KR101000948B1 - Method for Isolating Target Cells Using M-SERS dots - Google Patents

Abstract

The present invention relates to a method for separating cells by using M-SERS dots (Magnetic surface enhanced raman spectroscopic dots), and more specifically, (1) M-SERS dots conjugated with an antibody to a marker of a target cell Treatment with magnetic surface enhanced raman spectroscopic dots, (2) collecting magnetic cells positive for the marker by applying a magnetic force, and (3) targeting from the collected cells using a FACS (Fluorescence activated cell sorter) It relates to a method for separating cells comprising the step of separating the cells.

According to the present invention, when the target cells are separated using only FACS, the target cells can be separated with a higher yield, and can be successfully applied to separate rare cells existing in the tissue.

M-SERS dots, FACS, markers, cancer stem cells

Description

Method for Isolating Target Cells Using M-SERS dots}

The present invention relates to a method for separating target cells from a sample, and more particularly, (1) treating a sample with M-SERS dots conjugated with an antibody to a marker of the target cell, (2) applying a magnetic force Collecting cells that are positive for the marker, and (3) separating target cells from the collected cells using FACS.

Lung cancer is the leading cause of cancer deaths worldwide, and despite aggressive treatment, 5-year survival is relatively low. Adenocarcinoma is a common type of lung cancer and the rate is increasing rapidly (Yanagi, S et al. J Clin Invest 2007, 117, (10), 2929-40). It is important to identify the factors that cause lung cancer. Recently, the existence of cancer stem cells in lung cancer has been demonstrated by Kim et al. (Kim, C. et al. Cell 2005, 121, (6), 823-35; Wicha, MS et al. Cancer Res 2006, 66, ( 4), 1883-90; discussion 1895-6).

An important feature of cancer stem cells is their involvement in tumor development and self-renewal (Jordan, C. T et al. N Engl J Med 2006, 355, (12), 1253-61; Reya, T et al. Nature 2001, 414, (6859), 105-11; Yang, Y et al. PLoS ONE 2008, 3, (5), e2220). Among many stem cell types, especially bronchioalveolar stem cells (BASCs) have been reported to exhibit self-renewal and pluripotency (Kim, C. F et al. Cell 2005, 121, (6), 823-35). BASCs retain bronchiolar clara cells and alveolar cells in the distal lung, and their transformed counterparts cause adenocarcinoma (Kim, CF et al. Cell 2005, 121, ( 6), 823-35.). In addition, BASCs were found to be present in the BADJ (bronchoalveolar duct junction) (Kim, CF et al. Cell 2005, 121, (6), 823-35; Giangreco, A. et al. Am J Pathol 2002, 161, (1), 173-82; Ward, RJ et al. Annu Rev Pathol 2007, 2, 175-189). Kim et al. Demonstrated that BASCs are genuine lung epithelial stem cells that respond to tracheobronchial injury. In addition, these stem cells actively proliferate at the tumor site induced by K-ras. These studies have shown that BASCs induce tumor development and progression to initiate lung adenocarcinoma and act as cancer stem cells (Yanagi, S et al. J Clin Invest 2007, 117, (10), 2929-). 40; Yang, Y. et al. PLoS ONE 2008, 3, (5), e2220).

Although lung stem cells have been importantly studied, methods for isolating these cells have been inefficient and limited (Kim, CF et al. AmJ Physiol Lung Cell Mol Physiol 2007, 293, (5), L1092-8). CCSP ^pos , SP-C ^pos , CD34 ^pos and Sca-1 ^pos are known as markers for detecting and isolating BASCs by FACS (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40; Kim, CF et al. Cell 2005, 121, (6), 823-35; Kim, CF et al. AmJ Physiol Lung Cell Mol Physiol 2007, 293, (5), L1092-8) However, stem Population is very rare in total lung cells (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40; Kim, CF et al. Cell 2005, 121, (6) 823-35).

In an effort to separate rare cancer stem cells, the present inventors treated the sample with M-SERS dots conjugated with antibodies to the markers of the cancer stem cells before separating cancer stem cells from the sample using FACS. After collecting positive cells for the marker by applying magnetic force, cancer stem cells were isolated from previously collected cells using FACS, and cancer stem cells can be obtained with higher yield when cancer stem cells are separated using only FACS. It has been confirmed that the present invention has been completed.

