Med Oncol (2012) 29:205–211 DOI 10.1007/s12032-010-9788-5
ORIGINAL PAPER
Lung fluorescence imaging to evaluate tumor metastasis induced by AQP5 expression in murine model Zi-qiang Zhang • Zhu-xian Zhu • Chun-xue Bai
Received: 19 November 2010 / Accepted: 15 December 2010 / Published online: 31 December 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Our recent study, by up-regulation of AQP5 expression, showed enhanced proliferation and migration potential in lung cancer. However, so far none of the in vivo study of gene silencing of AQP5 has been tested. In this study, we tested roles of AQP5 on lung cancer metastasis potential by gene silencing of AQP5 in two lung cancer cell lines and tried to monitor lung metastases with EGFP marker. Lungs were imaged at different time points and allowed an accurate evaluation of tumor burden over time. Our results showed significantly decreased metastasis potential in AQP5 gene-silencing cells. Lung imaging confirmed the frequency of metastasis in mice. These data provide more evidence that AQP5 plays important roles in the metastasis potential of lung cancer. Lung fluorescence imaging provides rapid monitoring for tumor growth and metastasis, and it also offers quantitative and sensitive analysis of tumor growth and metastasis, compared to the traditional histology technique.
Zi-qiang Zhang, Zhu-xian Zhu and Chun-xue Bai contributed equally to this work. Z. Zhang (&) Department of pulmonary medicine, Qianfoshan Hospital of Shandong Province, Shandong University, 250014 Jinan, People’s Republic of China e-mail:
[email protected] Z. Zhu Department of nephropathy, Qianfoshan Hospital of Shandong Province, Shandong University, 250014 Jinan, People’s Republic of China C. Bai Department of pulmonary medicine, Zhongshan Hospital, Fudan University, 200032 Shanghai, People’s Republic of China
Keywords Lung imaging Green fluorescence protein (GFP) Metastasis Aquaporin (AQP)
Introduction Studies have demonstrated that AQP has unexpected functions other than just facilitating osmotic water transport [1, 2]. Recent studies [3, 4] suggested its potential role in promoting lung cancer development, and AQP5 expression was associated with poor prognosis in lung cancer. Our recent study [5], by up-regulation of AQP5 expression in lung cancer cell lines, showed enhanced proliferation and migration potential in lung cancer cells and provided evidence for AQP5-facilitated lung cancer proliferation and migration possibly through EGFR/ERK/ p38 MAPK signaling pathway partially. All the above studies suggested AQP5 as a unique biomarker is associated with malignancy in lung cancer. In this study, by gene silencing of AQP5, we further tested roles of AQP5 in lung cancer metastasis potential. Several studies [6–10] have attempted to optimize the methods for monitoring metastases including bioluminescence imaging of cancer cells. However, few reports can provide appropriate methods for monitoring metastases with fluorescence marker such as green fluorescent protein (GFP), because of technical problem for in vivo imaging. Generally, lung fluorescence imaging cannot be detected through soft tissue and skin by imaging system [6]. Moreover, obstacles of in vivo imaging for fluorescence detection are also limited by autofluorescence of mice. Yamamoto et al. [11] reported that when using real-time GFP imaging of lung metastasis by in vivo imaging, a skinflap window was needed to improve resolution attributed to autofluorescence, so it may be performed inefficiently and
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usually lead to inaccuracy results. Moreover, methods of macroscopic and microscopic evaluations for metastases, as they are currently practiced, usually lead to more qualitative than quantitative assessments of tumor spread. For the widely utility of GFP marker for gene-labeling in cells, we wondered whether lung fluorescence imaging by excised lung can provide an appropriate methods for monitoring metastases. In this study, an experimental bloodborne murine lung metastases model was established with intravenously administered EGFP–expressed cancer cells (SPC-A1 or LETP-A2 cell lines). The efficacy of lung fluorescence imaging for monitoring lung metastatic lesions was assessed and further to explore the effects of AQP5 RNAi on lung cancer metastasis.
