiological and biochemical processes. These sites include lymphocytes, epithelial cells of lactating mammary glands, breast cancer cells of epithelial origin, and the placenta. The placenta is one of the most important organs secreting prolactin; however, its role in placental biology has not to date been reviewed comprehensively. This review elaborates upon the various facets of prolactin hormone, including prolactin production and its post-translational modifications and signaling. Major emphasis is placed on placental prolactin and its potential roles, ranging from the role of prolactin in angiogenesis, preeclampsia, maternal diabetes, and anti-apoptosis, among others.

Lack of association between Screencell-detected circulating tumour cells and long-term survival of patients undergoing surgery for non-small cell lung cancer: A pilot clinical study.

Circulating tumour cells (CTCs) are cancer cells of epithelial origin that are present in peripheral blood samples. ScreenCell detection of CTCs and the association with long term survival in non-small cell lung cancer (NSCLC) patients was evaluated in the present study. A total of 33 patients undergoing surgical resection for NSCLC were recruited. Patients were followed up for 5-years post-operatively. Pre-operative patient bloods samples were processed using ScreenCell. CTCs were detected in 26 (79%) patients. In patients who were positive for CTCs, a total of 9 (35%) patients succumbed to the disease, whereas in patients negative for CTCs, a total of 4 (57%) patients succumbed to the disease (P=0.29). No association was identified between positive CTCs and poorer survival (Chi-squared 1.47, P=0.23; hazard ratio, 0.42; 95% confidence interval: 0.1-1.7). The presence of CTCs detected with ScreenCell does not influence prognosis in patients with NSCLC that was operated on. The high rate of CTC detection is encouraging in supporting this technology to aid early lung cancer diagnosis.

Abstract 2185: Cross-disciplinary optimization of nano-drug delivery to ovarian carcinoma and glioma cells

Abstract This study reports on the quantitative analysis of the diffusion and localization of a targeted drug delivery system (DDS) consisting of fluorescent labeled 0.01 µM paclitaxel-BODIPY 564/570 encapsulated in non-ionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel. This is a multi-tiered DDS that allows for enhanced stability, sustained and controlled delivery of embedded drugs to tumor sites, and reduced toxicity. We showed that cancer cells of epithelial origin, such as human ovarian epithelial carcinoma OV2008 and highly migratory mouse glioma G-26, overexpress MUC1, a mucin surface antigen, which effectively enhances the specific targeting capacity of chitosan. Utilizing this DDS we found that OV2008 carcinoma cells had ~2 times higher fluorescence intensity level than normal ovarian epithelial IMCC3 cells with a statistical significance at 5 min and 24 h incubation times suggesting that this DDS had a higher affinity for tumor cells. Therefore, we hypothesized that there is a difference in PTX diffusion towards epithelial origin tumor cells when compared to normal cells in both distance/location and time. Diffusion and localization of fluorescent labeled PTX was evaluated in vitro using confocal microscopy. The fluorescence intensity captured on the images was quantified with ImageJ software in OV2008 carcinoma cells and compared to IMCC3 normal cells at time intervals 5 min, 1 h, 24 h, 48 h, and 72 h. The fluorescence intensity image data was analyzed in multiple radial line segments separated into three different zones of 24 µm each to represent multiple diffusion distances. Our results showed that fluorescence intensity levels in the zones around the IMCC3 normal cells at 5 min and 1 h were significantly higher (p<0.05) than around OV2008 carcinoma cells correlating reciprocally with our finding of intracellular fluorescence intensity in these two cell lines. Therefore, the data evaluating fluorescence levels in the radial zones outside the cells indicated that the migrating DDS-PTX had already been taken up by the tumor cells. Furthermore, the normal cells which showed significantly lower intracellular levels of fluorescence had higher levels of fluorescence in the measured radial zones outside the cells. This PTX-BODIPY 564/570 diffusion data from in vitro studies is currently being used for computational modeling in the in vivo intracerebral model of G-26 glioma. It will be integrated with mathematical model simulations that describe the pharmacokinetic and pharmacodynamic (PK/PD) properties of this drug delivery system for the evaluation of its efficacy and to optimize drug delivery in vivo. Computational modelling is being done in Matlab using the treatment scenario of the post-surgical late-stage glioma. The simulation studies will be used to determine optimal drug concentrations, chitosan density, and localization. Citation Format: Rupin Singh, Rana Falahat, Eva Williams, Joseph O. Johnson, Norma Alcantar, Aleksandra Karolak, Katarzyna Rejniak, Marzenna Wiranowska. Cross-disciplinary optimization of nano-drug delivery to ovarian carcinoma and glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2185. doi:10.1158/1538-7445.AM2017-2185