Accordingly, an object of the present invention is to (1) treating a sample with M-SERS dots conjugated with an antibody to a marker of a target cell, (2) applying a magnetic force to collect cells positive for the marker, and (3) to provide a method for separating cells comprising the step of separating target cells from the collected cells using FACS.

The present invention treats a sample with M-SERS dots conjugated with an antibody to a marker of the target cell and separates the cells positive for the marker before separating target cells from the sample using FACS. Characterized in that the steps are carried out in advance.

In the present invention, 'sample' means various tissues or cells isolated therefrom, preferably various cancer tissues or cells isolated therefrom. The cancer tissue is not limited thereto, and may be, for example, breast cancer, cervical cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, bronchial cancer, ovarian cancer, nasopharyngeal cancer, laryngeal cancer, bladder cancer, or colon cancer tissue. In the present invention, the sample is preferably in the form of a suspension of cells isolated from the tissue.

'Target cell' means a cell to be separated, and preferably means a cell that is rarely present (ie, in a small amount) in a sample. The rarely present cells preferably refer to stem cells, and more preferably to cancer stem cells. Even more preferably it refers to BASCs (bronchioalveolar stem cells) present in very small amounts in lung cancer tissue.

'FACS (Fluorescence Activated Cell Sorter)' is a device that labels cells with fluorescent materials (eg FITC, PE, APC, etc.) and flows them through microtubules to sort cells or count the number of cells according to the presence or absence of fluorescence to be.

'M-SERS dots (Magnetic surface enhanced raman spectroscopic dots)' means nanoparticles containing a magnetic material and a Raman compound (application number: 10-2008-0078969, application date: August 12, 2008, the name of the invention : Magnetic / surface enhanced Raman scattering particles, preparation method thereof, and biosensor using the same). An embodiment of the M-SERS dot according to the present invention is shown in FIG. The M-SERS dot contains a number of magnetic materials (eg magnetite) in the central region. In addition, it contains a Raman compound such as benzenethiol (BT), 4-methylbenzenethiol (4-MT), the Raman compound is coated with silver, silver serves to improve the Raman signal of the compound. The silica shell of M-SERS dot shows excellent biocompatibility for viable cells. According to the present invention, M-SERS dots did not cause toxicity at various concentrations (Fig. 6 (D)). The appropriate concentration of M-SERS dots was determined to be 0.1-2.5 mg / ml, preferably 1-2 mg / ml, more preferably 1.15 mg / ml.

Markers of target cells can be referred to as known in the art. In the present invention, the target cells are preferably cancer stem cells, and the markers for each cancer stem cell are as follows.

Acute myeloid leukemia ^{(AMM): CD34 + / CD38} -

- Multiple authentication marrow (multiple myeloma): CD133 ^-

Breast cancer: CD44 ⁺ / CD24- ^{/ low}

Glioblastoma: CD133 ⁺

Colorectal Cancer: CD133 ⁺

Prostate Cancer: CD44 ⁺ / α ₂ β ₁ ^hi / CD133 ⁺

Melanoma: ABCB5 ⁺

The antibody to each marker may be a polyclonal antibody or a monoclonal antibody, with monoclonal antibodies being more preferred.

Monoclonal antibodies can be generated using known techniques (Kennettm McKearn and Bechtol (eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses , Plenum Press, 1980). Monoclonal antibodies immunize animals with marker proteins as immunogens, fusion of splenocytes of immunized animals with myeloma cells to produce hybridomas, select hybridomas that selectively recognize marker proteins, and select high It can be prepared by culturing the bridoma and separating the antibody from the culture medium of the hybridoma. In addition, the monoclonal antibody of the present invention can be prepared by injecting the above-mentioned hybridomas producing an anti-marker antibody selectively recognizing a marker protein into an animal and separating it from the ascites of the recovered animal after a certain period of time after the injection. Can be.

Conjugation of the antibody with the marker of the target cell and M-SERS dots can be carried out as known. The antibodies can be conjugated by mixing the M-SERS dots and then stirring them (see Example 7). M-SERS dots conjugated with antibodies are treated with 0.1-10%, preferably 0.5-5% BSA (Bovine serum albumin) diluted in a suitable buffer (eg PBS) or media to block nonspecific binding. desirable.

