Magnetic surface-enhanced Raman spectroscopic (M-SERS) dots for the identification of bronchioalveolar stem cells in normal and lung cancer mice

Biomaterials 30 (2009) 3915–3925

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Magnetic surface-enhanced Raman spectroscopic (M-SERS) dots for the identification of bronchioalveolar stem cells in normal and lung cancer mice Mi Suk Noh a, b, f,1, Bong-Hyun Jun c,1, Seongyong Kim b, d,1, Homan Kang b, c, Min-Ah Woo a, b, Arash Minai-Tehrani a, Ji-Eun Kim a, b, Jaeyun Kim c, g, Jooyoung Park c, Hwang-Tae Lim a, b, Se-Chang Park a, Taeghwan Hyeon c, g, Yong-Kweon Kim e, Dae Hong Jeong d, *, Yoon-Sik Lee c, *, Myung-Haing Cho a, b, h, ** a

Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea Interdisciplinary Program in Nano-Science and Technology, Seoul National University, Seoul 151-742, Republic of Korea c School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea d Department of Chemistry Education, Seoul National University, Seoul 151-742, Republic of Korea e School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-742, Republic of Korea f Korea Institute of Radiological and Medical Sciences, KIRAMS, Seoul 139-706, Republic of Korea g National Creative Research Initiative Center for Oxide Nanocrystalline Materials, Seoul 151-742, Republic of Korea h National Institute of Toxicological Research, KFDA, Seoul 122-704, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 January 2009 Accepted 26 March 2009 Available online 2 May 2009

Bronchioalveolar stem cells (BASCs) play an important role in the development of cancer. To study the characterization of BASCs, their isolation and purification are important. However, the cells are very rare in tissues and the available methods of isolating them are limited. The current study was performed to isolate BASCs in the murine lung using magnetic nanoparticle-based surface-enhanced Raman spectroscopic dots (M-SERS Dots). We used K-rasLA1 mice, a laboratory animal model of non-small cell lung cancer of human, and C57BL/6 mice having the same age as a control. We compared the BASCs between 2 species by FACS analysis with 4 markers of BASCs, CCSP, SP-C, CD34, and Sca-1. We found that BASCs were more abundant in the K-rasLA1 mice than in the C57BL/6 mice. Also, the M-SERS Dot-mediated positive selection of the CD34pos cells enabled the BASCs to be enriched to an approximately 4- to 5-fold higher level than that in the case without pre-separation. In summary, our study demonstrates the potential of using M-SERS Dots as a sorting system with very effective isolation of BASCs and multiplex targeting probe, showing that they may play an effective role in the study of BASCs in the future. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Bronchioalveolar stem cells Lung cancer Magnetic nanoparticles Cell sorting Multiple targeting Raman spectroscopy

1. Introduction The identification and isolation of cancer stem cells (CSCs) have challenged researchers for decades [1–4]. Recently, the existence of the putative CSCs, bronchioalveolar stem cells (BASCs), in lung cancer has been demonstrated [5–7]. BASCs, localized in the bronchioalveolar duct junction (BADJ), contain Clara cells and alveolar type II cells [6–9]. Moreover, the transformed counterparts of BASCs are known to cause adenocarcinoma. Kim et al. [6] demonstrated that BASCs are genuine lung epithelial stem cells that respond to bronchioalveolar damage.

* Corresponding authors. ** Corresponding author. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea. E-mail addresses: [email protected] (D.H. Jeong), [email protected] (Y.-S. Lee), [email protected] (M.-H. Cho). 1 Equal contribution to this work. 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.03.059

Simultaneously, the stem cells proliferate extensively at the site of Kras-induced tumors. In spite of the extensive studies of putative lung stem cells that have been carried out, the available methods of isolating these cells remain limited [10]. Up to the present, many technologies have been developed for effective cell isolation. Fluorescence-activated cell sorting (FACS) is one of the most widely used cell separation technologies [11–13]. The primary advantage of FACS is that it allows for the isolation of cell subsets with high purity based on the cell size and granularity. Also, the diverse fluorescence signals originating from antibodies that are bound at the cell surface make it possible to discriminate specific cell populations. Kim et al. [6] and Yanagi et al. [5] demonstrated several marker proteins for the detection and isolation of BASCs, the Clara cell specific protein (CCSP), surfactant protein C (SP-C), cell differentiation antigen (CD34), and stem cell antigen-1 (Sca-1). However, FACS has limitations in terms of the low viability of the recovered cells and its inability to process large numbers of cells [1,12].