Apoptosis as the basic mechanism of cytotoxic action of ursolic and pomolic acids in glioma cells

Pentacyclic triterpene acids are of great interest as compounds that exhibit selective cytotoxicity against malignant tumor cells. If earlier studies were carried out mainly in cancer cells of epithelial origin, in the present work the cytotoxic effect of ursolic and pomolic acids on the primary and permanent glioma cell lines was analyzed. Both compounds are toxic to oncotransformed cells and induce apoptosis in U-87 MG line. Using molecular docking, it has been shown that Akt1 and MDM2 may be potential targets of the studied triterpene acids. It has been suggested that ursolic and pomolic acids induce apoptosis in glioma cells through inhibition of the PI3K/Akt signaling pathway, and they can be considered as potentially promising agents for the treatment of glioblastoma.

In vitro enrichment of ovarian cancer tumor-initiating cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

<em>In vitro</em> Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

Cytotoxicity of nickel zinc ferrite nanoparticles on cancer cells of epithelial origin

In this study, in vitro cytotoxicity of nickel zinc (NiZn) ferrite nanoparticles against human colon cancer HT29, breast cancer MCF7, and liver cancer HepG2 cells was examined. The morphology, homogeneity, and elemental composition of NiZn ferrite nanoparticles were investigated by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy, respectively. The exposure of cancer cells to NiZn ferrite nanoparticles (15.6–1,000 μg/mL; 72 hours) has resulted in a dose-dependent inhibition of cell growth determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The quantification of caspase-3 and -9 activities and DNA fragmentation to assess the cell death pathway of the treated cells showed that both were stimulated when exposed to NiZn ferrite nanoparticles. Light microscopy examination of the cells exposed to NiZn ferrite nanoparticles demonstrated significant changes in cellular morphology. The HepG2 cells were most prone to apoptosis among the three cells lines examined, as the result of treatment with NiZn nanoparticles. In conclusion, NiZn ferrite nanoparticles are suggested to have potential cytotoxicity against cancer cells.

Separation of tumor cells with dielectrophoresis-based microfluidic chip

The present work demonstrates the use of a dielectrophoretic lab-on-a-chip device in effectively separating different cancer cells of epithelial origin for application in circulating tumor cell (CTC) identification. This study uses dielectrophoresis (DEP) to distinguish and separate MCF-7 human breast cancer cells from HCT-116 colorectal cancer cells. The DEP responses for each cell type were measured against AC electrical frequency changes in solutions of varying conductivities. Increasing the conductivity of the suspension directly correlated with an increasing frequency value for the first cross-over (no DEP force) point in the DEP spectra. Differences in the cross-over frequency for each cell type were leveraged to determine a frequency at which the two types of cell could be separated through DEP forces. Under a particular medium conductivity, different types of cells could have different DEP behaviors in a very narrow AC frequency band, demonstrating a high specificity of DEP. Using a microfluidic DEP sorter with optically transparent electrodes, MCF-7 and HCT-116 cells were successfully separated from each other under a 3.2 MHz frequency in a 0.1X PBS solution. Further experiments were conducted to characterize the separation efficiency (enrichment factor) by changing experimental parameters (AC frequency, voltage, and flow rate). This work has shown the high specificity of the described DEP cell sorter for distinguishing cells with similar characteristics for potential diagnostic applications through CTC enrichment.