If the sample is treated with M-SERS dots conjugated with an antibody against a marker of a target cell, the M-SERS dots will bind with cells expressing the marker present in the sample (ie, positive for the marker). Since the M-SERS dots contain a magnetic material, the cells bound to the M-SERS dots (that is, cells positive for the marker) move in accordance with the direction in which the magnetic force is applied and are easily collected when the magnetic force is applied. Can be.

M-SERS dots do not have any effect on FACS, so cells positive for previously collected markers can undergo FACS without any treatment. FACS can be carried out with reference to known methods using conventional apparatus. The marker of the cells to be introduced in the FACS may be the same or different from the marker used in the collection step described above.

In the past, FACS was used to separate cells present in tissues such as cancer stem cells.

The present invention treats a sample with M-SERS dots conjugated with an antibody to a marker of the target cell and separates the cells positive for the marker before separating target cells from the sample using FACS. Characterized in that the steps are carried out in advance.

According to the present invention, when the target cells are separated by only FACS, the target cells can be obtained with higher yield.

In particular, the isolation method of the present invention can be successfully applied to isolate cancer stem cells, and the isolated cancer stem cells can be used to develop cancer stem cell target therapy.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Reagents and Methods

Example 1 Animal Breeding

This example was performed on C57BL / 6 mice and K-ras ^LA1 mice at 14-16 weeks of age. C57BL / 6 mice were obtained from central laboratory animals (Seoul, Korea) and breeding K-ras ^LA1 mice from the National Cancer Center (Frederick, MD, USA). Animals were bred on a 12-hour day / night cycle in experimental animal facilities where temperatures and relative humidity were maintained at 23 ± 2 ° C and 50 ± 20%, respectively. This example was conducted with the approval of the Animal Experimentation Committee (SNU-070830-1) of Seoul National University.

Example 1-2 H & E Dyeing

Lung tissue was fixed in formalin buffered to 10% neutral, paraffinized and cut into 3 μm. For histological analysis, tissue sections were stained with hematoxylin and eosin. Cover slips were then sampled using DakoCytomation Faramount Aqueous Encapsulant (DakoCytomation, Denmark), and slides were observed using a light microscope (Carl Zeiss, Thornwood, NY, USA).

Example 3: Immunofluorescence Assay in Tissues

Lung tissue was fixed in formalin buffered to 10% neutral, paraffinized and cut into 3 μm. For immunofluorescence analysis, tissue sections were deparaffinized in xylene and rehydrated via alcohol gradient. Tissue sections were left standing in 150 ml proteinase K and washed and left standing in 3% hydrogen peroxide (AppliChem, Darmstadt, Germany) to quench endogenous peroxidase activity for 30 minutes. After washing with PBS, tissue sections were allowed to stand for 1 hour at room temperature with 1% BSA dissolved in PBS to block nonspecific binding sites. Primary antibodies were applied to tissue sections overnight at 4 ° C. The following day, tissue sections were washed and allowed to stand for 2 hours at room temperature with secondary fluorescent-conjugated antibody (1:50). After careful washing, the nuclei of lung tissue were stained by staining with 4, 6-diamidino-2-phenylndole (DAPI) (SantaCruz Biotechnology, CA, USA) for 5 minutes. Cover slips were then sampled with DakoCytomation Faramount Aqueous Encapsulant (DakoCytomation, Denmark) and slides were observed using CLSM (Confocal Laser Scanning Microscope; Carl Zeiss, Thornwood, NY, USA). Here, as the antibody for staining bronchial alveolar stem cells (BASCs), CD34 (Abcam, Cambridge, UK), Sca-1 (R & D systems, Minneapolis, MN, USA), CCSP (SantaCruz Biotechnology, CA, USA) and SP-C (SantaCruz Biotechnology, CA, USA) was used.

