Detection of cancer stem cells by EMT-specific biomarker-based peptide ligands.

The occurrence of epithelial-mesenchymal transition (EMT) within tumors, which enables invasion and metastasis, is linked to cancer stem cells (CSCs) with drug and radiation resistance. We used two specific peptides, F7 and SP peptides, to detect EMT derived cells or CSCs. Human tongue squamous carcinoma cell line-SAS transfected with reporter genes was generated and followed by spheroid culture. A small molecule inhibitor-Unc0642 and low-dose ionizing radiation (IR) were used for induction of EMT. Confocal microscopic imaging and fluorescence-activated cell sorting analysis were performed to evaluate the binding ability and specificity of peptides. A SAS xenograft mouse model with EMT induction was established for assessing the binding affinity of peptides. The results showed that F7 and SP peptides not only specifically penetrated into cytoplasm of SAS cells but also bound to EMT derived cells and CSCs with high nucleolin and vimentin expression. In addition, the expression of CSC marker and the binding of peptides were increased in tumors isolated from Unc0642/IR-treated groups. Our study demonstrates the potential of these peptides for detecting EMT derived cells or CSCs and might provide an alternative isolation method for these subpopulations within the tumor in the future.

Quantitative Measurement of the Thyroid Uptake Function of Mouse by Cerenkov Luminescence Imaging.

Cerenkov luminescence imaging (CLI) has been an evolutional and alternative approach of nuclear imaging in basic research. This study aimed to measure the 131I thyroid uptake of mouse using CLI for assessment of thyroid function. Quantification of 131I thyroid uptake of mice in euthyroid, hypothyroid and hyperthyroid status was performed by CLI and γ-scintigraphy at 24 hours after injection of 131I. The 131I thyroid uptake was calculated using the equation: (thyroid counts - background counts)/(counts of injected dose of 131I) × 100%. Serum T4 concentration was determined to evaluate the thyroid function. The radioactivity of 131I was linearly correlated with the CL signals in both in vitro and in vivo measurements. CLI showed a significant decrease and increase of 131I thyroid uptake in the mice in hypo- and hyperfunctioning status, respectively, and highly correlated with that measured by γ-scintigraphy. However, the percent thyroid uptake measured by CLI were one-fifth of those measured by γ-scintigraphy due to insufficient tissue penetration of CL. These results indicate that CLI, in addition to nuclear imaging, is able to image and evaluate the 131I thyroid uptake function in mice in preclinical and research settings.

STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo. An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [18F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV-TK/GCV therapy. Moreover, STAT3/NF-κB signaling targeting could further sensitize tumor cells to cisplatin. This study successfully established a theranositic approach to treat triple-negative breast cancer via STAT3-NF-κB responsive element-driven suicide gene therapy. This platform may also be an alternative strategy to handle with drug-resistant cancer cells.

Demonstration of Tightly Radiation-Controlled Molecular Switch Based on CArG Repeats by In Vivo Molecular Imaging.

PURPOSE: Promoters developed for radiogene therapy always show non-negligible transcriptional activities, even when cells are not irradiated. This study developed a tightly radiation-controlled molecular switch based on radiation responsive element (CArG) repeats for in vivo molecular imaging using the Cre/loxP system. PROCEDURES: Different numbers of CArG repeats were cloned as a basal promoter directly, and its pre- and postirradiation transcriptional activities were analyzed by luciferase assay. Nine CArG repeats (E9) were chosen for use as a radiation-controlled molecular switch for the Cre/loxP system, and the feasibility of the switch in vitro and in vivo was demonstrated by luciferase assay and bioluminescence imaging, respectively. RESULTS: The E9 promoter, which exhibits extremely low transcriptional activity, showed a 1.8-fold enhancement after irradiation with a clinical dose of 2 Gy. Both in vitro and in vivo results indicated that E9 is relatively inert but sufficient to trigger the Cre/loxP system. The luciferase activity of stable H1299/pSTOP-FLuc cells transfected with pE9-NLSCre and exposed to 2-Gy radiation can reach 44 % of that of the same cells transfected with pCMV-NLSCre and not subjected to irradiation. By contrast, no appreciable difference was observed in reporter gene expression in both H1299/pSTOPFluc cells and tumors transfected with pE4Pcmv-NLSCre before and after irradiation, because the strong basal transcriptional activity of the CMV promoter, which acts as a copartner of E4, masked the response of E4 to radiation. CONCLUSIONS: Our results provide detailed insight into CArG elements as a radiation-controlled molecular switch that can facilitate the development of radiogene therapy.