P1-02-02: Zoledronic Acid Reverses the Epithelial-Mesenchymal Transition While Inhibiting the Tumor Initiating Cell Population of Highly Tumorigenic Breast Cancer Cell Lines.

Abstract Breast cancer remains the second leading cause of cancer related death amongst women in the United States. This is largely due to metastasis of cancer cells from the primary tumor to other parts of the body, and to the putative cancer stem cell population. Both are driven by the epithelial-mesenchymal transition (EMT), a cellular process whereby cancer cells of epithelial origin lose their epithelial characteristics and acquire a mesenchymal phenotype. Cells which undergo EMT tend to be motile and invasive, and therefore can metastasize to other parts of the body. EMT has also been implicated in the generation of cells expressing the cancer stem cell phenotype. As metastatic disease and the cancer stem cell are difficult to eliminate, more effective therapy is required. Zoledronic acid, originally indicated for use in the treatment of osteoporosis, has been reported to inhibit the growth of breast cancer cells. The mechanism of this effect however has yet to be determined. In preliminary data studies, treatment with zoledronic acid was found to reduce activation of nuclear factor kappa-b, an established regulator of EMT. These findings suggest that treatment with zoledronic acid may reverse EMT in breast cancer cells, driving them to express a more epithelial phenotype. To test this hypothesis, the highly metastatic, triple negative breast cancer cell lines MDA-MB-231 and Hs578t, which largely express mesenchymal proteins, were used to measure cell viability and changes in protein and mRNA expression following treatment with zoledronic acid. Dose response analysis for inhibition of cell viability showed an IC50 of approximately 2μM in the Hs578t and 6 μM in the MDA-MB-231 cell line. Zoledronic acid treated cells displayed a decreased mesenchymal phenotype, as evidenced by reduced expression of mesenchymal markers N-cadherin (75% reduction, p<0.076; 65% reduction, p<0.005) and TWIST (67% reduction, p<0.08; 64% reduction, p<0.009) in MDA-MB-231 and Hs578t cells, respectively. This was accompanied by a subsequent increase in epithelial phenotype as evidenced by increased expression of epithelial marker E-cadherin (223% increase, p<0.006) in MDA-MB-231 cells. To further elucidate the effects on the mesenchymal and epithelial phenotypes of these cells, surface expression of CD24 and CD44 was measured by flow cytometry. While vehicle treated samples of both cell lines stained positive for CD44 and negative for CD24, zoledronic acid treatment decreased CD44 expression. As both increased the ratio of surface expression of CD44high/CD24low and mammosphere formation are characteristics of the breast cancer stem cell, effects of zoledronic acid on this subpopulation in Hs578t cells were determined. Cells pretreated with zoledronic acid were seeded under mammosphere conditions and allowed to propagate for 7 days. Zoledronic acid treated cells formed significantly fewer mammospheres (86% reduction, p<0.041), while the ones that formed were smaller in size. These findings suggest that zoledronic acid is able to reverse the epithelial mesenchymal transition, which may reduce the tumor initiating capacity of highly metastatic cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-02.

Downregulation of APOBEC3G by xenotropic murine leukemia-virus related virus (XMRV) in prostate cancer cells

BackgroundXenotropic murine leukemia virus (MLV)-related virus (XMRV) is a gammaretrovirus that was discovered in prostate cancer tissues. Recently, it has been proposed that XMRV is a laboratory contaminant and may have originated via a rare recombination event. Host restriction factor APOBEC3G (A3G) has been reported to severely restrict XMRV replication in human peripheral blood mononuclear cells. Interestingly, XMRV infects and replicates efficiently in prostate cancer cells of epithelial origin. It has been proposed that due to lack off or very low levels of A3G protein XMRV is able to productively replicate in these cells.FindingsThis report builds on and challenges the published data on the absence of A3G protein in prostate epithelial cells lines. We demonstrate the presence of A3G in prostate epithelial cell lines (LNCaP and DU145) by western blot and mass spectrometry. We believe the discrepancy in A3G detection is may be due to selection and sensitivity of A3G antibodies employed in the prior studies. Our results also indicate that XMRV produced from A3G expressing LNCaP cells can infect and replicate in target cells. Most importantly our data reveal downregulation of A3G in XMRV infected LNCaP and DU145 cells.ConclusionsWe propose that XMRV replicates efficiently in prostate epithelial cells by downregulating A3G expression. Given that XMRV lacks accessory proteins such as HIV-1 Vif that are known to counteract A3G function in human cells, our data suggest a novel mechanism by which retroviruses can counteract the antiviral effects of A3G proteins.