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Magnetic-activated cell sorting (MACS) is used to overcome the drawbacks of FACS. The advantage of MACS is to isolate the desired cells efficiently from a large number of cells within a short period of time [11,13]. MACS utilizes magnetic microbeads or nanoparticles to target the specific cells. After magnetic labeling, the cells are passed through a column which has a strong permanent magnet. Therefore, the magnetically labeled cells are retained in the column. Based on these characteristics, David et al. [13] introduced a method of purifying differentiated embryonic stem cells using MACS. In a recent study, magnetic nanoparticles were used for the discrimination of tumor cells from leukocytes in peripheral blood [14,15]. Magnetic nanoparticle-based surface-enhanced Raman spectroscopic dots (M-SERS Dots), which were developed by our multidisciplinary research group, constitute a new type of analytical tool for multiplexing and separating targeted cells with magnetic properties. M-SERS Dots could have diverse encoding chemicals with intrinsic Raman signals for multiple biomedical applications. Also, their detection limit is very low, because the signal can be enhanced by applying a silver coating [16,17]. The fact that stem cells are very rare in the lungs and the available methods of isolating cancer stem cells are limited, combined with the poor prognosis of lung cancer that involves cancer stem cells, prompted us to develop a novel isolation technology for BASCs in the lungs of normal and K-rasLA1 mice. In this study, M-SERS Dots were used to isolate BASCs effectively by the positive selection of the CD34 marker in the murine lung. BASCs were isolated effectively from diverse lung cells by M-SERS Dots, a powerful technique of magnetic separation, and FACS analysis. We were able to enrich BASCs in the CD34pos cell group with a high percentage. Herein, we report that M-SERS Dots can be employed for multiple targeting as well as the effective isolation of BASCs. This method of isolating rare cells in the tissue may serve as a platform technology to understand the characteristics of specific cells applicable to therapeutic strategies in the future. 2. Materials and methods 2.1. Animal experiment Animal experiments were carried out on 14–16 weeks C57BL/6 mice and K-rasLA1 mice. The C57BL/6 mice were purchased from Joongang Laboratory Animals (Seoul, Korea) and the breeding K-rasLA1 mice were obtained from the National Cancer Institute (Frederick, MD, USA). The animals were kept in a laboratory animal facility with the temperature and relative humidity maintained at 23  2  C and 50  20%, respectively, under a 12 h light/dark cycle. All of the methods used in this study were approved by the Animal Care and Use Committee at Seoul National University (SNU-070830-1).

cover slips were mounted using DakoCytomation Faramount Aqueous Mounting Medium (DakoCytomation, Denmark), and the slides were reviewed using a Confocal Laser Scanning Microscope (CLSM) (Carl Zeiss). The antibodies used for staining the bronchioalveolar stem cells were CCSP, and SP-C (Santa Cruz). The secondary antibodies are conjugated with anti-rabbit FITC and anti-goat TRITC (Zymed Lab, San Francisco, CA, USA). 2.4. IF assay in primary cells The total lung cells were extracted using a previously reported method. Mice were anesthetized and perfused with 10 mL PBS, followed by intra-tracheal instillation of 1 mL dispase (Becton Dickinson) and 1 mL 1% LMP agarose. Lungs were iced, minced and incubated in 0.001% DNAse (Sigma) and 2mg/mL collagenase/dispase (Roche) in PBS for 45 min at 37  C with shacking condition, filtered through 100 mm and 40 mm cell strainers (Fisher), and centrifuged at 800 rpm, 5 min at 4  C. Cells were resuspended in 10% FBS/PBS. Then, the cells were incubated on a Lab-tek glass chamber slide (Nalge Nunc International, Naperville, IN, USA) with DMEM-F12 media (Invitrogen-Gibco, Carlsbad, CA, USA) containing 10% FBS. After 3 days, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min and then washed with PBS 2 times. The fixed cells were treated with 0.25% TritonX-100/PBS for 10 min only for the purpose of staining the intracellular proteins, especially, CCSP and SP-C. After washing with PBS, the fixed cells were treated with 1% BSA in PBS for 30 min to block the nonspecific binding sites. Primary antibodies (CCSP, SP-C or CD34, Sca-1) were applied to the slides overnight at 4  C or 1 h at room temperature. CCSP and SP-C were diluted in 1% BSA (1:100), and CD34 (Abcam, Cambridge, UK) and Sca-1 (R&D Systems, Minneapolis, MN, USA) were diluted in 1% BSA (1:50). The following day, the slide of the cells was washed and incubated with secondary fluorescence-conjugated antibodies (1:50) for 1 h at room temperature. The concentration of anti-rabbit, anti-rat FITC (Zymed Lab) and anti-goat TRITC conjugated antibodies were same. For BASCs staining, primary cells were treated with anti-goat AF647 (Invitrogen) and anti-rat FITC (Zymed Lab) were used to same concentration of other secondary antibodies. After careful washing, the cells were treated with DAPI (SantaCruz) for nuclei staining. 2.5. Flow cytometric analyses of BASCs Single-cell suspensions were prepared from the lungs and perfused with PBS according to the method previously described [6]. The total lung cells were resuspended in 10% FBS/PBS at 3  106 cells/100 ml and stained with APC-conjugated anti-Sca-1 (0.18 mg/3  106 cells, eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD34 (3 mg/3  106 cells, eBioscience) and PE-conjugated anti-CD31 (PECAM-1, 1.5 mg/3  106 cells, eBioscience) and anti-CD45 (0.09 mg/3  106 cells, eBioscience) for 30 min in an ice box with dark condition. The antibodies were diluted in 1% BSA. Subsequent centrifugation of the samples was performed with washing, 10% FBS/PBS with several times. The stained cells were analyzed directly. For the application of the M-SERS Dots, the total lung cells were resuspended in 10% FBS/PBS at 3  106 cells/ 100 ml and treated with M-SERS Dots conjugated with CD34 marker for 2 h. The targeted cells were collected by applying a magnetic force. The supernatant was removed from the sample tube while holding magnet. Then, the cell population was incubated with FITC-conjugated anti-Sca-1 (1.5 mg/3  106 cells, eBioscience), and PE-conjugated anti-CD31, CD45 for 30 min with the same concentration and condition as described above. Flow cytometry was performed using a BDFACS AriaÔ (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed with BDFACS AriaÔ software. 2.6. Cytotoxicity assay

