KR101218798B1 - A method for imaging metastasis of cancer via primo-vessel - Google Patents

Abstract

The present invention relates to an imaging method capable of analyzing cancer metastasis pathways through Bonghan ducts. The method of the present invention comprises the steps of (i) microinjecting a quantum dot dye into cancer cells; (Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development; (Iv) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths after the incision around the cancer-generating site of the animal; (Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And (iii) comparing the optical color image with the normalized fluorescence image to determine whether fluorescence is present in the Bonghan duct. Since the method of the present invention is efficient and highly sensitive, it can be usefully used for screening metastasis inhibiting agents and metastasis mechanism of cancer through Bonghan duct.

Description

A method for imaging metastasis of cancer via primo-vessel}

The present invention relates to an imaging method capable of analyzing cancer metastasis pathways through Bonghan ducts.

Metastasis is the process by which cancer cells spread from the primary site to form new tumors in other organs. This is a complex, multi-step process that, in fact, considers it very poorly at the cellular level, causing a sudden death of the patient. Thus, inhibition and control of metastasis is the most promising approach for completing cancer treatment. Over the past three decades, research on cancer metastasis has exploded. Since tumor metastasis is believed to occur via blood vessels and lymphatic vessels (referred to as the vascular and lymphatic pathways, respectively), the focus of the study is on the identification of many molecules and genes related to anti-angiogenesis and anti-lymphocytic production. Has been tailored. However, the blocking of angiogenesis and lymphangiogenesis has not always been shown to be effective in clinical trials, and the process and mechanism of metastasis has not yet been fully elucidated. This calls for radically different ways of thinking about new ways of thinking about the process of cancer's spread and the technologies needed to access these themes.

Recently, new thread-like coronary structures have been found in the fascia surrounding superficial tumors (Yoo et al ., J. Acupunct. Meridian Stud ., 2 (2): 118-). 123, 2009). These coronary threads are not only a new kind of conduit, but are also clearly distinguished from blood vessels or lymphatic vessels. It is a major breakthrough that these new conduits are present both inside and outside the tumor tissue. Anatomical and histological observations have confirmed that this structure is in fact a pathological development of the primo-vascular system, which was rediscovered only 40 years after it was first discovered by Dr. Kim Bong-han (Soh et al ., J. Acupunct). Meridian Stud ., 2 (2): 93-106, 2009). The Bonghan tissue is a whole-scale web of thread-like tubular structure called primo-vessel, in which some liquid flows. The Bonghan duct has been detected on the surface of various tissues of mice, rats and rabbits and inside the central canal of blood vessels, lymphatic vessels, ventricles and spinal cords (Soh et al ., J. Acupunct. Meridian Stud ., 2) 2): 93-106, 2009. Although the overall circulatory network has not yet been identified, evidence for the liquid transport function of bony tissues has been found for yarn-like structures containing both liquid and very small cells with diameters of 1 to 2 μm. Observations were made through electron microscopy (Lee et al ., Microsc. Res. Tech . 70 (1): 34-43, 2007) .Measurement of flow rate using microinjection of dye (0.3 ± 0.1 mm / s) (Sung et al ., Naturwissenschaften , 95 (2): 117-124, 2008) and measurement of liquid viscosity using a microextraction method (1.4 ± 0.1 mPa s at room temperature) have been reported (Sung et al. al ., Phys . Rev. E: Stat ., Nonlinear , Soft Matter Phys ., 79 (2), 2009).

However, the function of the Bonghan duct is related to the meridians, and the research results of the degree of the flow of chi is unknown.

An object of the present invention is to provide an imaging method for cancer cell metastasis observation through Bonghan duct.

Still another object of the present invention is to provide a method for in vivo screening of a substance that inhibits cancer cell metastasis through the Bonghan duct using the imaging method.

The present inventors have proposed a hypothesis that cancer cells can be used for seeding and clustering of Bonghan ducts in addition to blood vessels and lymphatic vessels in addition to vascular and lymphatic vessels. The present invention was completed by demonstrating that cancer cells can metastasize to Bonso via Bonghan ducts using NIR QD) -electroporation and multispectral fluorescence imaging methods.

Definition of terms used in this document

Hereinafter, terms used in the present document will be described in more detail.

QD (Quantum dot , QD ) : A quantum dot is composed of a shell made of ZnS (zinc sulfide) and a centroid of about 2 to 10 nm (nanometer) in size. CdSe (cadmium selenide), CdTe (cadmium telluride), CdS (cadmium sulfide) are mainly used as the center of the quantum dot. Quantum dots have various properties that other materials do not have. Typically, quantum dots generate a strong fluorescence in a narrow wavelength band, and the light emitted by the quantum dots is unstable from the conduction band to the valence band. As the electrons in the state come down, the generated fluorescence has a very special property of generating shorter wavelength light as the particles of the quantum dot is smaller, and longer wavelength light as the particles are larger. Due to these characteristics, quantum dots have recently been used to label biological materials such as cells. In addition, it is excellent in natural color reproducibility, has recently been spotlighted as a material that can compensate for the shortcomings of the LED.