Materials and methods Cell culture and AQP5 gene silencing Lung cancer cell lines (SPC-A1 and LTEP-A2) with stable EGFP expression obtained from our previous study [5], which showed higher expression of AQP5 compared with other NSCLC cell lines such as NCI-H292 and A549, were selected for this AQP5 gene-silencing study. Cells, grown in RPMI medium (Hyclone, USA) supplemented with 10% FBS (Hyclone, USA), were cultured in a humidified atmosphere in 5% CO2 incubator at 37°C. In this study, AQP5-siRNA mediated by lentiviral vector was first constructed and tested. The siRNA was obtained from GenePharma (Shanghai, China) as described in our previous study [5]. Recombinant lentiviral vector containing AQP5 siRNA was confirmed by PCR and sequencing, then the positive recombinant lentiviral vector was named as Lv-siAQP5 and negative packed virus as control (Lv-NC). AQP5 gene silencing was performed following manufacturer’s protocols. SPC-A1 and LTEP-A2 cells were infected with specific and NC packed virus, respectively, and selected the stable cell lines with blasticidin. AQP5 protein after infection was detected by western blot in order to test the effects of AQP5-siRNA. SPC-A1/Lv-siAQP5, SPC-A1/NC, LTEP-A2/Lv-siAQP5, and LTEP-A2/NC cells were injected intravenously and established the murine metastasis models separately. Then tumor metastasis was monitored at different time points after injection. Pictures were taken by phase-contrast microscopy (Leica, Germany).
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(Chinese Academy of Sciences, China) and were bred and housed in pathogen-free barrier with water and food ad libitum. Litter mate male mice at age of 8 weeks were used in this study. Animal protocol was approved by the ethical committee on animal experiments of the University of Fudan animal care committee, Shanghai, China. Tumor metastasis in vivo In this study, 1 9 106 SPC-A1or LTEP-A2 cells were injected intravenously into nude mice. Mice were then monitored for potential lung tumor colonization and metastases by in vivo fluorescence imaging [12]. Metastatic lesions were identified by fluorescence signals (Night OWL LB 981 in vivo imaging system, Berthold, Germany) and analyzed by WinLight32 Software. Mice were also killed after 2 and 4 weeks, and lungs were harvested for hemotoxylin/eosin staining and AQP5 immunofluorescence staining. The metastatic lesions were identified by histology and fluorescence analysis: Paraffin-embedded sections were stained with hematoxylin and eosin. Lung frozen sections were observed directly or for fluorescence detection by standard procedures as previously described [5]. Lung tumor colonies and fluorescence imaging analysis were performed. Histology Lungs were fixed in 4% paraformaldehyde and embedded in paraffin or OCT for paraffin or frozen slices, respectively. Paraffin-embedded slices were stained with hematoxylin and eosin. Cells or frozen lung tissue sections of adult mice immunofluorescence staining were done as previously described [5]. Pictures were taken by phasecontrast microscopy (Leica, Germany). Immunofluorescence All specimens were formalin-fixed and paraffin-embedded, cut in 4–5 lm thickness. The sections were processed for immunofluorescence analysis as done previously [5]. Images of immunofluorescence staining were photographed under microscope (Leica, Germany). Staining for AQP5 was performed following manufacturer’s protocols, and the following primary antibodies were used: rabbit monoclonal anti-AQP5 (CHEMICON, USA) and rabbit monoclonal antibody with Cy3 marker (cell signaling, USA) was used as second antibody. Antibodies were diluted to 1:100 according to manufacture instructions.
Mice Statistics Experiments were done in weight- and sex-matched male nude mice [BALB/CA-nu/nu] (6-week-old), which were obtained from Shanghai Experimental Animal Center
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Statistics were conducted by SPSS 10.0 software. Values are represented as mean ± SD. Statistical analysis was
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performed by t test and one-way analysis of variance (ANOVA) and LSD post hoc multiple comparison test. All P values were two-sided, and differences at P \ 0.05 were considered statistically significant.
Results Cell lines and AQP5 gene silencing Stable EGFP-expressing cell lines (SPC-A1 and LTEP-A2, obtained from our previous study [5]) were imaged by phase-contrast microscope. Figure 1a showed EGFP protein expression in cell lines, and significant EGFP protein expression was found. In the AQP5 siRNA study, SPC-A1 and LTEP-A2 cells were infected with Lv-siAQP5 or Lv-NC and stably infected cell lines were selected. AQP5 mRNA and protein expression was down-regulated by lentiviral-mediated AQP5-siRNA. By real-time PCR and western blot analysis, AQP5 mRNA and protein expression was detected. Results showed AQP5 mRNA was downregulated by 91.2 and 92.48%, and AQP5 protein was down-regulated by 83.02 and 89.22% in SPC-A1 and LTEP-A2 cells, respectively, compared to parent cells (Fig. 1b).
