IN SITU IMAGING OF BREAST CANCER CELLS USING GREEN SEMICONDUCTOR QUANTUM DOTS

Xu Hengyi,Zoraida P.Aguilar ,Su Huaipeng,Benjamin J.Jones,John.D.Dixon,Xiong Yonghua,Wei Hua,Andrew Y.Wang

(1.State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang,330047,P.R.China;2.Ocean Nano Tech LLC,2143 Worth Lane,Springdale,AR 72764,USA)

INTRODUCTION

The breast cancer is the second most common type of the cancer,the fifth most common cause of cancer deaths in the United States and the world and the most common cause of cancer deaths in women.Knowing the accurate stage of this disease is critical for patient survival.Sufficient treatment during the early stages of the breast cancer can prevent the spread and the transfer of the cancer cells to other parts of the body.Thus,one of the most recent interests for the breast cancer cell detection is highly sensitive and specific immunocy-tochemical staining which can be used to directly take the cancer cell image as accurate detection.

Highly sensitive and specific immunocy-tochemical,and molecular methods[1-2]can detect disseminated tumor cells(DTCs)in the bone marrow and circulating tumor cells(CTCs)in the peripheral blood at the single cell level in the presence of millions of normal cells.In the peripheral blood,CTCs are detectable months to years after complete removal of the primary tumors,thus indicating that the secells can circulate different metastatic sites[3].

The National Institutes of Health(NIH)estimates the cost of cancer in 2008 is＄228.1 billion.Solid tumors derived from epithelial tissues,such as breast,prostate,lung or colorectal carcinomas are the most frequent forms of the cancer.Of the various cancer types reported,the breast cancer is the second most common type of the cancer and the fifth most common cause of the cancer death in the United States and the world(WHO).There are 11.38 million people alive who has a history of the cancer that in cluds those who are cured and with active disease in 2006 in the USA[4].In 2004,the breast cancer caused 519 000 deaths worldwide(7% of cancer deaths;almost 1%of all deaths,WHO).

The breast cancer is composed of distinct diseases with different outcomes.Usually,clinical and pathological factors are used to determine the prognosis of patients.Currently,most of the diagnostic predictions are a combination of prognostic factors to adapt adjuvant treatment based on the prognosis prediction,such as the Saint Gallen Guidelines[5],National Institute of Health Guidelines[6],and Nottingham Prognostic Index Guidelines[7]as well as the Adjuvant Online Decision-Making Tools[8].

Knowing the accurate stage of the breast cancer is critical for patient survival.Sufficient treatment during the early stages of breast cancer can prevent the spread and transfer of the cancer cells to other parts of the body.But the cancer cells need to be detected at an early stagein order to start life-saving treatment.Owing to inaccurate prognosis predictions,however,a substantial proportion of breast cancer patients receive adjuvant systemic therapy without gaining any benefit[9].There are just several cancer cells per milliliter in the blood during the early stages making these cells difficult to find and differentiate from millions of white and red blood cells,thus sensitive and accurate detection methods are needed for the diagnosis of the early stages of the breast cancer.

Nanoparticles can be used for accurate immunocy-tochemical imaging of breast cancer cells.Among the nano particles currently in use,quantum dot(QD)nano particles have received considerable attention due to their inherent advantages[10-11].QDs have unique size-dependent physical properties,such as broad absorption spectrum,precision small bandwidth emission wavelength,thus enhancing chemical and photochemical stabilities,the endocytosis,the cooperative binding activity,and multi-functionalities for the targeted delivery and imaging[12-14].QD immunocy-to chemical imaging of breast cancer cells provides ultra bright signals that persist the unlimited time for observations.

This paper presents the development of early stage in situ imaging of breast cancer cells(SKBR3)using green QDs as fluorescent signal generator.This study chooses a HER2 over expressing cell line,SK-BR3,and uses the anti-HER2/neu antibody as the specific recognizing antibody.The HER2 gene(HER2/neu and ErbB2 gene)is amplified by 20%—30% at the early-stage of breast cancer which makes it overactive[15].HER2 passes through the cell membrane and sends signals from the cell outside to the inside.However,in the cancer,HER2 sends signals without being stimulated by mitogen promoting invasion,survival,and growth of new vessels of cells(angiogenesis)[16].HER2 over expression can also confer resistance to cancer drugs[17].Thus,the detection of breast cancer cells using HER2 over expression has clinical advantages.

