Endothelial progenitor cells (EPCs) as gene carrier system for rat model of human glioma

Background

Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.

Methods and Results

Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.

Conclusion

EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.     

Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

Purpose

Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.

Methods

HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.

Results

cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.

Conclusions

These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.     

Quantitative Immunohistochemical Analysis Reveals Association between Sodium Iodide Symporter and Estrogen Receptor Expression in Breast Cancer

Background

Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.

Methods

hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.

Results

Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.

Conclusion

The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted ¹³¹I radio-ablative therapy is aimed.     

Optimization of Multimodal Imaging of Mesenchymal Stem Cells Using the Human Sodium Iodide Symporter for PET and Cerenkov Luminescence Imaging

Purpose

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

Methods

First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, ¹²⁴I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using ^99mTcO₄ ⁻ radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell''s differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI.

Results

The expression of both imaging reporter genes was functional and specific. An elution of ^99mTcO₄ ⁻ from the cells was observed, with 31% retention after 3 h. After labeling cells with ¹²⁴I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using ¹²⁴I small-animal PET, CLI and BLI.

Conclusions

This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.     

An Exploratory Study on 99mTc-RGD-BBN Peptide Scintimammography in the Assessment of Breast Malignant Lesions Compared to 99mTc-3P4-RGD2

Purpose

This study aimed to explore the diagnostic performance of single photon emission computed tomography / computerized tomography (SPECT/CT) using a new radiotracer ^99mTc-RGD-BBN for breast malignant tumor compared with ^99mTc-3P4-RGD₂.

Methods

6 female patients with breast malignant tumors diagnosed by fine needle aspiration cytology biopsy (FNAB) who were scheduled to undergo surgery were included in the study. ^99mTc-3P4-RGD₂ and ^99mTc-RGD-BBN were performed with single photon emission computed tomography (SPECT) at 1 hour after intravenous injection of 299 ± 30 MBq and 293 ± 32 MBq of radiotracers respectively at separate day. The results were evaluated by the Tumor to non-Tumor ratios (T/NT). ^99mTc-RGD-BBN and ^99mTc-3P4-RGD₂ SPECT/CT images were interpreted independently by 3 experienced nuclear medicine physicians using a 3-point scale system. All of the samples were analyzed immunohistochemically to evaluate the integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) expression. The safety, biodistribution and radiation dosimetry of ^99mTc-RGD-BBN were also evaluated in the healthy volunteers.

Results

No serious adverse events were reported in any of the patients during the study. The effective radiation dose entirely conformed to the relevant standards. A total of 6 palpable malignant lesions were detected using ^99mTc-RGD-BBN SPECT/CT with clear uptake. All malignant lesions were also detected using ^99mTc-3P4-RGD₂ SPECT/CT. The results showed that five malignant lesions were with clear uptake and the other one with barely an uptake. 4 malignant cases were found with both αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) and 1 case with only integrin αvβ3 positive expression (GRPR negative).

Conclusion

^99mTc-RGD-BBN is a safe agent for detecting breast cancer. ^99mTc-RGD-BBN may have the potential to make up for the deficiency of ^99mTc-3P4-RGD₂ in the detection of breast cancer with only GRPR positive expression (integrin α_vβ₃ negative). The preliminary application of ^99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.     

99mTc-3P4-RGD2 Scintimammography in the Assessment of Breast Lesions: Comparative Study with 99mTc-MIBI

Purpose

To compare the potential application of ^99mTc-3P-Arg-Gly-Asp (^99mTc-3P₄-RGD₂) scintimammography (SMM) and ^99mTc-methoxyisobutylisonitrile (^99mTc-MIBI) SMM for the differentiation of malignant from benign breast lesions.

Method

Thirty-six patients with breast masses on physical examination and/or suspicious mammography results that required fine needle aspiration cytology biopsy (FNAB) were included in the study. ^99mTc-3P₄-RGD₂ and ^99mTc-MIBI SMM were performed with single photon emission computed tomography (SPECT) at 60 min and 20 min respectively after intravenous injection of 738±86 MBq radiotracers on a separate day. Images were evaluated by the tumor to non-tumor localization ratios (T/NT). Receiver operating characteristic (ROC) curve analysis was performed on each radiotracer to calculate the cut-off values of quantitative indices and to compare the diagnostic performance for the ability to differentiate malignant from benign diseases.