Fig. 1 AQP5 gene silencing (a) summarized stable EGFP-expressing cells (SPC-A1and LTEP-A2 cell lines), which were imaged by phasecontrast microscope and fluorescence microscope, and significant EGFP protein expression (green) was found. Quantitative real-time RT-PCR and western blot analysis for the examination of AQP5 mRNA and protein, respectively, showed AQP5 mRNA and protein expression was down-regulated by AQP5 siRNA, significantly (n = 4–6, *P \ 0.01) (b) (Color figure online)
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Comparison of fluorescence imaging in vitro and in vivo First, cell lines were evaluated by fluorescence imaging in vitro (Fig. 2a). In another set of experiment, mice were killed when metastasis models were established with SPC-A1 or LTEP-A2 cells injected through tail veins. Then, lungs were excised for fluorescence imaging evaluation by fluorescence signals (Night OWL LB 981 in vivo imaging system, Berthold, Germany), and results showed in Fig. 2b. All data were analyzed by WinLight32 Software (Berthold, Germany). And results showed there were robust correlations between in vitro and in vivo evaluation of fluorescence imaging. Similar results were demonstrated between these two different cell lines.
Fig. 2 Comparison of fluorescence detection in vitro and in vivo. In the in vitro study, SPC-A1 and LTEP-A2 cell lines with stable EGFR protein expression were evaluated by fluorescence imaging (a). In another set of experiment, mice were killed when metastasis models were established. Lungs were excised for fluorescence imaging evaluation (b). The ratio of in vitro to in vivo fluorescence value between these two different cell lines showed no significant difference (n = 6, *P [ 0.05). There are robust correlations in fluorescence evaluation between different cell lines (c)
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Evaluating metastasis potential by lung fluorescence imaging As mentioned previously, nude mice were used to study tumor cell invasion and metastasis. Murine metastasis models were established with SPC-A1 or LTEP-A2 cells. Figure 3a showed excised lungs at different time points before or after metastasis models were established. Lung metastases were monitored for potential lung tumor colonization and metastases by lung fluorescence imaging (Fig. 3b). Metastatic lesions were identified by fluorescence signals and analyzed. Mice were killed 2 and 4 weeks later, after metastasis models were established. Lung fluorescence evaluation was performed by standard procedures. Results demonstrated that down-regulation of AQP5 resulted in significantly decreased metastasis potential on 2 and 4 weeks (Fig. 3c).
Fig. 3 Lung fluorescence imaging to evaluate metastasis potential in vivo. Mice metastasis models were established. a showed excised lungs at different time points before or after metastasis models were established. Lung metastases were monitored for potential tumor colonization and metastases by lung fluorescence imaging. Metastatic lesions were identified by fluorescence signals and analyzed. Results showed down-regulation of AQP5 by RNAi resulted in significantly decreased metastasis potential on 2 and 4 weeks after models were established (b) (n = 6, # P \ 0.05, *P \ 0.01)
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Evaluating metastasis potential by histology analysis Histology analysis showed a number of well-demarcated tumor metastasis nodules in lungs. Tumor colony analysis in lung was performed and results showed AQP5-siRNA resulted in significantly decreased metastasis on 2 and 4 weeks after models were established, which suggested lower AQP5 expression lead to declined metastasis of lung cancer cells (Fig. 4a). Immunofluorescence staining in tumor lesion showed significantly decreased AQP5 expression in AQP5-siRNA group, compared with the control group (Fig. 4b). Moreover, a positive relationship was noted between lung fluorescence imaging and histology analysis in evaluating lung cancer metastasis potential. So the robust correlations between these two methods for metastasis evaluation provided evidence for the potential utility of
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Fig. 4 Histology analysis to evaluate metastasis potential in vivo. Histology analysis showed a number of well-demarcated tumor metastasis nodules in lungs. AQP5 siRNA resulted in significantly decreased metastasis on 2 and 4 weeks, which suggested downregulation of AQP5 expression lead to declined metastasis of lung cancer cells (a) (n = 6–8, #P \ 0.05, *P \ 0.01). Immunofluorescence
staining showed significantly decreased AQP5 expression in AQP5 siRNA group, compared to control group (b). Moreover, correlation analysis demonstrated a positive relationship between lung fluorescence imaging and histology analysis in the evaluation of metastasis (P \ 0.01)
lung fluorescence imaging. Moreover, our results showed more advantages of fluorescence imaging over traditional histology approaches such as increased biological information, short imaging time, and so on.