Non-small cell lung cancer invasion and metastasis promoted by MMP-26

Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase (MMP) family and is widely expressed in cancer cells of epithelial origin. MMP-26 has been shown to contribute to tumor development and to the restoration of tissue injury. In this study, in order to identify the functions of MMP-26 that contribute to the biological phenotype and behavior of human lung carcinoma A549 cells, we established an MMP-26 low-expressing tumor cell model using RNA interference (RNAi) transfection. These cells were used to investigate the role of MMP-26 in tumor progression. The MTT, colony forming, adhere-nce and spreading, wound-healing and Transwell chamber invasion assays were performed to analyze the invasion ability of pshRNA-MMP26-transfected A549 cells. Semi-quantitative reverse transcription polymerase chain reaction, Western blotting and double immunofluorescent staining were employed to detect the relationship between MMP-26 and MMP-9. Results showed that the adhesive rate was down-regulated in pshRNA-MMP26-transfected cells, compared to the controls. Silencing of the MMP-26 gene significantly retarded the invasiveness of A549 cells in Transwell insert invasion assays. The cells proliferated in the three-dimensional (3D) culture system. A549 cells transfected with the pshRNA-MMP26-C plasmid mainly developed reticular structures in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. The mRNA and protein expression of MMP-9 in pshRNA-MMP26-transfected cells were significantly lower than those of the controls. Double immunofluorescence labeling and focal laser scanning microscopy showed that MMP-26 was colocalized with MMP-9 in the controls. In conclusion, we successfully established an MMP-26 low-expressing cell model and confirmed that MMP-26 contributed to A549 cell invasion and migration in vitro. We also demonstrated that MMP-26 plays an important role in local invasion at least in part through coordination with MMP-9.

Image-Guided Surgery: Breast Cancer Detection Using Monoclonal Antibodies Conjugated to a Near-Infrared Fluorescent Dye in a Syngeneic Rat Model.

Abstract Background: Incomplete tumor resections occur frequently in patients undergoing breast-conserving surgery. As the surgeon can only rely on palpation and visual inspection, real-time visualization of cancer cells during surgery is needed to increase the number of complete tumor resections. Near-infrared fluorescence (NIRF) imaging using an intra-operative camera system is a novel technique to assess the extent of disease during surgery. Advantages of NIRF light (700-900 nm) include high tissue penetration up to several centimeters deep and low autofluorescence providing sufficient signal-to-noise ratio. Currently, several NIRF dyes are available for chemical conjugation to molecules that can target tumors, e.g. antibodies. This study aimed to investigate whether monoclonal antibodies conjugated to a NIRF dye could detect primary breast carcinomas in a syngeneic rat model.Methods: The hormone-dependent syngeneic breast cancer rat model EMR86 and its derived cell line MCR86 were used. Tumors were induced by orthotopic implantation of fresh EMR86 tumor fragments of 1 mm3 into the flanks at four sites in female WAG/Rij rats (Harlan, the Netherlands). The mouse monoclonal antibody MG1 was used for tumor cell detection. MG1 (IgG2a) recognizes a membrane epitope of rat cancer cells of epithelial origin and is developed by our research group. MG1 was conjugated to the NIRF dye CW-800 (LI-COR, USA) and dye/protein ratio was determined using absorbance measurements (UltroSpec, Amersham, UK). Fluorescence imaging of cells, animals and organs was performed using IVIS Spectrum (Caliper, USA) and Odyssey (LI-COR, USA) equipment.Results: Conjugated antibodies MG1-CW800 bound specifically to cultured MCR86 cells. The signal intensity was linearly correlated with the number of cells and concentration of MG1-CW800 (R2=0.99, p<0.00001). Addition of unconjugated MG1 lowered the fluorescent signal in a competitive binding assay, showing that labeling did not influence binding properties. Intravenous injection of unconjugated MG1 (200 μg/rat) in 3 Wag/Rij rats and subsequent immunohistochemical analysis of tissue sections revealed a specific and exclusive binding of MG1 to the EMR86 tumors and no difference in staining intensity between 24 and 48hrs after injection. Intravenous injection of conjugated MG1-CW800 (200 μg/rat) demonstrated a clear fluorescent demarcation of EMR86 tumors and the surrounding tissue after spectral unmixing (paired t-test 8.17, p<0.00001). Mean fluorescence did not differ between 24 and 48hrs after injection (t=1.85, p=0.11). However, a two-fold increase of the dye/protein ratio led to a two-fold increase of the in vivo fluorescence (t=-6.40, p=0.0001).Conclusion: Intravenous injection of monoclonal antibodies MG1 conjugated to the NIRF dye CW800 enabled detection of primary breast carcinomas in the syngeneic hormone-dependent EMR86 rat model. This study demonstrates proof-of-concept for future labeling of clinically relevant antibodies (e.g. HER2/neu, EGF, VEGF). Translation of this technique to breast cancer patients can lead to intra-operative identification of optimal resection margins in order to increase the comple tumor resection rate. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5006.