2.2. H&E staining The lung tissues were fixed in 10% neutral buffered formalin, paraffin processed, and sectioned at 3 mm. For the histological analysis, the tissue sections were stained with hematoxylin and eosin (H&E). Then, the cover slips were mounted using DakoCytomation Faramount Aqueous Mounting Medium (DakoCytomation, Copenhagen, Denmark), and the slides were reviewed using a light microscope (Carl Zeiss, Thornwood, NY, USA). 2.3. Immunofluorescence (IF) assay in tissues For the IF assay, the fixed lung tissue sections were deparaffinized in xylene and rehydrated through an alcohol gradient. The tissue sections were incubated in 150 ml proteinase K, washed and incubated in 3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 30 min to quench the endogenous peroxidase activity. After washing in PBS, the tissue sections were incubated with 1% BSA in PBS for 1 h at room temperature to block the nonspecific binding sites. Primary antibodies were applied to the tissue sections overnight at 4  C. CCSP and SP-C antibodies were diluted in 1% BSA (1:50). The following day, the tissue sections were washed and incubated with secondary fluorescence-conjugated antibodies, FITC (green) and TRITC (red), (1:50) for 2 h at room temperature. After careful washing, the tissue sections were treated with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) briefly (5 min) for nuclei staining. Then, the

Primary cells from fresh murine lung tissues were seeded in 96-well tissue culture dishes at 50,000 cells per well. They were cultured for 3 days, and the cells were attached to the surface of the dishes. They were treated with M-SERS Dots of 10 ml in 100 ml DMEM-F12 media (Invitrogen-Gibco, contained 10% FBS) with diverse concentrations of the nanoparticles for 2 h. After the treatment with the M-SERS Dot solution, the cytotoxicity was measured by the MTT [3-(4,5-dimethylthiazo-2-yl)2,5-diphenyltetrazolium bromide, Sigma–Aldrich, St. Louis, MO, USA] assay. MTT solution 20 ml (5 mg/ml) was added to the well and incubated with the cells for 4 h at 37  C. The cells were treated with 100 ml of dimethylsulfoxide (DMSO, Sigma– Aldrich) and the absorbance was quantified using an ELISA reader (BioRad, Hercules, CA, USA). 2.7. Preparation of antibody conjugated M-SERS Dots M-SERS Dots containing 18-nm magnetite in the core were coated with Ag nanoparticle and silica [17]. A 100-ml portion of CD34 antibodies (0.1 mg/ml) or Sca1 antibodies (0.1 mg/ml) was added to the M-SERS Dots containing the encoding chemical benzenethiol (BT) or 4-methylbenzenethiol (4-MT) dispersed in PBS, and then the resulting mixture (1.15 mg/ml, 1 ml M-SERS Dot) was stirred for 1.5 h at 25  C [17,18]. The resulting M-SERS dots were centrifuged and washed with PBS solution. The M-SERS dots were treated with bovine serum albumin [1%(wt) in PBS solution] for 30 min at 25  C and then washed with PBS solution.

M.S. Noh et al. / Biomaterials 30 (2009) 3915–3925 2.8. Multiple detection in primary cells with M-SERS Dots After the extraction of the primary cells of the fresh murine lung tissues, the total lung cells were incubated in a Lab-tek glass chamber slide (Nalge Nunc International) in a CO2 incubator for 3 days. Then, the cells were fixed with paraformaldehyde (4%) for 15–30 min at 25  C. Three day-incubated cells which are attached on the surface of chamber slide are better to perform multiple target detections because several washings are enough to remove most of the nonspecifically bound M-SERS Dots. The fixative agent was removed by centrifugation, and then the cells were washed with PBS solution. Simultaneously, the primary cells were incubated with M-SERS DotBT/CD34 (1.15 mg/ml) or M-SERS Dots4-MT/Sca-1 (1.15 mg/ml), diluted to 10% in total media, DMEM/F12 with 10% FBS, at 4  C overnight for the targeting study. After incubation, the cells were washed with PBS solution containing Tween 20 (0.1 wt%) several times. 2.9. Raman measurement of M-SERS Dots The Raman measurements were performed using a micro-Raman system (JYHoriba, Labram 300) equipped with an integral microscope (Olympus BX41, NA ¼ 0.95, 100). In this system, the 514.5 nm laserline from an Ar ion laser (MellesGriot, 35-MAP321) was used as an excitation source and the Raman scattering signals were collected in 180 scattering geometry. 2.10. Data analysis All results are given as means  s.e.m. The results were analyzed by Student’s ttest (Graphpad software, San Diego, CA). *P < 0.05 was considered significant and **P < 0.01 highly significant compared to corresponding control.