QDs based imaging of breast cancer cells involves anti-HER2/neu antibody for labeling with the over expressed HER2 on the surface of SKBR3 cells.The complete assay involves SK-BR3+ Ab-biotin+ streptavid in～QDs.The SK-BR3 cells are grown in the culture and exposed to the biotin labeled antibodies followed by the exposure to streptavidin labeled QDs that utilize the strong and stable biotin-streptavidin interaction.Fluorescent images of the complete assay for SK-BR3 cells are evaluated on a microscope with a UV light source.QDs with different wavelengths of the emission are tested to assess the different imaging effects of QDs at different sizes and to establish the possibility of using multicolor cancer cells.

1 MATERIAL AND METHODS

1.1 Synthesis and characterization of green QDs

Green water soluble carboxyl fun ctionalized Cd Se/ZnScore/shell QDs(catalog# QSH 530;λemission 530 nm,molar extinction coefficient 1.5×105M/cm)are synthesized in the house at Ocean Nanotech,LLC,and Springdale,AR.After synthesis following standard protocols,UV-visible absorption spectra are acquired with a HP UV-visible absorption spectrophotometer and photoluminescence spectra are recorded on a PE spectr of luorometer. Transmission electron microscopy(TEM)samples are prepared by dropping a chloroform solution of Cd Se/Cd S QDs on an agar carbon-coated copper grid(400mesh)and the solvent is evaporated.TEM images are obtained at 100 K magnification with a Philips CM 20 TEM operating at 200 k V.The PLQYs of Cd Se/Cd SQDs are measured according to the literature.Rhodamine 6G in ethanol is chosen as the reference standard(PLQY 95%)[18-19].All optical measurements are performed at room temperature under ambient conditions.

1.2 Surface functionali zation of QDs with strep-tavidin

To prepare the functionalized QDs, the streptavidin is conjugated to QDs by using sulfo-N HSand EDC covalent coupling chemistry.The QDs(4μM)are activated by adding and incubating with sulfo-NHS(sulfo-N-hydrox ysuccinimide)at a molar ratio 1 000∶1 and EDC(1-ethyl-3,3-dimethy lamino pro pyl carbodiimide hydrochloride)at molar ratio 1 000∶1 for 5 min in boratebuffer,p H 7.4,after which 2 mg of the streptavidin is added,vortexed thoroughly,and reacted for 2 h at the room temperature.At the end of 2 h,the reaction is quenched by adding 5μL of quenching buffer(Cat.# QB,Ocean Nano Tech,USA)and mixed for more 10 min.The streptavidin～QD conjugates are stored at 4°C about 12 h and purified by ultra centrifugation using a Beckman ultracentrifuge(Beckman,USA).

The surface functionalized streptavidin～ QD conjugates are evaluated with a horizontal submerged gel electrophoresis apparatus(Mini-Sub-Cell GT,Bio-Rad,USA)using a 1.5% (w/v)agarose gel in Tris-acetate-EDTA(TAE)buffer,p H 8.5.For each well,20μL of the streptavidin～ QD conjugates at 100 nM is mixed with 5μL of 5× TAE loading buffer(5× TAE,25% (v/v)glycerol,0.25% (w/v)orange-Gat p H 8.5)before being loaded on the gel.The gel is resolved at 100 V for 30 min(Power Pak Basic,Bio-Rad,USA)and the photo is taken with a 2 s exposure using a gel imaging system(Alpha Imager HP 2006,Alpha Innotech,USA).