Example 4: Immunofluorescence Measurement in Primary Cells

All lung cells were extracted according to known methods (Kim, CF et al. Cell 2005, 121, (6), 823-35). Cells were then allowed to stand on Lab-tek glass chamber slides (Nalge Nunc International, Naperville, IN) in DMEM-F12 medium (Invitrogen-Gibco, Carlsbad, Calif., USA). After several days, cells were fixed for 15 minutes at room temperature with 4% formaldehyde and washed twice with PBS. 0.25% TritonX-100 / PBS was treated for 10 minutes to stain intracellular proteins, in particular CCSP and SP-C. After washing with PBS, treatment with 1% BSA dissolved in PBS for 30 minutes to block nonspecific binding sites. Primary antibodies (CCSP, SP-C or CD34, Sca-1) were applied on slides overnight at 4 ° C. or 1 hour at room temperature. The next day, tissue sections were washed and allowed to stand for 1 hour at room temperature with secondary fluorescent-conjugated antibody (1:50). After careful washing, the nuclei of lung tissue were stained by staining with 4, 6-diamidino-2-phenylndole (DAPI) (SantaCruz Biotechnology, CA, USA) for 5 minutes. Cover slips were then sampled using DakoCytomation Faramount Aqueous Encapsulant (DakoCytomation, Denmark), and slides were observed using a Confocal Laser Scanning Microscope (Carlisle Zeiss, Thornwood, NY, USA).

Example 5 Flow Cytometry of BASCs

Single cell suspensions were prepared from lungs and injected into PBS as known (Kim, CF et al. Cell 2005, 121, (6), 823-35). Total lung cells resuspended in PBS / 10% FBS to 3 × 10 ⁶ / 100㎕ for 30 minutes APC- konju ligated wherein -Sca-1, FITC- and PE- konju ligated anti -CD34 konju ligated wherein during -CD31, Staining with CD45 (eBioscience).

For application of the M-SERS dots, total reproduce the lung cells to 3 × 10 ⁶ / 100㎕ in PBS / 10% FBS-suspended and treated for 2 hours with CD34 and the ligation konju M-SERS dots. Target cells were magnetically collected for several minutes using a rod magnet. Cell populations were then stained with FITC-conjugated anti-Sca-1 and PE-conjugated anti-CD31, CD45 for 30 minutes. Flow cytometry was performed using BDFACS Aria ^™ (BD Biosciences, USA) and data was analyzed with BDFACS Aria ^™ software (BD Biosciences, USA).

Example 6: Cytotoxicity Test

Cell viability was measured by 3- (4, 5-dimethylthiazo-2-yl) 2 and 5-diphenyltetrazolium bromide (MTT) assay. Primary cells derived from lung tissues of C57BL / 6 mice were seeded at 50,000 cells per sphere in 96-neck tissue culture dishes and incubated for several days. After treatment with various concentrations of M-SERS Dot, cytotoxicity was measured by MTT (3- [4, 5-Dimethylthiazol-2-yl] -2, 5-diphenyl-tetrazolium bromide). 20 μl of MTT (Sigma) solution (5 mg / ml) was added and left at 37 ° C. for 4 hours. Cells were treated with 10 μl DMSO (dimethylsulfoxide, Sigma) and absorbance was quantified using an ELISA reader (BioRad, USA).

Example 7: Conjugation of M-SERS dots with Antibody

100 μl of antibody CD34 (0.1 mg / ml, Abcam, Cambridge, UK) or Sca-1 (0.1 mg / ml, R & D systems, USA) was added to the dispersion and the resulting mixture (1.15 mg / ml, 1 ml M- SERS dots) was stirred at 25 ° C. for 2 hours. The M-SERS dots thus formed were centrifuged and washed with PBS solution. M-SERS dots were treated with bovine serum albumin (1 wt% in PBS dose) at 25 ° C. for 30 minutes and then washed with PBS solution.

Example 8 Multiple Detection in Primary Cells with M-SERS Dots

After primary cells were isolated from lung tissue of K-ras ^LA1 mice, total lung cells were allowed to stand for 3 days in Lab-tek glass chamber slides (Nalge Nunc International, Naperville, IN) in a CO ₂ incubator. Cells were then fixed with formaldehyde (4%) at 25 ° C. for 15-30 minutes. The fixative was removed by centrifugation and the cells washed with PBS solution. For targeting analysis, M-SERS dot ^{BT / CD34} (1.15 mg / ml) and M-SERS dot ⁴ diluted primary cells in total media DMEM / F12 (Invitrogen-Gibco, Carlsbad, CA, USA) at 10% each ^{-MT / Sca-1} (1.15 mg / ml) was left overnight at 4 ° C. After standing, cells were washed several times with PBS solution containing Tween20 (0.1 wt%).

