Liang-Ting Lin

Evaluation of the Therapeutic and Diagnostic Effects of PEGylated Liposome–Embedded 188Re on Human Non–Small Cell Lung Cancer Using an Orthotopic Small-Animal Model

Non–small cell lung cancer (NSCLC) is a highly morbid and mortal cancer type that is difficult to eradicate using conventional chemotherapy and radiotherapy. Little is known about whether radionuclide-based pharmaceuticals can be used for treating NSCLC. Here we embedded the therapeutic radionuclide 188Re in PEGylated (PEG is polyethylene glycol) liposomes and investigated the biodistribution, pharmacokinetics, and therapeutic efficacy of this nanoradiopharmaceutical on NSCLC using a xenograft lung tumor model and the reporter gene imaging techniques. Methods: Human NSCLC NCI-H292 cells expressing multiple reporter genes were used in this study. 188Re was conjugated to N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA) and loaded into the PEGylated liposome to form a 188Re-liposome. The tumor growth rates and localizations were confirmed using bioluminescent imaging and SPECT/CT after the 188Re-BMEDA or 188Re-liposome was intravenously injected. The accumulation of the nanodrug in various organs was determined by the biodistribution analysis and the nano-SPECT/CT system. The pharmacokinetic and dosimetric analyses were further determined using WinNonlin and OLINDA/EXM, respectively. Results: The biodistribution and nano-SPECT/CT imaging showed that PEGylated 188Re-liposome could efficiently accumulate in xenograft tumors formed by NCI-H292 cells that were subcutaneously implanted in nude mice. Pharmacokinetic analysis also showed that the retention of 188Re-liposome was longer than that of 188Re-BMEDA. In an orthotopic tumor model, ex vivo γ counting revealed that the uptake of 188Re-liposome was detected in tumor lesions but not in surrounding normal lung tissues. Moreover, we evaluated the therapeutic efficacy using bioluminescent imaging and showed that the lung tumor growth was suppressed but not eradicated by 188Re-liposome. The life span of 188Re-liposome–treated mice was 2-fold longer than that of untreated control mice. Conclusion: The results of biodistribution, pharmacokinetics, estimated dosimetry, nano-SPECT/CT, and bioluminescent imaging suggest that the PEGylated liposome–embedded 188Re could be used for the treatment of human lung cancers.

Synergistic effects of glycated chitosan with high-intensity focused ultrasound on suppression of metastases in a syngeneic breast tumor model.

Stimulation of the host immune system is crucial in cancer treatment. In particular, nonspecific immunotherapies, when combined with other traditional therapies such as radiation and chemotherapy, may induce immunity against primary and metastatic tumors. In this study, we demonstrate that a novel, non-toxic immunoadjuvant, glycated chitosan (GC), decreases the motility and invasion of mammalian breast cancer cells in vitro and in vivo. Lung metastatic ratios were reduced in 4T1 tumor-bearing mice when intratumoral GC injection was combined with local high-intensity focused ultrasound (HIFU) treatment. We postulate that this treatment modality stimulates the host immune system to combat cancer cells, as macrophage accumulation in tumor lesions was detected after GC-HIFU treatment. In addition, plasma collected from GC-HIFU-treated tumor-bearing mice exhibited tumor-specific cytotoxicity. We also investigated the effect of GC on epithelial–mesenchymal transition-related markers. Our results showed that GC decreased the expression of Twist-1 and Slug, proto-oncogenes commonly implicated in metastasis. Epithelial-cadherin, which is regulated by these genes, was also upregulated. Taken together, our current data suggest that GC alone can reduce cancer cell motility and invasion, whereas GC-HIFU treatment can induce immune responses to suppress tumor metastasis in vivo.