Results

The mean T/NT ratio of ^99mTc-3P₄-RGD₂ in malignant lesions was significantly higher than that in benign lesions (3.54±1.51 vs. 1.83±0.98, p<0.001). The sensitivity, specificity, and accuracy of ^99mTc-3P₄-RGD₂ SMM were 89.3%, 90.9% and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of ^99mTc-MIBI in malignant lesions was also significantly higher than that in benign lesions (2.86±0.99 vs. 1.51±0.61, p<0.001). The sensitivity, specificity and accuracy of ^99mTc-MIBI SMM were 87.5%, 72.7% and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for ^99mTc-3P₄-RGD₂ SMM (area = 0.851) was higher than that for ^99mTc-MIBI SMM (area = 0.781), but the statistical difference was not significant.

Conclusion

^99mTc-3P₄-RGD₂ SMM does not provide any significant advantage over the established ^99mTc-MIBI SMM for the detection of primary breast cancer. The T/NT ratio of ^99mTc-3P₄-RGD₂ SMM was significantly higher than that of ^99mTc-MIBI SMM. Both tracers could offer an alternative method for elucidating non-diagnostic mammograms.     

Imaging characteristics, tissue distribution, and spread of a novel oncolytic vaccinia virus carrying the human sodium iodide symporter

Introduction

Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.

Methods

GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide ¹³¹I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via ¹²⁴I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both ¹²⁴I-PET and 99m-technecium gamma-scintigraphy.

Results

GLV-1h153 successfully facilitated time-dependent intracellular uptake of ¹³¹I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10⁹ plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via ¹²⁴I-PET and 99m-technecium-scintigraphy.

Conclusion

GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.     

Comparison of 99mTc-3PRGD2 Integrin Receptor Imaging with 99mTc-MDP Bone Scan in Diagnosis of Bone Metastasis in Patients with Lung Cancer: A Multicenter Study

Purpose

^99mTc-3PRGD2, a promising tracer targeting integrin receptor, may serve as a novel tumor-specific agent for single photon emission computed tomography (SPECT). A multi-center study was prospectively designed to evaluate the diagnostic accuracy of ^99mTc-3PRGD2 imaging for bone metastasis in patients with lung cancer in comparison with the conventional ^99mTc-MDP bone scan.

Methods

The patients underwent whole-body scan and chest tomography successively at both 1 h and 4 h after intravenous injection of 11.1 MBq/Kg ^99mTc-3PRGD2. ^99mTc-MDP whole-body bone scan was routinely performed within 1 week for comparison. Three experienced nuclear medicine physicians blindly read the ^99mTc-3PRGD2 and ^99mTc-MDP images. The final diagnosis was established based on the comprehensive assessment of all available data.

Results

A total of 44 patients (29 male, 59±10 years old) with suspected lung cancer were recruited from 4 centers. Eighty-nine bone lesions in 18 patients were diagnosed as metastases and 23 bone lesions in 9 patients were benign. In a lesion-based analysis, ^99mTc-3PRGD2 imaging demonstrated a sensitivity, specificity, and accuracy of 92.1%, 91.3%, and 92.0%, respectively. The corresponding diagnostic values for ^99mTc-MDP bone scan were 87.6%, 60.9%, and 82.1%, respectively in the same patients. ^99mTc-MDP bone scan had better contrast in most lesions, whereas the ^99mTc-3PRGD2 imaging seemed to be more effective to exclude pseudo-positive lesions and detect bone metastases without osteogenesis.

Conclusion

^99mTc-3PRGD2 is a novel tumor-specific agent based on SPECT technology with a promising value in diagnosis of bone metastasis in patients with lung cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01737112","term_id":"NCT01737112"}}NCT01737112     

Experimental Study of Nasopharyngeal Carcinoma Radionuclide Imaging and Therapy Using Transferred Human Sodium/Iodide Symporter Gene

Purpose

The aim of this study was to design a method of radionuclide for imaging and therapy of nasopharyngeal carcinoma (NPC) using the transferred human sodium/iodide symporter (hNIS) gene.