Example 9 Detection of M-SERS Dots by Micro Raman Spectroscopy

Raman measurements were performed using a micro-Raman system (JY-Horiba, Labram 300) equipped with an integral microscope (Olympus BX41, NA = 0.95, x100). In this system a 514.5 nm laser line from an argon ion laser (Melles-Griot, 35-MAP321) was used as the excitation source, and Raman scattering signals were collected with a 180 ° scattering geometry.

result

1. Morphology of normal and lung cancer tissue

BASCs are assumed to be cancer stem cells in a mouse model of lung adenocarcinoma induced by carcinogenic K-ras (Yang, Y. et al. PLoS ONE 2008, 3, (5), e2220). To measure the amount of BASCs in late lung adenocarcinoma in K-ras ^LA1 , a 14-16 week old mouse model was used instead of a 4-8 week old mouse model. K-ras mutations are associated with tumor progression in a transgenic mouse model (Johnson, L et al. Nature 2001, 410, (6832), 1111-6; Yamashita, N. et al. Cancer 1995, 75 , (6 Suppl), 1527-33). A, b and (C) in FIG. 1 (A) show normal lungs of C57BL / 6 mice. C57BL / 6 mice are the backgroud of transgenic mouse K-ras ^LA1 (Johnson, L et al. Nature 2001, 410, (6832), 1111-6). K-ras ^LA1 mice had already advanced cancer significantly at that age (a, b in Fig. 1 (B)). Adenocarcinoma developed in the lung tissue of Kras ^LA1 mice as indicated by the red circle in FIG. 1 (D).

2. Detection of BASCs in vitro and in vivo

For detection of BASCs, several markers were examined for expression patterns in primary cells. Double staining was performed with two markers in primary cells of normal mice, intracellular markers (CCSP and SP-C) and surface markers (CD34 and Sca-1). At the surface of primary cells, CD34 and Sca-1 positive cells were detected and CCSP and SP-C positive cells were observed in the intracellular region as confirmed by CLSM (FIGS. 2A and 2B).

Tumor BASCs can be derived from a cell population of adult lung similar to fetal lung epithelial progenitor cells. In addition, BASCs have been identified in BADJ in lung tissue (Kim, CF et al. Cell 2005, 121, (6), 823-35; Kim, CF et al. AmJ Physiol Lung Cell Mol Physiol 2007, 293, (5) , L1092-8; Giangreco, A. et al. Am J Pathol 2002, 161, (1), 173-82; Yang, Y. et al. PLoS ONE 2008, 3, (5), e2220). The cell population is positive for Clara and AT2 cell markers (Wuenschell, CW et al. 1996, 44, (2), 113-23). Thus, tissues of normal and K-ras ^LA1 mice were stained with CCSP and SP-C markers capable of detecting BASCs and compared for expression. 3A shows an image of lung tissue derived from C57BL / 6 mice. The dashed black rectangle is enlarged to track the BASCs in the BADJ area. Immunofluorescence (IF) distinguished double positive regions showing green and red (i in FIG. 3A). Interestingly, cancer-advanced K-ras ^LA1 mice contained more cell populations of BASCs than normal mice (FIG. 3B i). 3C shows the number of double positive cells in four randomly selected BADJ regions.

3. Normal and K-ras 14-14 weeks old ^LA1  Isolation and Comparison of BASCs from Mice

To determine whether BASCs are increased in advanced cancers of K-ras ^LA1 mice, the amount of BASCs was measured by FACS analysis. The prior art discloses that stem cell groups in the distal lung region are increased at the onset of cancer (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40; .Kim, CF et al. AmJ Physiol Lung Cell Mol Physiol 2007, 293, (5), L1092-8). The BASCs sought to determine whether they affect not only the onset of the cancer, but also later cancer. K-ras ^LA1 mice have lung cancer from birth (Johnson, L. et al. Nature 2001, 410, (6832), 1111-6). To this end, we first selected a 14-16 week old mouse model with advanced cancer. In addition, whole lung cells were extracted from lung tissue and the amount of BASCs was compared. Here, CD34 and Sca-1 were the surface markers of BASCs and thus were used for the separation of BASCs (Kim, CF et al. Cell 2005, 121, (6), 823-35).