Low-fluence rate, long duration photodynamic therapy in glioma mouse model using organic light emitting diode (OLED)

BACKGROUND The treatment of gliomas poses significant clinical challenges due to resistance to chemo and radiation therapy, and treatment side effects. Metronomic photodynamic therapy (mPDT), which involves long treatment time with low fluence rate and multiple or continuous photosensitizer administrations, has potential in treating gliomas without threatening the quality of life and has been demonstrated in rats and rabbits. mPDT in small animals such as mouse is not yet shown due to lack of lightweight illumination device for long periods of time. METHODS We presented low fluence rate (3mW/cm(2)) and long duration (3.7h) PDT treatment in a nude mouse model of human glioblastoma by using organic light emitting diode (OLED) with single dose of 5-aminolevulinic acid (ALA) administration as photosensitizer. Tumor volume was measured using bioluminescent imaging and the animal survival time was recorded. Additionally, we have performed limited PDT dosimetric measurements of PpIX fluorescence, tumor oxygenation and hemoglobin concentration in 3 PDT mice. RESULTS For animals with similar pre- and immediate post-light tumor volume, the averaged total survival time of PDT mice is 40.5±9.2 days that are significantly longer than the control mice (26.0±2.0 days). The post-light survival time of PDT mice is 14.3±5.9 days that are marginally longer than the control group (8.0±0.0 days). In the dosimetric measurement, good maintenance of PpIX fluorescence in one PDT mouse has relatively improved survival time, compared with the other two PDT mice (i.e., 24 days versus 16 and 17 days). CONCLUSIONS This pilot study demonstrated the feasibility of low-fluence rate and long treatment time of ALA-PDT using OLED without anesthetization of animals. The response of PDT treated animals with similar pre- and post-light tumor volume is encouraging to show a longer survival time than the controls. The dosimetric indices such as photosensitizer fluorescence and tissue oxygenation would help understand the possible treatment barriers for further improvement of treatment plans.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7 days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2′-fluoro-1-beta-D-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo.

A Comparative Study of Primary and Recurrent Human Glioblastoma Multiforme Using the Small Animal Imaging and Molecular Expressive Profiles.

PurposeGlioblastoma multiforme (GBM) is the most malignant brain tumor with the characteristics of highly infiltrative growth and recurrent rate. In this study, we used animal imaging and molecular expressive profiles to investigate the characteristics of the primary tumor (GBM-3) cells and recurrent tumor (S1R1) cells from different GBM patients.ProceduresBioluminescent imaging and 3T magnetic resonance imaging (MRI) were used for assessing the orthotopical tumor development of GBM cells harboring a polycistronic reporter gene system. Western blot analysis and quantitative polymerase chain reaction were used to compare the molecular expressive profiles of two types of GBM cells.ResultsS1R1 cells exhibited apparent invasive ability compared to GBM-3 cells using in vitro invasion assay. In vivo bioluminescent imaging showed that intracranial tumors are formed by both types of GBM cells, but the bioluminescent signal was also detected in the lumbar region at late-stage tumor formed by S1R1 cells. The MRI showed that intracranial tumors formed by S1R1 cells were highly infiltrative compared to that formed by GBM-3 cells. Additionally, these two GBM types expressed different patterns of molecules associated with tumor development. Moreover, the suppressive effects of interleukine-23 (IL-23) on xenograft tumors formed by both GBM types were detected using bioluminescent imaging.ConclusionThe current data suggest that the in vivo growth behaviors and therapeutic responses of the primary and recurrent human GBMs were comparable using the reporter gene imaging, and different molecular expressive profiles exist between these two GBM types.

mPlum-IFP 1.4 fluorescent fusion protein may display Förster resonance energy transfer associated properties that can be used for near-infrared based reporter gene imaging.

Abstract. Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Förster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovativefluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo.

Involvement of let-7 microRNA for the therapeutic effects of Rhenium-188-embedded liposomal nanoparticles on orthotopic human head and neck cancer model

Human head and neck squamous cell carcinoma (HNSCC) is usually treated by surgical resection with adjuvant radio-chemotherapy. In this study, we examined whether the radiopharmaceutical 188Re-liposome could suppress the growth of HNSCC followed by an investigation of molecular mechanisms. The orthotopic HNSCC tumor model was established by human hypopharyngeal FaDu carcinoma cells harboring multiple reporter genes. The drug targeting and therapeutic efficacy of 188Re-liposome were examined using in vivo imaging, bio-distribution, pharmacokinetics, and dosimetry. The results showed that 188Re-liposome significantly accumulated in the tumor lesion compared to free 188Re. The circulation time and tumor targeting of 188Re-liposome were also longer than that of free 188Re in tumor-bearing mice. The tumor growth was suppressed by 188Re-liposome up to three weeks using a single dose treatment. Subsequently, microarray analysis followed by Ingenuity Pathway Analysis (IPA) showed that tumor suppressor let-7 microRNA could be an upstream regulator induced by 188Re-liposome to regulate downstream genes. Additionally, inhibition of let-7i could reduce the effects of 188Re-liposome on suppression of tumor growth, suggesting that let-7 family was involved in 188Re-liposome mediated suppression of tumor growth in vivo. Our data suggest that 188Re-liposome could be a novel strategy for targeting HNSCC partially via induction of let-7 microRNA.