3. Results 3.1. Histopathology of normal and lung cancer tissues To understand the expression level of BASCs in the normal C57BL/6 and K-rasLA1 mice in terms of lung cancer development, we chose to use the model mice at the age of 14–16 weeks. Fig. 1A and C show the representative normal lung of the C57BL/6 mice. Fig. 1B and D are from the K-rasLA1 mice. The white circle is the tumor area with significantly progressed cancer (Fig. 1B). For the histological analysis, the tissue sections were stained with H&E. The histopathological study demonstrated the presence of adenocarcinoma in the form of a red circle in the lung tissues of the K-rasLA1 mice (Fig. 1D).

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3.2. Detection of BASCs in vitro and in vivo For the detection of the BASCs, we examined the expression pattern of several markers under in vitro system. We carried out dual staining of intracellular markers (CCSP and SP-C) and surface markers (CD34 and Sca-1), in the primary cells of lung tissues. CD34 and Sca-1 markers were detected at the surface of the cell population with the colors green and red of conjugated fluorescence, respectively (Supplementary material, S 1A). CCSP and SP-C markers were observed in the intracellular region at the primary cells with green and red colors, respectively, as determined by CLSM (Supplementary material, S 1B). Also, we detected BASCs at the BADJ of the lung tissues with CCSP and SP-C to compare the double positive cells between the normal and K-rasLA1 mice. For this purpose, the tissues of the normal and K-rasLA1 mice were stained with CCSP (green) and SP-C (red) markers. As shown in Fig. 2A, the normal lung of the C57BL/6 mice was stained with the two markers (CCSP and SP-C). The square area of black dots was amplified to observe the BASCs in the BADJ. The double positive cells were distinguished by their combined green and red color by immunofluorescence (Fig. 2A-i). The white arrows indicate the double positive cells. Fig. 2B shows the staining results of the K-rasLA1 mice with the CCSP and SP-C markers. Interestingly, the K-rasLA1 mice contained a higher BASC population than the normal mice significantly (white arrows, Fig. 2B-i). To compare the number of BASCs that are positive to both the CCSP and SP-C antibodies, we investigated the BADJ areas in the lung tissue. As shown in Fig. 2C, the double positive cells were counted at 4 random selections of the BADJ areas at 400  magnification. From the graph, we demonstrated that the model mice of lung cancer had a larger amount of BASCs than the normal mice. 3.3. Separation and comparison of BASCs in normal and K-rasLA1 mice In order to verify the potential difference between the BASCs in the lung tissues of the C57BL/6 and K-rasLA1 mice, the amount of BASCs was measured by FACS analysis. We hypothesized that BASCs

Fig. 1. Histopathology of normal and lung cancer tissues. Experiments were carried out on 14–16 week C57BL/6 mice and K-rasLA1 mice. (A) and (C) are from the normal lung tissues, while (B) and (D) are from the lung cancer tissues. In (B) the K-rasLA1 mice have numerous visible tumor lesions (marked in yellow circle). (C) and (D) show the histopathological characteristics of the normal lung and K-rasLA1 mice, respectively. The red circle in (D) indicates the tumor in the lungs of the K-rasLA1 mice. The scale bar represents 50 mm.

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Fig. 2. Identification of BASCs and quantitative analysis of BASCs in normal and lung cancer tissues. The BASCs are located at the bronchioalveolar duct junction (BADJ). The black squares in (A) and (B) indicate the BADJ region, which was amplified to provide a better view. The green (FITC) and red (TRITC) fluorescences were from CCSP and SP-C, respectively. The blue fluorescence (DAPI) was used to stain the nuclei. The images in (A) are from the normal lungs of the C57BL/6 mice, whereas those in (B) are from the lung cancer tissue of the K-rasLA1 mice. (a) CCSP stained image, (b) bright field image, (c) SP-C stained image, (d) DAPI stained image, and (e) merged image (400). The amplified images are from the black square, where the white arrows indicate the BASCs (double positive cells, 800). AS: alveolar sac, TB: terminal bronchiole. (C) is the graph of double positive cells with CCSP and SP-C markers in normal and lung cancer mice. The double staining was determined by counting 4 randomly chosen BADJ areas per section at 400  magnification (*P < 0.05 is compared to normal, mean  s.e.m., n ¼ 4).

might be more abundant in the lung cancer tissue than that in the normal lung. The surface markers of BASCs, CD34 and Sca-1 were testified to the expression level by the FACS analysis (Supplementary material, S 1B). The positive marker was conjugated with FITC. Also, the hematopoietic and endothelial lineage cells were excluded from the total lung cells in order to purify the BASCs using CD45 and Pecam-1(CD31) conjugated with PE, by fluorescent

labeling. The application of the positive markers, CD34 and Sca-1 for better FACS analysis and targeting by M-SERS Dot is very important to achieve a high yield and purification. As shown in the Supplementary material, S 2A and B, a comparison was made of the ratio of BASC like cells between the CD34 and Sca-1 markers in normal mice. The results show that the positive cell population of Sca-1 markers is lower than the cell population of the CD34