1.3 Cell ref reshment and subculture

HER2-overexpressing human breast cancer cell line(SK-BR3)used in this research is originally obtained from the American Type Culture Collection(ATCC,Manassas,VA)and is donated by Dr.Lily Yang from Emory University.Liquid nitrogen frozen SK-BR3 cells are thawed in 37°C water bath before transferring into a flask containing RPMI-1640 medium (Invitrogen,Carlsbad,CA,USA)supplemented with 10%fetal bovine serum(FBS,Hy Clone,Logan,UT,USA)and 1% of streptomycin/penicillin antibiotics(Hy Clone,Logan,UT,USA).The flask is placed in a humidified atmosphere with 5%CO2 at 37°Cin a cell culture incubator(Sanyo,Japan).The media is replaced once every three days.The cells are cultured about 80%—90%confluence before the harvest.During the harvest,the cells are washed two times with DPBS(Hy Clone, Logan,UT, USA)followed by adding 1 m L of trypsin(Hy Clone,Logan,UT,USA)to detach the cells from the flask.The trypsin is neutralized by adding 5 mL of fresh RPMI-1640 medium to the flask and is centrifuged at 250×g for 5 min,where g is the gravitational acceleration.The pelleted cells are resuspended in fresh RPMI-1640 and then counted with a Z1 cell counter(Beckman,USA)after 105cells are inoculated on a cover placed on a 6-well cell culture plate.The cells are sub-cultured under the same conditions until the cells are on the glass cover to reach 80%—90%confluence.

1.4 Antibody modif ication

Anti-HER2/neu antibody is purchased from Ray Biotech,Inc.(Norcross,GA)and modified with biotin for binding with streptavidin～ QDs.The conjugation process is performed using the hydrazide method.Briefly,1 mg anti-HER2/neu antibody is dissolved in MESbuffer at 5 mg/mL and 25μL of biotin hydrazide solution(50 mM)is added in a centrifuge tube.After thorough mixing,12.5μL of EDC solution(100 mg/mL)is added and mixed thoroughly.The solution is incubated for 2 h at the room temperature.The conjugates are purified by using the dialysis.

1.5 In situ cell imaging with QDs

The SK-BR3 cells grown on the glass cover are washed with 1 mL DPBS three times.After washing,1 mL of DPBS containing 1 mg/mL of BSA(Bovine Serum Albumin)is added to each well,followed by 1μg of biotin labeled anti-HER2/neu antibody.The plate is placed on a vortex(VWR,USA)and gently mixed in a 37°C chamber for 30 min.The wells are washed with 1 mL blocking buffer(Ocean catalog#BBB)five times with 1 min shaking sufficiently to remove non-specifically attached antibodies.After washing and blocking,1 mL DPBS with 1 mg/mL BSAis added to each well to which 10 picomole of streptavidin-QD conjugate is added.The plate is vortexed and incubated under the previous condition for 30 min.The blocking and washing steps are repeated three times.The glass cover slide is taken out gently and placed on a microscope.

QD labeled cells are observed on an Emscope fluorescence microscope(Emscope,USA)using 200×magnification.Thecells are observed using UV light and digital photographs are taken at various sections of the slides.

2 RESULTSAND DISCUSSION

Early stage detection of breast cancer cells is critical for patient survival.Sufficient treatment during the early stages of the breast cancer can lead to higher survival rate as well as prevent the spread of cancer cells to other parts of the body.The most common method for detecting cancer cells is immuno fluorescence assays and immuno histochemical staining.QDs as labels of antibodies for fluorescent signal generation have become a viable alternative to enzyme and fluorescent dyes[1-2,20-22]. In this project,we used only one kind of the antibody,anti-HER2/neu antibody labeled with biotin in combination with streptavidin-QD conjugates for signal generation.The in situ images of breast cancer cells are observed and recorded using a fluorescence microscope with a UV light source.

2.1 Size,surface charge,and optical properties of QDs

The physical properties of QDs are established after synthesized.As shown in Fig.1,the TEM image shows that the Cd Se/ZnScore/shell QDs are approximately 7 nm in diameter without the surface coating.This number is confirmed with a Zetatracker(Zetatrac Company,USA).After surface coating with a triblock polymer,the diameter increases to 14 nm with a zeta potential of 31 mV.

Fig.1 TEM image of Cd Se/ZnScore/shell QDs

After surfacecoa ting modification,the water soluble Cd Se/ZnS core/shell QDs exhibite the same optical properties,such as UV-vis absorption and fluorescence emission. The quantum yield＞ 50% indicates that the modification process involving a polymer coating for water soluble conversion has little effect on the brightness of QDs(see Fig.2).

2.2 Surface functionalizat ion of QDs and gel electrophoresis analysis

Fig.2 UV-vis absorption(left sloping curves)and fluorescence emission spectra (symmetric peaks at 530 nm)of QDs in water

The conjugation of the streptavidin to the QDs is confirmed with agarosegel electrophoresis shown in Fig.3.The QDs without the streptavidin label are expected to run more rapidly toward the positive electrodebecause of their smaller size and high negative charges.The streptavidin-QDs are expected to run slower because of their larger size and reduced charges due to protein shielding. The results of the gel electrophoresis reveal that carboxyl group modified QDs(Ocean catalog# SHP)have greater migration distances which are in agreement with their strongly negative zeta potential and the smaller size.