Enhancement of tumor initiation and expression of KCNMA1, MORF4L2 and ASPM genes in the adenocarcinoma of lung xenograft after vorinostat treatment.

Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDHbr lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDHbr cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.

Rational design of a triple reporter gene for multimodality molecular imaging.

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

P21-driven multifusion gene system for evaluating the efficacy of histone deacetylase inhibitors by in vivo molecular imaging and for transcription targeting therapy of cancer mediated by histone deacetylase inhibitor.

UNLABELLED: Overexpressed histone deacetylase (HDAC) activity has been linked with tumor initiation and progression that prompt the development of histone deacetylase inhibitors (HDACIs) as anticancer agents. HDACI was reported to be able to activate p21 promoter through the SP1 binding sites in the proximal region of p21(WAF1/CIP1) promoter. In this study, we established a p21(WAF1/CIP1) promoter-driven triple-fused reporter gene system (p21-3H) to evaluate the efficacy of HDACI and the ganciclovir (GCV)-mediated anticancer effect contributed by HDACI-induced and p21-driven truncated herpes simplex virus-1 thymidine kinase sr39 mutant (ttksr39) in vitro and in vivo. METHODS: The p21-3H construct was generated and stably or transiently transfected into H1299 cell lines. These cells were treated with trichostatin A or vorinostat (suberoylanilide hydroxamic acid [SAHA]) to evaluate the activation of p21 promoter-driven reporter gene expression by in vitro confocal fluorescence microscopy, luciferase assay, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil ((3)H-FEAU) cellular uptake, in vivo bioluminescence imaging, and 9-(4-(18)F-fluoro-3-hydroxymethylbutyl) guanine ((18)F-FHBG) small-animal PET imaging. The therapeutic efficacy on p21-3H-expressing tumor xenografts was assessed by daily administration with SAHA (100 mg/kg intraperitoneally) or GCV (20 mg/kg) for 9 d, followed by tumor volume measurement. RESULTS: On treatment with trichostatin A or SAHA, H1299 cells carrying p21-3H showed a significant increase of luciferase activity, cellular uptake of (3)H-FEAU (Moravek), and DsRed expression. In vivo tumor xenografts carrying p21-3H also showed increased luciferase activity by luminescent imaging and enhanced accumulation of (18)F-FHBG by small-animal PET imaging. Furthermore, when cells transfected with p21-3H or p21/PstI-3H (which lacks p53-binding sites) were treated, the increase of luciferase activity was similar in both groups, indicating that HDACI-induced p21 promoter activation is independent of p53. Both in vitro and in vivo results showed improved therapeutic effect by combined treatment of GCV and HDACI. CONCLUSION: We have established an HDACI-inducible, p21-driven reporter system that has the potential for evaluating the anticancer effect of HDACIs on cancer cells by multiple molecular imaging modalities. Furthermore, ttksr39 in a p21-3H reporter construct provides a potential combination with thymidine kinase-mediated gene therapy to optimize the therapeutic benefit of HDACI.

Evaluation of lentiviral-mediated expression of sodium iodide symporter in anaplastic thyroid cancer and the efficacy of in vivo imaging and therapy.