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markers. Supplementary material, S 2C, shows the results corresponding to 4 independently repeated tests using FACS analysis. The relative amounts of BASCs between the normal and K-rasLA1 mice were determined by analyzing the cell populations of Sca1posCD34posCD45negCD31neg (APCþ/FITCþ/PE) using FACS analysis (Fig. 3). The total number of cells was 3  106. After collecting the CD34posCD45negCD31neg cells (FITCþ/PE), the Sca-1pos cells (APCþ) were isolated from them. Finally, the Sca-1posCD34posCD45negCD31neg cells (APCþ/FITCþ/PE) were identified as BASCs. As shown in Fig. 3A and B, the amount of BASCs in the KrasLA1 mice was higher than the cell population of the normal mice (C57BL/6: CD34posCD45neg CD31neg – 0.6%, Sca-1pos – 26.1% in CD34posCD45neg CD31neg cells, K-rasLA1: CD34posCD45neg CD31neg – 1.3%, Sca-1pos – 35.0% in CD34posCD45neg CD31neg cells). Fig. 3A and B is the representative data from 6 independently repeated experiments. The image of IF was obtained to verify the BASCs from the sorted cells (CD34posSca-1posCD45neg CD31neg) with the dual

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positive markers, CCSP and SP-C (Fig. 3C and D). Therefore, we confirmed the presence of BASCs with 4 markers of CD34posSca-1posCCSPposSP-Cpos. The results showed that the BASCs existed more abundantly in the K-rasLA1 mice than in the normal lung. 3.4. Analysis of BASCs by M-SERS Dots Fig. 4A shows the structure of the M-SERS Dots that have magnetic nanoparticles in their core area. Ag nanoparticles were used to enhance the Raman signal. Also, they have a silica shell which provides them with good biocompatibility with live cells (Fig. 4A). Fig. 4B shows TEM images of M-SERS Dots. Fig. 4C shows the typical Raman peaks for the encoding chemicals, benzenethiol (BT) and 4-methylbenzenethiol (4-MT). The peak of the M-SERS Dots was sufficient to discriminate the encoding chemicals when they are bound to the biomolecules. Therefore, M-SERS Dots constitute a novel nanoprobe for the multiplex detection and

Fig. 3. Isolation of BASCs from normal and lung cancer tissues by FACS analysis. (A) and (B) exhibit the typical scatter plots of the FACS analysis from the 14–16 weeks’ old C57BL/6 mice and K-rasLA1 mice, respectively. The primary cells (3  106 cells/100 ml) from the lung tissues were stained with CD34-FITC, CD45-PE, Pecam-1(CD31)-PE, and Sca-1-APC markers for 30 min. The analysis of the Sca-1-APC positive cells only (26.1%, 35.0% in red dots) from the CD34posCD45negCD31neg cells (0.6%, 1.3% in total lung cells) was performed to isolate BASCs. (A) and (B) are representative data from 5 independent analyses. Blue dots: total lung cells, Red dots: CD34posCD45negCD31neg cells, the right peak area in the lower graph: Sca-1pos cells in the CD34posCD45negCD31neg group. In (C) and (D), the cells sorted by FACS from the C57BL/6 and K-rasLA1 mice were further stained with CCSP (a, FITC), and SP-C (b, AF647). c, DAPI staining; d, merged image.

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Fig. 4. Structure and biological properties of M-SERS Dots. (A) The schematic structure of the M-SERS Dots with encoding chemicals and silica layer bound with specific antibodies such as CD34 and Sca-1. (B) TEM image of M-SERS Dots. The scale bar represents 50 nm. (C) Raman spectra of M-SERS Dots encoded with 4-methylbenzenethiol (4-MT) and benzenethiol (BT) from top to bottom. (D) Cell viability assay in primary cells of lung tissues with diverse concentrations of M-SERS Dots (**P < 0.01 is compared to control, mean  s.e.m., n ¼ 3). Based on the cell viability assay, the optimal concentration of the M-SERS dots was determined to be 1.15 mg/ml.

separation of biocompounds in high-throughput screening (HTS) systems. Also, the cell viability of M-SERS Dots is a very important property if they are to be applied to live organisms. The M-SERS Dots showed no toxicity at various concentrations below the concentration of 2.3 mg/ml in primary cells of total lung tissues (Fig. 4D). Through the MTT assay with the M-SERS Dots, their most appropriate concentration was found to be 1.15 mg/ml. In the all experiments of this study, 1.15 mg/ml of M-SERS Dots were used. To demonstrate their capability for the specific and multiple targeting of BASCs, CD34 and Sca-1 were chosen and conjugated

to the M-SERS Dots. The encoding chemicals were BT and 4-MT. The BT encoded M-SERS Dots were conjugated with the CD34 antibody (M-SERS DotBT/CD34) and the 4-MT encoded M-SERS Dots were conjugated with the Sca-1 antibody (M-SERS Dot4-MT/Sca-1). When the primary cells were mixed with the M-SERS Dots, the cells targeted by both the M-SERS DotsBT/CD34 and M-SERS Dots4-MT/Sca-1 could be detected by Raman spectroscopy (Fig. 5). Fig. 5A shows the optical image of the primary cells in the lung tissues. The spots in images i, ii, and iii were the areas detected by Raman spectroscopy. Areas i and ii are from the inside of the cells.