Expression of matrix metalloproteinase-26 promotes human glioma U251 cell invasion in vitro and in vivo

MMP-26 is a novel member of the MMP family and is widely expressed in cancer cells of epithelial origin. Published research shows that MMP-26 contributes to tumor development and to the restoration of tissue injury. In this study, in order to identify the functions of MMP-26 that contribute to the biological phenotype and behavior of non-epithelial human glioma U251 cells, we established an MMP-26 overexpressing tumor cell model using gene transfection. We then used these cells to investigate the role of MMP-26 in tumor progression. Adherence and spreading assay, wound healing assay, Boyden chamber invasion assay, and in vivo tumorigenicity assay were performed to analyze the invasion ability of MMP-26 transfected U251 cells. Microvessel density analysis and tumor cell induced angiogenesis assay were employed to detect the function of MMP-26 in angiogenesis. Results showed that the spreading cell ratio of MMP-26 transfected cells was significantly higher than parental U251 cells. The relative migration distance of MMP-26 transfected cells on Matrigel was significantly higher than that of parental U251 cells. The Boyden chamber assay showed that MMP-26 could significantly enhance the ability of U251 cells to invade through Matrigel. MMP-26 could also enhance the local invasion ability of U251 cells in vivo. There was a significant increase of the microvessel density of tumor tissue derived from MMP-26 transfected U251 cells. The vessel numbe induced by MMP-26 transfected U251 cells in nude mice was also significantly higher than that induced by parental U251 cells. In conclusion, we successfully established an MMP-26 overexpressing cell model and confirmed that MMP-26 contributed to U251 cell invasion and migration in vitro. We also demonstrated that MMP-26 plays an important role in local invasion, and angiogenesis.

Molecular principles of cancer invasion and metastasis (Review)

The main threat and the reason for most cancer deaths are not the primary neoplasias, but secondary tumors, the metastases. Drastic phenotypic and biochemical changes occur during the metamorphosis of a normal tissue cell into an invasive cancer cell. These alterations concern various areas such as growth factor signaling, cell-cell adhesion, gene expression, motility or cell shape. Cancer cells of epithelial origin can even shed their typical qualities and characteristics and adopt a mesenchymal-like phenotype. This is often referred to as an epithelial-mesenchymal transition. Various oncogenes, tumor suppressor genes and metastasis suppressor genes are known to affect the invasiveness and the metastatic potential of tumor cells. Cells of the innate and adaptive immunity, adjacent stroma cells as well as chemokines and their receptors also play a vital role in the spread of cancer cells. Furthermore, the micro-environment, vascularization and the supply with special cytokines affect the above-mentioned changes. Finally, some researchers claim that tumors consist of two types of cells - transit amplifying cells and cancer stem cells. Only the latter are thought to be able to proliferate indefinitely and thus they might be the cells that successfully spread and initially build most of the clinically relevant metastases. This review article describes some of the molecular principles which underlie those changes as well as covers some aspects of current research.