Abstract 3548: Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit

Glioblastoma multiforme (GBM) is classified the fourth grade of human glioma that indicates the most malignant brain tumor with poor five-year survival (5%). Accumulated evidence showed that GBM cells were resistant to various therapeutic strategies. Recently, use of arsenic trioxide (ATO) to induce apoptosis and to increase the radiosensitivity was reported for the benefit in treating GBMs. However, the responsible signaling cascades are still remain investigation. MicroRNA, a category of RNA interference, regulates its downstream genes post transcriptionally by binding to the 3′untraslated region (UTR) of its targets. In this study, we used S1 and S1R1 GBM cells which were derived from the primary and the recurrent lesion respectively. Firstly, we identified that an oncogenic microRNA, miR-182, was down-regulated after the treatment of ATO in both GBM cells. In addition, one of the miR-182 targeted coding genes, Sestrin 2 (SESN2), plays an important role in relieving intracellular oxidative stress and participates in TP53 tumor suppressive pathway. Use of luciferase-based reporter assay confirmed that miR-182 directly bound to SESN2 3′UTR and interrupted the further translation. Intriguingly, the basal level of miR-182 in the primary S1 GBM was significantly lower than the recurrent S1R1 GBM, supporting the oncogenic evidence in the previous studies. The counteracted SESN2 expression was also determined by real-time qPCR and western blot. We then hypothesized that ATO may retarded the growth of GBM through a miR-182-SESN2 related regulatory axis. Overexpression of miR-182 in S1 GBM resulted in a decrease of SESN2 and insensitive to ATO treatment. On the opposite, inhibition of miR-182 in S1R1 GBM dramatically retarded the growth rate and sensitized the cells to ATO. Moreover, pretreatment of an antioxidant, N-acetyl-cysteine (NAC), rescued the miR-182 suppression, suggesting that ATO-induced oxidative stress may be dominant in this responsive axis. Concordant expression of miR-182/96/182 cassette was revealed, indicating the transcriptional regulation was unveiled. Further investigation on the detailed machinery still remain elucidating. Based on our findings, we displayed that ATO-induced oxidative stress triggered a tumor suppressive response through a miR-182-SESN2 dependent machinery. Citation Format: Liang-Ting Lin, Shih-Hwa Chiou, Yi-Jang Lee. Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit. [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 3548. doi:10.1158/1538-7445.AM2014-3548

Abstract 1243: Cofilin mediates actin destabilization and shape change in replicative senescence and stress-induced premature senescence

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cellular senescence is an irreversible growth arrest mechanism that prevents indefinite proliferation of mammalian cells. Change of morphology is a common characteristic of senescent cells, while the underlying mechanism has not been completely addressed. In this study, we found that the flattened and enlarged cell shape in aged WI-38 fibroblast was accompanied by reduced actin cytoskeletal organization and up-regulated cofilin-1, a member of actin depolymerization factor family. Up-regulation of cofilin-1 was also detected in various tissues (lung, kidney, heart and liver) of one-year old BALB/c mice compared to that of 6-week old mice. Forced expression of cofilin-1 in early passaged cells accelerated senescence, while knockdown of cofilin-1 in late passaged cells inhibited senescence. Hydrogen peroxide mediated stress-induced premature senescence (SIPS) also induced cofilin-1 in human lung cancer cell lines, and this phenomenon was suppressed by knockdown of cofilin-1. Up-regulation of cofilin-1 in SIPS was independent of p53, while p27kip1 was essential for cofilin-1 induced senescence. Knockdown of p27kip1 in cells treated with hydrogen peroxide or in that with over-expressed cofilin alleviated senescence. Moreover, forced expression of cofilin-1 was sufficient to induce senescence in human lung cancer cells and repress the growth of xenograft tumor in NOD-SCID mice. Taken together, we found that cofilin-1 is able to mediate the cell senescence that is important for repressing the cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2011-1243