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However, area iii is from the outside of the cells. Fig. 5B corresponds to the mapping of the M-SERS DotsBT/CD34 (red, 1006 cm1 band), and Fig. 5C to the mapping of the M-SERS Dots4-MT/Sca-1 (green, 396 cm1 band). The results show that the spot in image i exhibits the Raman signal of BT, whereas that in image ii exhibits the 4-MT signal in the same targeted cell. However, the spot in image iii exhibits no Raman signal. Thus, it was demonstrated that the M-SERS Dots can target several biomolecules at the same time through their specific and selective binding to the surface markers. Therefore, the M-SERS Dots have the capability of multiplex detection in vitro. 3.5. Application of M-SERS Dots to sort BASCs To isolate the BASCs efficiently, we applied the M-SERS Dots to FACS analysis. In Scheme 1, the method of using the M-SERS Dots to separate the BASCs is illustrated. In this study, the CD34 marker, a positive marker of BASCs, was chosen to conjugate the M-SERS Dots for effective positive selection. The cells targeted with the M-SERS DotsBT/CD34 were BASC like cells. The total lung cells were mixed with

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the M-SERS DotsBT/CD34 in 10% FBS/PBS. CD34pos cells in 10% FBS/PBS were able to be collected easily by applying a magnetic force to the M-SERS Dots with magnetite nanoparticles in core. Untargeted cells could be removed from the supernatant. In this way, only the cells targeted with the M-SERS DotsBT/CD34 were applied to the FACS analysis with the Sca-1 marker conjugated with FITC. For the precise sorting of the BASCs, the separation of the BASC like cells by means of the M-SERS DotsBT/CD34 was very important. The powerful effect of magnetic separation was demonstrated by Raman signal of the M-SERS Dots. Fig. 6 shows the Raman spectra of the encoding chemical, BT in the isolated cells. Fig. 6A shows the optical image of the cells separated by the M-SERS DotsBT/CD34. The spots in images i, ii and iii are on the inside of the cells, whereas those in image iv are on the outside of the cells. Fig. 6B shows the mapping image of the Raman signal from the M-SERS DotsBT/CD34. The Raman spectra of images i, ii, and iii correspond to the signal of the M-SERS DotsBT/CD34 on the cells, however, image iv shows no Raman signal from the M-SERS DotsBT/CD34 (Fig. 6C). This result illustrates that the M-SERS Dots have the great ability to target BASCs and separate CD34pos cells by magnetism. BASCs (CD34posSca-1posCD45negCD31neg

Fig. 5. In vitro multiple detections of stem cell markers by M-SERS Dots. The BASCs have two surface markers, CD34 and Sca-1. In the primary cells from the total lung cells, the BASCs were detected with the M-SERS DotsBT/CD34 (1006 cm1 band, 1.15 mg/ml) and M-SERS Dots4-MT/Sca-1 (396 cm1 band, 1.15 mg/ml). The cells were fixed with paraformaldehyde (4%) for 15–30 min at 25  C. Simultaneously, the cells were incubated with M-SERS DotsBT/CD34 and M-SERS Dots4-MT/Sca-1 at 4  C overnight. (A) Bright field optical image of primary cells. (B and C) Intensity maps of 1006 and 396 cm1 Raman bands of M-SERS DotsBT/CD34 (Red) and M-SERS Dots4-MT/Sca-1 (Green), respectively. (D) The designated SERS spectra at several positions are shown as white dots (i, ii and iii). i; M-SERS DotsBT/CD34 (Red), ii; -SERS Dots4-MT/Sca-1 (Green), iii; the background area of the cell image having no signal. The whole area was scanned in 20  20 steps, and the Raman signals were collected for 1 min in each step.

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Scheme 1. Isolation of BASCs using M-SERS Dots. The total lung cells were extracted from lung tissues and treated with an M-SERS dotBT/CD34 solution (1.15 mg/ml) for 2 h at 25  C. The targeted cells, CD34pos cells, moved in the direction of the magnetic force and, in this way, it was possible to remove the other unnecessary cells. Then, the collected cells (CD34pos cells) were applied to FACS analysis in order to isolate the BASCs. Another marker, Sca-1, was conjugated with FITC. The negative markers CD45 and CD31 bound with PE. Finally, BASCs (CD34posSca1posCD45negCD31neg) were extracted from the CD34pos cells.

cell population) were able to be sorted from diverse lung cells using the proposed method, as summarized in Scheme 1. 3.6. Efficiency of novel isolation system As shown in Fig. 6, the utility of the M-SERS Dots in terms of their high selectivity, high sensitivity, and high magnetic force was confirmed. Herein, we investigated the ability of the M-SERS Dots to isolate BASCs from K-rasLA1 and C57BL/6 mice (Fig. 7). For comparison, all experiments were performed with total lung cells from the same mice simultaneously, each of 3  106 cells. Fig. 7A demonstrates that the CD34posCD45negCD31neg cell population (FITCþ/PE) was 2.9% with the typical method of FACS analysis. Among this population, the amount of Sca-1pos cells (APCþ) was 12.9%. Therefore, the total proportion of BASCs was approximately 0.37% (2.9  12.9/100 ¼ 0.37). Fig. 7B shows the application of the