Methods

A stable NPC cell line expressing hNIS was established (CNE-2-hNIS). After ¹³¹I treatment, we detected proliferation and apoptosis of NPC cells, both in vitro and vivo. In vivo, the radioactivity of different organs of nude mice was counted and ^99mTc imaging using SPECT was performed. The apparent diffusion coefficient (ADC) value changes of tumor xenografts were observed by diffusion-weighted magnetic resonance imaging (DW-MRI) within 6–24 days of ¹³¹I treatment. The correlation of ADC changes with apoptosis and proliferation was investigated. Post-treatment expression levels of P53, Bax, Bcl-2, Caspase-3, and Survivin proteins were detected by western blotting.

Results

¹³¹I uptake was higher in CNE-2-hNIS than in CNE-2 cells. The proliferation and apoptosis rate decreased and increased respectively both in vitro and vivo in the experimental group after ¹³¹I treatment. The experimental group tumors accumulated ^99mTc in vivo, leading to a good visualization by SPECT. DW-MRI showed that ADC values increased in the experimental group 6 days after treatment, while ADC values were positively and negatively correlated with the apoptotic and Ki-67 proliferation indices, respectively. After treatment, CNE-2-hNIS cells up-regulated the expression of P53 and Survivin proteins and activated Caspase-3, and down-regulated the expression of Bcl-2 proteins.

Conclusions

The radionuclide imaging and therapy technique for NPC hNIS-transfected cell lines can provide a new therapy strategy for monitoring and treatment of NPC.     

Agreement of Mammographic Measures of Volumetric Breast Density to MRI

Background

Clinical scores of mammographic breast density are highly subjective. Automated technologies for mammography exist to quantify breast density objectively, but the technique that most accurately measures the quantity of breast fibroglandular tissue is not known.

Purpose

To compare the agreement of three automated mammographic techniques for measuring volumetric breast density with a quantitative volumetric MRI-based technique in a screening population.

Materials and Methods

Women were selected from the UCSF Medical Center screening population that had received both a screening MRI and digital mammogram within one year of each other, had Breast Imaging Reporting and Data System (BI-RADS) assessments of normal or benign finding, and no history of breast cancer or surgery. Agreement was assessed of three mammographic techniques (Single-energy X-ray Absorptiometry [SXA], Quantra, and Volpara) with MRI for percent fibroglandular tissue volume, absolute fibroglandular tissue volume, and total breast volume.

Results

Among 99 women, the automated mammographic density techniques were correlated with MRI measures with R² values ranging from 0.40 (log fibroglandular volume) to 0.91 (total breast volume). Substantial agreement measured by kappa statistic was found between all percent fibroglandular tissue measures (0.72 to 0.63), but only moderate agreement for log fibroglandular volumes. The kappa statistics for all percent density measures were highest in the comparisons of the SXA and MRI results. The largest error source between MRI and the mammography techniques was found to be differences in measures of total breast volume.

Conclusion

Automated volumetric fibroglandular tissue measures from screening digital mammograms were in substantial agreement with MRI and if associated with breast cancer could be used in clinical practice to enhance risk assessment and prevention.     

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

Background  

Real-time quantitative PCR (RQ-PCR) forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC) genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1) was used a target gene to compare the effect of choice of EC on the estimate of gene quantity.     

Theranostic Studies of Human Sodium Iodide Symporter Imaging and Therapy Using 188Re: A Human Glioma Study in Mice

Objective

To investigate the role of ¹⁸⁸Re in human sodium iodide symporter (hNIS) theranostic gene-mediated human glioma imaging and therapy in model mice.

Methods

The human glioma cell line U87 was transfected with recombinant lentivirus encoding the hNIS gene under the control of cytomegalovirus promoter (U87-hNIS). The uptake and efflux of ¹⁸⁸Re were determined after incubating the cells with ¹⁸⁸Re. ¹⁸⁸Re uptake experiments in the presence of various concentrations of sodium perchlorate were carried out. In vitro cell killing tests with ¹⁸⁸Re were performed. U87-hNIS mediated ¹⁸⁸Re distribution, imaging and therapy in nude mice were also tested.