Fig.3 Gel electrophoresis data(100 V for 30 min)for streptavidin-QDs

The polarities of electrodes are such that the top is negative and the bottom is positive.The bands from left to right correspond to carboxylated QDsλ emission 530 nm(Lane 1)and streptavidin-QDsλ emission 530 nm(Lane 2).The binding of streptavidin on the surface of the QDs leads to an increase in the net size,thus the migration on the gel during electrophoresis is slower compared with the free QDs.

2.3 Breast cancer cell(SK-BR3)culture

The breast cancer cells grow fast as shown by the change of the medium on the second day of the culture.The cells are multi-angular as a attachment result to the bottom of the culture flasks.On the fourth day,the cells already reach 90%confluence so they are harvested and inoculated on the glass cover placed on a six-well culture plate.Three days after the inoculation,the glass cover shows 80%—90%confluence exhibiting a natural multi-angular cell shape that has characteristic of cells in situ(see Fig.4).

Fig.4 Digital photograph of SK-BR3 cells grown on glass cover

The cells are multi-angular and irregular in the shape with the good light reflection(white light source at 200×magnification).

2.4 In situ cancer cell imaging using green QDs

The breast cancer cells in the complete assay consisting of SK-BR3+ 1°Ab-biotin+ streptavidin-QDs appeared as bright green colored irregular structures in situ under UV illuminated condition(see Fig.5).The brighter fluorescence indicates that most of QDs are attached to the cell surface because anti-HER2/neu is an antibody against the cancer cell surface protein.Even after 48 h of the storage in a humidified atmosphere,most of the cells still keep their shape and the bright green fluorescence.QD labeled fluorescent cells are stableat 4°Cin excess of 48h.The control using FITC(data not shown)shows that the color is lighter and diminished after several hours of the storage. Lower detection limits are achieved during biological assays using QDs because of their brighter signals compared with the organic dye.

Initial studies indicate that there are less bright signals in the incomplete assays that do not have 1°Ab-biotin. Using the blocking buffer#BBB with shaking and incubation for 1 min that is repeated three times.It can eliminate the signals from the incomplete assay.Thus,before finalizing the in situ imaging of the breast cancer cells using green QDs, non-specific binding(NSB)on the incomplete as well as the complete assays are successfully eliminated using blocking buffer#BBB.

Fig.5(a)shows that in a complete assay involving SK-BR3+1°Ab-biotin+streptavidin-QDs under white light and Fig.5(b)shows that under the UV light,QDs haveλemission 530 nm green on a microscope at 200×magnification.

Fig.5 Digital photographs of microscope images of breast cancer cells(SK-BR3)

The streptavidin-QDs targeted biotin molecules bound to antibodies are specific to HER2 proteins on the surface of SK-BR3 breast cancer cells.This results in images with bright fluorescent cells on the microscope when using a UV light source.Compared QD-cell imaging along side imaging with fluoresce in(FITC)dye QD imaging is 90%brighter.The use of QDs can significantly enhance the signals because of their high quantum yield and stability.Furthermore,the QDimaged cells remain bright even after 48 h of the storage indicating stability of the QDs for imaging cells.Thus,the studies show that QD-labeled antibodies have a promise for the sensitive and specific imaging,and the detection of cancer cells.

3 CONCLUSION

Instead of direct conjugation of the antibodies to QDs,it is possible to use streptavidin-biotin as a bridge for assays.This can beused for different kinds of imaging combined with commercially available antibodies.The biotin-Ab and the streptavidin-QD combination can be used as a tool for various assays,northern blotting,western blotting,cell labeling,and other applications.Ocean′s super blocking buffer#BBB of ocean eliminates the non-specific signals.Thus,the use of blocking buffer# BBB will eliminate non-specific signals that can mninimize false positive results.Future work will focus on the optimization of the various assay parameters,the elimination of nonspecific signals from biological fluids,quantification,as well as multi-cell/multi-color QD detection. All the parameters for QD-cell imaging analysis areoptimized for the diagnosis of the early stage breast cancer cell.

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