M-SERS Dots to FACS analysis. The cell population was already identified as CD34pos cells by means of the M-SERS DotsBT/CD34. Therefore, the Sca-1posCD45negCD31neg cell population might be BASCs (2.3%). The isolated cells were confirmed to be BASCs by dual staining with CCSP and SP-C. Together, we demonstrated that even extremely rare BASCs in the lungs of the K-rasLA1 mice could be enriched by the positive selection of the CD34 marker achieved by the M-SERS Dots. The percent of BASCs in the CD34pos cells after separation was about 4- to 5-fold higher than that before separation (Table 1). Also, when this method was applied to the normal mice (Fig. 7D), the CD34posCD45negCD31neg cell population was 1.9%. Among this population, the proportion of Sca-1pos cells was 11.0%. Therefore, the total proportion of BASCs was approximately 0.21% (1.9  11.0/100 ¼ 0.21). In Fig. 7E, the BASC population was 1.7% in CD34pos cells. The isolated cells were also further analyzed by dual staining with CCSP and SP-C in order to verify whether they

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Fig. 6. Raman image of CD34pos cells targeted with M-SERS DotsBT/CD34. The CD34pos cells were separated by targeting with M-SERS DotsBT/CD34. (A) Bright field image of the CD34pos cells. The white dots in i, ii, iii, and iv are the positions detected by Raman spectroscopy. (B) Intensity map of Raman signal at 1006 cm1 band of M-SERS DotsBT/CD34 (Red). (C) Several SERS spectra of M-SERS DotsBT/CD34 at the measured positions in the CD34pos cells by Raman spectroscopy. The i, ii, and iii signals are from the areas targeted with M-SERS DotsBT/CD34 on the surface of the cells. iv; The background area of the cell image has no signal of M-SERS DotsBT/CD34. The whole area was scanned in 20  20 steps, and the Raman signals were collected for 2 min in each step.

were real BASCs (Fig. 7F). Therefore, the BASCs in the normal lung were able to be enriched more than 5-fold by positive selection (Table 1). The results demonstrated that the application of M-SERS Dots to the enrichment and isolation of rare cell populations of BASCs could be achieved with high selectivity and sensitivity. 4. Discussion Recently, numerous investigations have proved that most cancers arise from the malignant transformation of multi-potent tissue-specific adult stem cells and their early progenitors into cancer progenitor cells, which are designated as CSCs [12,19–22]. These putative CSCs may be responsible for the generation of tumors in animal models in vivo [5,6,11,12,19]. Also, the cell population of CSCs can provide critical functions in cancer initiation and progression into metastatic and recurrent disease states [19]. In this study, we investigated the putative cancer stem cells, BASCs in lung tissues. Lung cancer is the leading cause of death, and the 5-year survival rate remains relatively poor, despite the extensive range of medical therapy now available. Adenocarcinoma is a common type of lung cancer and its occurrence is increasing rapidly [5]. Therefore, understanding BASCs is very important in the therapy of lung cancer. We hypothesized that BASCs might contribute to the development of lung adenocarcinomas. Therefore, we compared the amounts of BASCs between K-rasLA1 and C57BL/6 aged 14–16 weeks old mice. First of all, the histopathological morphology of the lung cancer cells in the K-rasLA1 mice was demonstrated (Fig. 1). Then, double positive cells of CCSP and SP-C

markers were counted at the BADJ in the lung tissues. Thus, we were able to prove that the BASCs in the K-rasLA1 mice were more abundant than the cell population in the normal mice (Fig. 2). In the FACS analysis of the BASCs in the K-rasLA1 and normal mice, we demonstrated that the amount of BASCs in K-rasLA1 is higher than that in C57BL/6. Therefore, our results indicate that the amount of BASCs is increased in lung cancer tissue (Fig. 3). On the other hand, previous investigations reported the isolation of CSCs from numerous cancer types and established their functional properties ex vivo and in vivo [12,19,22–24]. However, the available methods of isolating BASCs and demonstrating their stem cell properties are limited, because the cell population is very small [10]. Up to the present, FACS analysis has been utilized for the detection of BASCs. Using the specific antibodies of the surface markers, CD34 and Sca-1, it was possible to isolate very small populations of BASCs [5,6,10]. However, FACS has critical problems for the sorting of rare cell populations on a large scale, such as the time-consuming nature of this procedure and the potential damage to the isolated cells [11–13]. Magnetic separation is an easy technique for the purification and enrichment of the desired cells in comparison to the other non-magnetic conventional techniques [15,25]. However, MACS has been used for the separation of cells that are positive to only one marker, because of the difficulty to isolate cells by controlling the magnetic force [13,26]. In order to improve this sorting technique, we developed a sorting assay of BASCs using M-SERS Dots (Scheme 1). We combined the two sorting assay methods, FACS and MACS, into one system, which we refer to as M-SERS Dots for the separation of BASCs.

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Fig. 7. Effective extraction of BASCs using M-SERS Dots in models of lung cancer and normal mice. (A) Typical scatter plot of FACS analysis from total lung cells in K-rasLA1 mice by staining for CD34-FITC, CD45-PE, and Pecam-1(CD31)-PE (2.9% in total lung cells), then analysis of Sca-1-APC positive cells (12.9% in red dots) from CD34posCD45negCD31neg cells. The proportion of BASCs was approximately 0.37% (2.9  12.9/100 ¼ 0.37). (B) Scatter plot of FACS analysis from CD34pos cells by M-SERS Dots, the percent of Sca-1 positive cells (green, 2.3%). (C) Stained cells from sorted cells after analysis (B). (D) Typical scatter plot of FACS analysis from total lung cells in C57BL/6 mice obtained by the same method of (A). The proportion of BASCs was approximately 0.21% in the total lung cells (1.9  11.0/100 ¼ 0.21). (E) Scatter plot of FACS analysis from CD34pos cells by M-SERS Dots (green, 1.7%). (F) Stained cells from sorted cells after analysis (E). (C) and (F) are cell images stained with CCSP (a, FITC) and SP-C (b, AF647); c, DAPI; d, merged image (800).