Isolation of BASCs was performed as known (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40; Kim, CF et al. Cell 2005, 121, (6), 823- 35; Yang, Y. et al. PLoS ONE 2008, 3, (5), e2220). Lung cells were prepared and hematopoietic and endothelial lineages were removed by CD45 and Pecam-1 (CD31). Residual cell populations were sorted by CD34 and Sca-1 markers. There are two protocols for classifying BASCs by CD34 and Sca-1 (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40; Kim, CF et al. Cell 2005, 121 , (6), 823-35). Kim et al. First used Sca-1 markers and isolated BASCs using CD34 markers from Sca-1 marker positive cell populations (Kim, CF et al. Cell 2005, 121, (6), 823-35). As a result, BASCs rich in cell populations classified as Sca-1 ^pos CD34 ^pos CD45 ^neg CD31 ^neg were included (Kim, CF et al. Cell 2005, 121, (6), 823-35). Yanagi et al. Also isolated BASCs using the same markers. However, the CD34 marker was used first, and the BASCs population was classified using the Sca-1 marker in the cell population positive for the CD34 marker (Yanagi, S. et al. J Clin Invest 2007, 117, (10), 2929-40). ). From these results, the present inventors attempted to propose a protocol capable of separating BASCs. Thus, the amount of total lung cells positive for CD34 and SCa-1 markers in normal mice was compared (FIG. 4). As a result, fewer cell populations positive for the Sca-1 marker were present in the cell population positive for the CD34 marker. For this reason, in this example, CD34 markers were selected before Sca-1 markers to separate BASCs.

A comparison of the amount of lung cancer stem cells (BASCs) between normal and K-ras ^LA1 mice was confirmed by the cell population of Sca-1 ^pos CD34 ^pos CD45 ^neg CD31 ^neg . According to FACS analysis, the amount of BASCs in K-ras ^LA1 mice was higher than in normal mice as shown in FIG. 5A. In addition, immunofluorescence demonstrated that the sorted cells (Sca-1 ^pos CD34 ^pos CD45 ^neg CD31 ^neg ) were BASCs with CCSP and SP-C markers (FIG. 5B). From the results in FIGS. 5A and 5B, BASCs were present in greater amounts in late lung cancer than normal lung. Thus, BASCs are associated with late lung cancer in K-ras ^LA1 mice. That is, BASCs were increased not only in the early stages of lung cancer but also in later stages.

4. Characteristics of M-SERS dot

M-SERS dot is a nanoparticle having a few magnetite (magnetite) in the center region (Fig. 6 (A)). In addition, the signal by Raman spectroscopy of the coding compound is enhanced with a silver coating. Silica envelope is a good tool with biocompatibility for viable cells (Figs. 6 (A), (B)). Therefore, M-SERS dot is expected as a novel nanoprobe for multiplex detection and separation of biological components in high throughput screening (HTS) system. As shown in FIG. 6 (C), the intensity of the coding compound in M-SERS dots was detected prior to application onto a real system with multiple detection and sorting of positive cells. Cell viability of the M-SERS dot is important for the material of living organisms. M-SERS Dot did not cause toxicity at various concentrations (Figure 6 (D)). Through MTT measurement of the magnetic nanoparticles, an appropriate concentration of M-SERS dots could be determined to be 1.15 mg / ml.

CD34 and Sca-1 were conjugated to the M-SERS Dot for detection of BASCs to confirm that M-SERS dots can specifically target BASCs. As the coding material, benzenethiol (BT) and 4-methylbenzenethiol (4-MT) were used. When primary cells were targeted with M-SERS dots, dual target cells of M-SERS dot ^{BT / CD34} and M-SERS dot ^{4-MT / Sca-1} were detected by Raman spectroscopy (Fig. 7 (B), (C). ), (D)). As a result, M-SERS dots were able to efficiently target biological components with several markers through specific binding to surface markers. Thus, the M-SERS dot has multiple detection capabilities in vitro.