Anaplastic thyroid carcinoma (ATC) is one of the most deadly cancers. With intensive multimodalities of treatment, the survival remains low. ATC is not sensitive to (131)I therapy due to loss of sodium iodide symporter (NIS) gene expression. We have previously generated a stable human NIS-expressing ATC cell line, ARO, and the ability of iodide accumulation was restored. To make NIS-mediated gene therapy more applicable, this study aimed to establish a lentiviral system for transferring hNIS gene to cells and to evaluate the efficacy of in vitro and in vivo radioiodide accumulation for imaging and therapy. Lentivirus containing hNIS cDNA were produced to transduce ARO cells which do not concentrate iodide. Gene expression, cell function, radioiodide imaging and treatment were evaluated in vitro and in vivo. Results showed that the transduced cells were restored to express hNIS and accumulated higher amount of radioiodide than parental cells. Therapeutic dose of (131)I effectively inhibited the tumor growth derived from transduced cells as compared to saline-treated mice. Our results suggest that the lentiviral system efficiently transferred and expressed hNIS gene in ATC cells. The transduced cells showed a promising result of tumor imaging and therapy.

Specific activation of sodium iodide symporter gene in hepatoma using alpha-fetoprotein promoter combined with hepatitis B virus enhancer (EIIAPA).

BACKGROUND: This study aimed to develop a novel tumor-specific promoter gene linking sodium iodide symporter (NIS) gene to specifically target hepatocellular carcinoma in a mouse tumor model. MATERIALS AND METHODS: A tumor-specific chimeric promoter for alpha-fetoprotein gene (AFP) was combined with hepatitis B virus (HBV) enhancer II to investigate radioiodine uptake in vitro and in vivo in hepatoma (HepG2) and nonhepatoma (ARO) cell lines after transfer of hNIS gene. A lentiviral vector carrying the hNIS gene was employed in vitro and in vivo. Radionuclide imaging was acquired for 30 min at 60 min after administration of 1241 to monitor hNIS gene expression in vivo using microPET. RESULTS: The highest radioiodide uptake of ARO and HepG2 clones which stably expressed hNIS gene were 87- and 208-fold higher than that of parental cells, respectively. After infection of lentivirus, hNIS gene controlled by cytomegavirus (CMV) promoter was expressed in both ARO and HepG2 cells, and hNIS gene induction by EIIAPA promoter was higher than by CMV promoter in HepG2 cells but not in ARO cells. A similar result was observed in vivo, hNIS controlled by CMV promoter was highly expressed in both HepG2 and ARO tumors. The HepG2 tumor multi-infected with LV-EIIAPA-hNIS virus specifically, but the ARO tumor did not activate the EIIAPA promoter and further express the hNIS protein. CONCLUSION: Transduction of the hNIS gene controlled by the novel EIIAPA chimeric promoter successfully induces iodide transport in hepatoma.

Cre/loxP system controlled by specific promoter for radiation-mediated gene therapy of hepatoma.

BACKGROUND: To specifically target malignant cells, cancer gene therapy needs to combine highly selective gene delivery with highly specific gene expression. In this study, hepatitis B virus (HBV) enhancer II was combined with alpha-fetoprotein (AFP) promoter which selectively controls the Cre/loxP system in hepatoma. MATERIALS AND METHODS: pE4luc, pE4Tk, EIIAPA-Cre and E4CMV-STOP-Tk constructs and AFP promoter combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. RESULTS: The E4 enhancer showed the highest luciferase gene expression at a dose range of 5 approximately 7 Gy after 60 hours irradiation. The EIIAPA chimeric promoter which controls the Cre/loxP system provided high specificity only to the hepatoma cells. In addition, the E4 response to radiation encoded more Herpes simplex virus thymidine kinase (HSV1-Tk) protein and killed more tumor cells. CONCLUSION: The chimeric EIIAPA promoter can precisely control the Cre/loxP switch and the radiation effect on the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of hepatocellular carcinoma (HCC).