M-SERS Dots have excellent properties for biological assays. The application of a magnetic force was sufficient to separate cells such as BASCs (Figs. 4 and 5). Also, the M-SERS Dots have the advantages of SERS Dots, i.e., biocompatibility with low toxicity, sensitivity for targeting, selectivity and the ability to target multiple markers with diverse encoding chemicals (Fig. 4). The multiple targeting capabilities of the M-SERS Dots for the detection of BASCs were demonstrated in this study (Fig. 5). The Raman signal of the encoding chemical in the M-SERS Dots was effective to prove their specific binding to the BASCs (Figs. 5 and 6). The magnetic separation of the M-SERS Dots targeted with CD34pos cells was confirmed by the Raman signal of the encoding chemical, BT (Fig. 6). In addition, the M-SERS Dots can produce many sensitive SERS signals with different kinds of encoding chemical. Also, the MSERS Dots can be easily handled when a magnetic force is applied.

Table 1 The efficiency of isolation of BASCs using M-SERS Dots after positive selection.

C57BL/6 K-ras

Number of experiments

CD34pos before separation CD34pos, Sca-1pos cells (%)

CD34pos after separation CD34pos, Sca-1pos cells (%)

4 7

0.54  0.20 0.64  0.27

2.15  0.30 3.03  1.19

The CD34pos cells were isolated from the total lung cells (3  106 cells) from the C57BL/6 (n ¼ 4) and K-rasLA1 mice (n ¼ 7). The BASCs (CD34posSca-1posCD45negCD31neg) were very rare the only application of FACS analysis (CD34pos before separation). However, the FACS analysis with the M-SERS DotsBT/CD34 (CD34pos cells after separation) was more effective and enabled the BASCs to be enriched approximately 4- to 5-fold. Mean  s.e.m., n ¼ 4 (for C57BL/6 mice) and n ¼ 7 (for K-ras LA1 mice).

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The cells targeted by M-SERS Dots conjugated with specific antibodies moved in the direction of the magnetic bar. The M-SERS Dots were used for the positive selection of specific cells using the CD34 marker (Scheme 1). The positive selection of specific markers was very effective to enrich the desired cell population with high purity. The disadvantage of positive selection was the loss of the desired cells at the separation matrix column [1]. However, the M-SERS Dots allowed this problem to be solved by means of a strong magnetic force. In the application of our novel method to the sorting of BASCs in the K-rasLA1 and C57BL/6 mice, the BASCs are over 4- to 5-fold percent in the CD34pos cell population (Fig. 7 and Table 1). The FACS data obtained from the general method were applied to the total lung cells. Thus, we calculated the ratio of BASCs in the CD34posCD45negCD31neg population using Sca-1pos cells. Meanwhile, after the application of the M-SERS DotsBT/CD34, the Sca-1posCD45negCD31neg cells were identified as BASCs present in the CD34pos cell population. The identification of the cells was further confirmed with IF, after they were stained with CCSP and SP-C (Fig. 7). Thus, we demonstrated that our novel method combining FACS and M-SERS Dots could enrich BASCs effectively. Specially, the M-SERS Dots were very effective for the isolation of rare cells with specific targeting ability and multiplex targeting. In this study, we developed the simplified application of M-SERS Dots as a sorting system, showing that they may apply to an effective tool in the study of CSCs in the future. 5. Conclusion The present work focused on the identification and isolation of BASCs, which are putative lung cancer stem cells. To understand the characteristics of BASCs, we compared the amount of BASCs in K-rasLA1 and normal mice. We demonstrated that BASCs existed more abundantly in the tissues of lung cancer than those of normal. Also, we developed a successful and effective cell sorting system using M-SERS Dots with high percentages of BASCs. Moreover, with the aid of positive selection, our method of isolating BASCs can significantly reduce the sorting time from large numbers of total lung cells. Therefore, M-SERS Dots can play an important role in cancer diagnosis, multiple detection and fast isolation of desired cells and biological molecules. Acknowledgements This work was supported by the NANO Systems InstituteNational Core Research Center (NSI-NCRC), Korea Science and Engineering Foundation (KOSEF). M.H.C. was also partially supported by grants from the KOSEF (M20702000006-08N020000610) of the Ministry of Science and Technology in Korea. M.S.N. was supported by a scholarship from KOSEF. Also, M.S.N., M.A.W., J.E.K. are grateful for the award of a BK21 fellowship. We also wish to thank Carla F. Bender Kim who contributed important information for the isolation of the bronchioalveolar stem cells. Appendix. Supplementary material Supplementary material associated with this article can be found in the online version, at doi:10.1016/j.biomaterials.2009.03.059.

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