5. Application of M-SERS to the Classification of BASCs

BASCs are a very rare population in total lung cells. As shown in FIG. 5, FACS analysis shows that BASC is present in very small amounts in total lung cells. In the present invention, M-SERS dots are introduced for effective classification of BASC. As shown in FIG. 7, M-SERS dots can target specific cells with high sensitivity and high specificity. In addition, because of the magnetite, it is also possible to easily move the target cells using a rod magnet. M-SERS dots were applied as a sorting system as an effective tool for isolating BASCs by separating CD34 ^pos cells.

8 illustrates a protocol using M-SERS dots to separate BASCs. Total lung cells were extracted from lung tissue as known and M-SERS dot ^{BT / CD34} solution was treated with total lung cells at 25 ° C. for 2 hours. M-SERS dots were conjugated with CD34 antibody for 1 hour 30 minutes before mixing with lung cells. 1% BSA was then treated for 30 minutes to inhibit nonspecific targeting. Cells targeted to M-SERS dot ^{BT / CD34} are BASC like cells. M-SERS dot ^{BT / CD34} resolution that separates cells positive for the CD34 marker was detected by Raman spectroscopy (FIG. 9). Cells targeted with M-SERS dot ^{BT / CD34} could have mobility for the direction of magnetic force. Thus, CD34 ^pos cells could collect in one corner in the same direction as the rod magnets within a few minutes. The cells thus collected could be subjected to FACS analysis for isolation of abundant BASCs. Another marker, Sca-1, was used to detect BASCs. The Sca-1 marker is conjugated with green fluorescence and the other markers CD45 and Pecam-1 (CD31) are conjugated with red fluorescence. Finally, only a population of Sca-1 ^pos CD34 ^pos CD45 ^neg CD31 ^neg could be classified from this new protocol.

6. Efficacy in a Novel Separation System

M-SERS dots are sufficient to isolate specific cells with high selectivity, high magnetic force and high sensitivity. In addition, magnetic particles did not interfere with FACS analysis (data not shown).

As shown in Figs. 10 (A) and (B), the introduction of M-SERS dots in FACS analysis can enrich very rare BASC in the steady-state lung tissue of K-ras ^LA1 mice. The protocol using M-SERS dots is about 4-5 times higher (Table 1). Cells targeted to M-SERS dot ^{BT / CD34} and isolated with Sca-1 markers by FACS analysis demonstrated BASCs with immunofluorescence by CCSP and SP-C (FIG. 10 (C)).

In addition, the separation method of the present invention in which M-SERS dots were introduced was applied to normal mice. Populations of BASCs were separated by more than five times higher yields than conventional methods (FIGS. 11 (A), (B), Table 1). Cells sorted as shown in FIG. 11 (C) were positive for CCSP and SP-C markers.

From these results, M-SERS dots proved useful for separating BASCs.

Yield of separation method of the present invention Number of experiments Yield of BASCs on Separation by FACS Only Yield of BASCs in Separation of the Invention C57BL / 6 4 0.54 ± 0.20 2.15 ± 0.30 K-ras 7 0.64 ± 0.27 3.03 ± 0.19

Figure 1 shows the shape of lung tissue derived from normal and lung cancer mice, (A) (C) shows normal lung tissue, (B) (D) shows lung cancer tissue, (C) and (D) Shows the results of histological analysis of normal and lung cancer lung tissue, respectively.

Figure 2a shows the result of staining primary cells derived from normal lung tissue with intracellular markers capable of detecting BASCs (a: CCSP staining image (green), b: bright field image, c: SP-C) Stained image (red), d: DAPI, e: mixed image of CCSP and SP-c).

Figure 2b shows the result of staining primary cells derived from normal lung tissue with a surface marker capable of detecting BASCs (a: CD34 staining image (green), b: bright field image, c: Sca-1 staining) Image (red), d: DAPI, e: mixed image of CD34 and Sca-1).

Figure 3a shows the results of the identification of BASCs in normal lung tissue of C57BL / 6, the black dotted square is the area of the BADJ, the bottom picture is an enlarged view of the area (a ~ e: 400 times magnification, a: CCSP Stained image, b: Dig stained image (AS: alveolar sacs, TB: terminal bronchia), c: SP-C stained image, d: DAPI stained image, e: mixed image, f ~ i: 800Ｘ magnification area, f: CCSP stained image, g: magnified image of BADJ region, h: SP-C stained image, I: mixed image of f and g).