Upregulated expression of kappa light chain by Epstein–Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-κB and AP-1 pathways

B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1-targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-κB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-κB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins.

Quiescent Chronic Myelogenous Leukemia (CML) Cells Are Resistant to BCR-ABL Inhibitors but Preferentially Sensitive to BMS-214662, a Farnesyltransferase Inhibitor (FTI) with Unique Quiescent-Cell Selective Cytotoxicity.

The major concern in the treatment of CML is resistance to the approved agent imatinib mesylate at all stages of disease, most commonly due to mutations in BCR-ABL (but other mechanisms have also been identified). Experimental agents such as dasatinib (BMS-354825), a novel, oral kinase inhibitor that targets BCR-ABL and SRC kinases, or AMN107, which targets BCR-ABL but not SRC, were designed to address all or parts of these mechanisms and are currently under clinical testing. A second concern in CML is persistence of BCR-ABL-positive cells or ‘residual disease' in the majority of patients on imatinib therapy, including those with complete cytogenetic responses. Bone marrow studies reveal that the residual disease resides at least in part in the primitive CD34+ progenitor compartment, suggesting that imatinib may not be effective against these cell populations (Bhatia et al, Blood 101:4701, 2003). Moreover, several imatinib-resistant ABL kinase domain mutations have been detected in CD34+/BCR-ABL+ progenitors (Chu et al, Blood 105:2093, 2005), a scenario for eventual disease relapse. A hallmark of CD34+ primitive CML progenitors is quiescence (Elrick et al, Blood 105:1862, 2005). We hypothesized that BCR-ABL inhibitors like imatinib may not be effective in killing CML cells in this non-proliferative state. This was tested by comparing cytotoxicity of imatinib or dasatinib in proliferating K562 cells and in cells forced into quiescence by nutrient depletion. Proliferating K562 cells were effectively killed by imatinib (IC50 250–500 nM) and dasatinib (IC50 <1.00 nM). However, cells in quiescent cultures were far more resistant (imatinib IC50 >5000 nM; dasatinib IC50 >12 nM), suggesting that these inhibitors may be less effective in eradicating quiescent CD34+ progenitors. BMS-214662 is a FTI in Phase I clinical development. Unlike many other FTI, BMS-214662 exhibits potent cytotoxic activity against a variety of human tumor cells, and uniquely, its cytotoxicity is highly selective against non-proliferating cancer cells of epithelial origin (Lee et al, Proceedings of the AACR 42:260s, 2001). We now demonstrate similar selectivity in K562 CML cells. BMS-214662 was 68-fold more potent in killing quiescent (IC50 = 0.7 uM) than proliferating K562 cells (IC50 = 47.5 uM). Because BCR-ABL inhibitors and BMS-214662 target distinct cell populations (proliferating vs quiescent), there may be a positive therapeutic interaction when these agents are used in combination. In vitro studies in quiescent K562 cultures demonstrated that the combination of BMS-214662 and dasatinib, at concentrations readily achievable in the clinic, produced supra-additive cytotoxicity (% cell kill: dasatinib alone = 0%, BMS-214662 alone = 21%, combination = 71%). In vivo studies in K562 xenografts implanted SC in mice also showed that the combination of BMS-214662 and dasatinib produced a superior anti-leukemic activity than either dasatinib alone (P=0.0157) or BMS-214662 alone (P=0.0002). These results highlight the potential utility of BMS-214662 for targeting the quiescent progenitor compartment which, in combination with targeted agents such as dasatinib, address both BCR-ABL-dependent and -independent mechanisms of resistance, and may produce more durable responses and suppress the emergence of resistance.