Results

U87-hNIS cell line was successfully established. The uptake of ¹⁸⁸Re in U87-hNIS cells increased up to 26-fold compared to control cells, but was released rapidly with a half-life of approximately 4 minutes. Sodium perchlorate reduced hNIS-mediated ¹⁸⁸Re uptake to levels of control cell lines. U87-hNIS cells were selectively killed following exposure to ¹⁸⁸Re, with a survival of 21.4%, while control cells had a survival of 92.1%. Unlike in vitro studies, U87-hNIS tumor showed a markedly increased ¹⁸⁸Re retention even 48 hours after ¹⁸⁸Re injection. In the therapy study, there was a significant difference in tumor size between U87-hNIS mice (317±67 mm³) and control mice (861±153 mm³) treated with ¹⁸⁸Re for 4 weeks (P<0.01).

Conclusion

The results indicate that inserting the hNIS gene into U87 cells is sufficient to induce specific ¹⁸⁸Re uptake, which has a cell killing effect both in vitro and in vivo. Moreover, our study, based on the function of hNIS as a theranostic gene allowing noninvasive imaging of hNIS expression by ¹⁸⁸Re scintigraphy, provides detailed characterization of in vivo vector biodistribution and level, localization, essential prerequisites for precise planning and monitoring of clinical gene therapy that aims to individualize gene therapy concept.     

A Novel Cancer Testis Antigen,A-Kinase Anchor Protein 4 (AKAP4) Is a Potential Biomarker for Breast Cancer

Background

Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma.

Methodology/Findings

We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein.

Conclusions

Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.     

Role of hypoxia and glycolysis in the development of multi-drug resistance in human tumor cells and the establishment of an orthotopic multi-drug resistant tumor model in nude mice using hypoxic pre-conditioning

Background  

The development of multi-drug resistant (MDR) cancer is a significant challenge in the clinical treatment of recurrent disease. Hypoxia is an environmental selection pressure that contributes to the development of MDR. Many cancer cells, including MDR cells, resort to glycolysis for energy acquisition. This study aimed to explore the relationship between hypoxia, glycolysis, and MDR in a panel of human breast and ovarian cancer cells. A second aim of this study was to develop an orthotopic animal model of MDR breast cancer.     

The linkage between β1 integrin and the actin cytoskeleton is differentially regulated by tyrosine and serine/threonine phosphorylation of β1 integrin in normal and cancerous human breast cells

Background  

Structural requirements for the β1 integrin functions in cell adhesion, spreading and signaling have been well documented mainly for fibroblasts. In this study, we examined the reason for the reduced surface expression of β1 integrin in human breast cancer MCF-7 cells compared to normal human breast epithelial (HBE) cells, both of which adhered to collagen type IV.     

Targeted Gene Delivery to MCF-7 Cells Using Peptide-Conjugated Polyethylenimine

Specific and effective delivery of drugs and genes to cancer cells are the major issues in successful cancer treatment. Recently, targeted cancer gene therapy has been emerged as a main technology for the treatment of different types of cancers. Among various synthetic carriers, polyethylenimine is one of the most well-known polymers for gene delivery. In this study, we conjugated phage-derived peptide (DMPGTVLP) to polyethylenimine (10 kDa) via disulfide bonds for targeted gene delivery into breast adenocarcinoma cells (MCF-7). As negative-control cells, we used non-related hepatocellular carcinoma cells (HepG2). Peptide-conjugated polyplex exhibited low cytotoxicity and significantly increased the transfection efficiency in comparison with unmodified polyethylenimine. Therefore, the peptide-modified vector can be used as a good targeting agent for gene or drug delivery into breast adenocarcinoma cells.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0208-6) contains supplementary material, which is available to authorized users.KEY WORDS: breast cancer, gene therapy, phage-derived peptide, polyethylenimine     

Comparison of rNIS and hNIS as reporter genes for noninvasive imaging of bone mesenchymal stem cells transplanted into infarcted rat myocardium