Figure 3b shows the results of identifying BASCs in lung cancer tissue of K-ras ^LA1 , the black dotted square is the area of the BADJ, the bottom photo shows the area enlarged (a ~ e: 400 kW, a: CCSP Stained image, b: Dig stained image (AS: alveolar sacs, TB: terminal bronchia), c: SP-C stained image, d: DAPI stained image, e: mixed image, f ~ i: 800Ｘ magnification area, f: CCSP stained image, g: magnified image of BADJ region, h: SP-C stained image, I: mixed image of f and g).

3C shows the number of CCSP and SP-C double positive cells identified in four BADJ regions randomly selected from normal lung of C57BL / 6 and lung cancer tissue of K-ras ^LA1 .

Figure 4 shows the results of FACS analysis on normal lung primary cells to identify markers effective for detecting BASCs (A: CD34 staining, B: Sca-1 staining, C: CD34 or Sca-1 stained BASCs Graph showing the number of.

5A shows BASCs isolated from normal lung of C57BL / 6 and lung cancer tissue of K-ras ^LA1 by FACS (A: normal mouse, B: K-ras ^LA1 mouse).

5B shows immunofluorescence analysis of BASCs isolated from normal lung of C57BL / 6 and lung cancer tissue of K-ras ^LA1 by FACS (A: normal mouse, B: K-ras ^LA1 mouse; a: CCSP staining image , b: SP-C stained image, c: DAPI stained image, d: mixed image).

Figure 6 shows the structure and biological properties of the M-SERS dots (A: M-SERS dot configuration diagram, B: TEM image, C: Raman signal, survival rate of lung primary cells by concentration of M-SERS dot).

Figure 7 shows the multiple detection of M-SERS dots in vitro (A: bright field image of primary cells, B and C: M-SERS dot ^{BT / CD34} (red) and M-SERS dot ^{4-MT /)} Mapping image of Raman signal from SERS intensity of ^Sca-1 (green), D: SERS spectrum at position shown by yellow box).

8 is a flowchart illustrating a method of separating BASCs in which M-SERS dots are introduced according to the present invention.

9 shows Raman images of CD34 ^pos cells targeted with M-SERS dot ^{BT / CD34} (A: brightfield image of CD34 ^pos cells, B: from SERS intensity of M-SERS dot ^{BT / CD34} (Red)). Mapping image of the emerging Raman signal, C: SERS spectrum at the position indicated by the yellow box).

Figure 10 shows BASCs isolated from lung cancer tissue of K-ras ^LA1 using the separation method of the present invention (A: BASCs separated by FACS, B: BASCs separated by the separation method of the present invention, C: the present invention) Immunofluorescence analysis of BASCs separated by the separation method (a: CCSP staining image, b: SP-C staining image, c: DAPI staining image, d: mixed image).

Figure 11 shows BASCs isolated from normal lung tissue of C57BL / 6 using the separation method of the present invention (A: BASCs separated by FACS, B: BASCs separated by the separation method of the present invention, C: the present invention) Immunofluorescence analysis of BASCs separated by the separation method (a: CCSP staining image, b: SP-C staining image, c: DAPI staining image, d: mixed image).

Claims (6)

(1) treating the sample with M-SERS dots (Magnetic surface enhanced raman spectroscopic dots) conjugated with an antibody to a marker of a target cell, (2) applying a magnetic force to collect cells positive for the marker, And (3) separating target cells from the collected cells using FACS (Fluorescence activated cell sorter). The method of claim 1, The target cell is characterized in that the cancer stem cells. 3. The method of claim 2, The cancer stem cell is characterized in that the BASCs (bronchioalveolar stem cells). The method of claim 1, If the target cell is BASCs, the marker is CD34. The method of claim 1, M-SERS dots is characterized in that the treatment at a concentration of 0.15 to 2.5mg / ml. The method of claim 1, M-SERS dots conjugated with the antibody to the marker of the target cell is treated with bovine serum albumin (BSA), characterized in that non-specific binding is blocked.

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