metastasis and also provided an accurate assessment of gene treatment or even bio-therapies over time. In vivo bio-photonic imaging has introduced a sensitive and rapid alternative to monitor and track tumor development from tumors cells implanted into animal models [13–20], or tumor formed in transgenic animals [21], and even can be used to detect transgene expression [22–25] in various live animal models. Moreover, fluorescence protein including GFP/EGFP permits sensitive fluorescence detection and quantification of cells specifically transfected to emit visible light for in vivo imaging. So GFP/EGFP has been widely used as a bio-marker in cell or animal model researches by far. However, fluorescence in vivo imaging in metastatic tumor models is used infrequently because of technical limitations in detecting metastasis, primarily because of technical limitations in detecting metastasis such as autofluorescence. In our previous study [5], we have tried to explore lung imaging for EGFP fluorescence detection and demonstrated the underlying application of lung fluorescence imaging to monitor metastasis in an experimental animal model. The present study established experimental bloodborne lung metastasis model in mice receiving EGFP-expressing lung cancer cells intravenously and monitored the development of lung metastatic lesions by lung fluorescence imaging.
Discussion Various studies have suggested that AQPs are associated with cell proliferation and migration. Our recent study [5], by up-regulation of AQP5 expression, also showed increased metastasis potential in lung cancer. However, so far none of the in vivo study of gene silencing of AQP5 expression has been tested. In this in vivo study, we applied lung fluorescence imaging to monitor lung cancer metastasis and response to gene silencing of AQP5 by siRNA technique. We developed two cell lines with stable EGFP expression from standard human tumor cell lines including SPC-A1 and LETP-A2. In vitro or in vivo fluorescence detection was performed by a specialized system (Night OWL LB 981 in vivo imaging system, Berthold, Germany). Fluorescence monitoring of tumor growth and metastasis was compared to traditional histology analysis. Our results showed that fluorescence imaging offered a rapid and sensitive monitor of neoplastic
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Results showed cells expressing EGFP in lung metastasis model could be successfully detected. Lung imaging demonstrated excellent effects for lung metastasis evaluation, compared with histology analysis. Moreover, lung imaging demonstrated significant advantage for the evaluation of lung cancer proliferation and metastasis in latter stage, without the interference of necrosis and edema in total tissue mass. This study also demonstrated there are robust correlations between lung fluorescence imaging analysis and histology methods and further illustrated several distinct advantages of lung fluorescence imaging over traditional histology analysis in tumor monitoring: The high sensitivity of fluorescence and relatively rapid detection were effectively demonstrated. Helping to identify very small metastatic lesions not detectable in vivo contributed toward a more accurate assessment of tumor spread, thus helping to provide more biological information, short imaging time, and so on. In fact, tumor metastasis involves a series of complex host–tumor interactions [10, 26, 27]. Studies have suggested that AQP-facilitated water permeability in cell protrusions enhances their formation and thus the rate of cell migration. AQP expression and tumor cell water permeability are potentially important determinants for tumor spread and metastasis. Our previous study demonstrated significantly increased water permeability in AQP5-expressing tumor cells, which may be one mechanism for enhanced metastatic potential. And another possibility of enhanced malignancy tendency associated with AQP5 expression may be partially due to up-regulation of MUC5AC mucin production. Our results showed that lung metastases significantly decreased with the down-regulation of AQP5 expression with gene silencing. So this in vivo study provides more evidence that AQP5 plays important roles in lung cancer development. Taken together, the data extend findings from our previous work [5] that AQP5 plays important roles in lung cancer development and provide more evidence for the potential roles of AQP5 in lung cancer metastasis. Moreover, by validating lung imaging with direct comparison to tumor histology analysis, demonstrating the potential utility of lung fluorescence imaging in evaluation of cancer metastasis, especially fluorescence protein marker, which usually disturbed by technical problem such as tissue barrier or autofluorescence. Acknowledgments We thank Dr. J Deng (University of Texas M. D. Anderson Cancer Center, USA) for valuable advice.
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