The purpose of this study was to investigate and compare the feasibility of rat sodium iodide symporter (rNIS) and human sodium iodide symporter (hNIS) as reporter genes for noninvasive monitoring of rat bone marrow mesenchymal stem cells (rBMSCs) transplanted into infarcted rat myocardium. rBMSCs were isolated from rat bone marrow. Adenovirus (Ad) was reconstructed to contain rNIS-enhanced green fluorescent protein (eGFP) or hNIS-eGFP. The transfection efficiency of Ad/eGFP/rNIS and Ad/eGFP/hNIS to rBMSCs was measured by real-time polymerase chain reaction, flow cytometry, Western blot, and immunofluorescence staining. The transfected rBMSCs were transplanted into infarcted rat myocardium followed by a single-photon emission computed tomography (SPECT) study with (99m)Tc-pertechnetate as the radiotracer and by autoradiography. The isolated rBMSCs were CD29, CD44, and CD90 positive and CD34, CD45, and CD11b negative. The expression of rNIS and hNIS in the transfected rBMSCs at both gene and protein levels was obviously higher than that without transfection. The myocardium of rats transplanted with transfected rBMSCs could be visualized by SPECT owing to the accumulation of (99m)Tc-pertechnetate in rBMSCs mediated by exogenous NIS genes. The accumulation of (99m)Tc-pertechnetate in myocardium mediated by rNIS was higher than that by hNIS, which was also confirmed by autoradiography. Both rNIS and hNIS are useful reporter genes to monitor BMSCs transplanted into infarcted myocardium in vivo with rNIS being superior to hNIS as the reporter gene.     

Effect of a second nitroimidazole redox centre on the accumulation of a hypoxia marker: synthesis and in vitro evaluation of 99mTc-labeled bisnitroimidazole propylene amine oxime complexes

Up to now, most of the hypoxia markers contain only one nitroimidazole redox centre, such as Oxo[[3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioximato] (3-)-N,N',N″,N″']-technetium ((99m)Tc-1, BMS181321). Introducing a second nitroimidazole redox centre may enhance the hypoxic accumulation of the markers. In the present work, four (99m)Tc-1 (BMS181321, containing one 2-nitroimidazole) analogues, that is, (99m)Tc-2 (containing two 2-nitroimidazoles), (99m)Tc-3 (containing one 4-nitroimidazole), (99m)Tc-4 (containing two 4-nitroimidazoles) and (99m)Tc-5 (containing both a 2-nitroimidazole and a 4-nitroimidazole) were synthesized, and the hypoxic accumulation was evaluated in vitro using murine sarcoma S180 cells. (99m)Tc-3 and (99m)Tc-4 displayed no significant anoxic/normoxic differentials, whereas (99m)Tc-1 (BMS181321), (99m)Tc-2 and (99m)Tc-5 showed high anoxic cellular uptakes. The anoxic uptake of (99m)Tc-2 reached up to 59.0±0.9% at 4h, which was 2.4 times as that of (99m)Tc-1. (99m)Tc-2 displayed high hypoxic accumulation, indicating that introducing a second nitroimidazole redox centre, that is, 2-nitroimidazole, affected the hypoxic accumulation. Consequently, (99m)Tc-2 may serve as a viable candidate for hypoxia marker. This finding may eventually lead to the development of compounds containing multi-redox centres as hypoxia markers.     

The Added Diagnostic Value of Dynamic Contrast-Enhanced MRI at 3.0 T in Nonpalpable Breast Lesions

Objective

To investigate the added diagnostic value of 3.0 Tesla breast MRI over conventional breast imaging in the diagnosis of in situ and invasive breast cancer and to explore the role of routine versus expert reading.

Materials and Methods

We evaluated MRI scans of patients with nonpalpable BI-RADS 3–5 lesions who underwent dynamic contrast-enhanced 3.0 Tesla breast MRI. Initially, MRI scans were read by radiologists in a routine clinical setting. All histologically confirmed index lesions were re-evaluated by two dedicated breast radiologists. Sensitivity and specificity for the three MRI readings were determined, and the diagnostic value of breast MRI in addition to conventional imaging was assessed. Interobserver reliability between the three readings was evaluated.

Results

MRI examinations of 207 patients were analyzed. Seventy-eight of 207 (37.7%) patients had a malignant lesion, of which 33 (42.3%) patients had pure DCIS and 45 (57.7%) invasive breast cancer. Sensitivity of breast MRI was 66.7% during routine, and 89.3% and 94.7% during expert reading. Specificity was 77.5% in the routine setting, and 61.0% and 33.3% during expert reading. In the routine setting, MRI provided additional diagnostic information over clinical information and conventional imaging, as the Area Under the ROC Curve increased from 0.76 to 0.81. Expert MRI reading was associated with a stronger improvement of the AUC to 0.87. Interobserver reliability between the three MRI readings was fair and moderate.

Conclusions

3.0 T breast MRI of nonpalpable breast lesions is of added diagnostic value for the diagnosis of in situ and invasive breast cancer.