Multispectral fluorescent imaging system : A multispectral fluorescent imaging system refers to an imaging apparatus capable of simultaneously visualizing fluorescence emitted from various wavelength bands.

Optical color image : An optical color image refers to an image obtained by photographing reflected light by irradiating natural light to an anatomical site.

Dark current image : An image of light emitted from the same wavelength range without irradiating any light, and used to remove noise of the photographing apparatus itself.

Normalized fluorescent image : Normalized fluorescent image is a fluorescence image generated by pure quantum dots generated by considering intrinsic fluorescence and dark current images in the original fluorescence image.

Primo-vessel : Bonghan duct is a third vascular structure that is distinct from blood vessels and lymphatic vessels. It is white and has a rugged surface with a bundle structure, and its biological function is not known yet. It is known to flow.

Bonghanjeol (primo node) : Bonghanjeol refers to the intersection of two or more Bonghan ducts.

Electroporation : Electroporation is a method in which a certain width of current is applied to a cell at regular intervals to form a hole in a cell membrane, and a specific material flows through the hole according to the flow of current generated therein. It is a kind of intracellular substance delivery method that delivers substances into the cytoplasm.

Nanoinjection (nanoinjection) : Nanoinjection refers to a technique that can introduce a specific substance into the cell while inducing minimal fluctuations to the cell membrane using a nanometer injector. Currently, a nanoinjector using carbon nanotubes is used. (Chen et al ., Proc. Natl. Acad. Sci. USA , 104 (20): 8218-8222, 2007).

Osmotic method (osmotic method) : One method for non-invasive delivery of quantum dots into living cells, the complex is formed by mixing quantum dots-stereptavidin and biotin-labeled protein in a high molar ratio of 1: 1 molar ratio After this, the cells are placed in a cell in a low osmotic environment, and the pinocytic vesicles are lysed by osmotic pressure to deliver the complex intracellularly. (Courty et al ., Methods Enzymol ., 414: 211-228, 2006).

Lipofection : A method that allows a substance captured by a lipid bilayer or mycel to be delivered intracellularly by the cell's negative cell and / or phagocytosis. It is used to deliver water-soluble substances such as nucleic acids and, in the case of micelles, to deliver fat-soluble substances intracellularly.

Summary of the Invention

In one embodiment of the invention,

(Iii) microinjecting the quantum dot dye into cancer cells;

(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;

(Iv) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths after the incision around the cancer-generating site of the animal;

(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And

(Iii) comparing the optical color image with the normalized fluorescence image to determine whether fluorescence is present in the Bonghan duct, and providing an imaging method of metastasis of cancer cells through the Bonghan duct.

In this case, the micro-injection is preferably performed by nanoinjection (nanoinjection), lipofection (lipfection), osmotic method (electrosmotic method) or electroporation (electroporation), but is not limited thereto, using an electroporation method Most preferably. In the case of using the electroporation method, the pulse width of the applied pulse is 50 to 150 μs, the pulse interval is 50 to 150 μs, the number of pulses is 15 to 25 times, and the pulse amplitude is preferably 0.5 to 2.0 kV / cm. Most preferably, the width is 100 μs, the pulse interval is 100 μs, the number of pulses is 18 and the pulse amplitude is 1.0 kV / cm, but is not limited thereto.

The quantum dot dye is preferably CdSe (cadmium selenide), CdTe (cadmium telluride), CdS (cadmium sulfide), CdTeSe (cadmium telluride selenide) alloy quantum dots, and most preferably, but not limited to CdTeSe .

Any animal other than the human may be any animal that has an immune response suppressed, but is preferably athymic nude mouse or nude rat.

In another embodiment of the present invention,

(Iii) microinjecting the quantum dot dye into cancer cells;

(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;

(Iii) administering an anti-cancer metastasis candidate to the animal inducing cancer;

(Iii) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths by dissection around the cancer-generating site of the animal to which the candidate is administered and the control animal to which the candidate is not administered ;

(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And

(Iii) screening in vivo a method for inhibiting cancer cell metastasis through the Bonghan duct, comprising selecting a candidate substance whose fluorescence in the Bonghan duct identified on the optical color image is less than that in the Bonghan duct of the control group. to provide.

In this case, the micro-injection is preferably performed by nanoinjection (nanoinjection), lipofection (lipfection), osmotic method (electrosmotic method) or electroporation (electroporation), but is not limited thereto, using an electroporation method Most preferably. In the case of using the electroporation method, the pulse width of the applied pulse is 50 to 150 μs, the pulse interval is 50 to 150 μs, the number of pulses is 15 to 25 times, and the pulse amplitude is preferably 0.5 to 2.0 kV / cm. Most preferably, the width is 100 μs, the pulse interval is 100 μs, the number of pulses is 18 and the pulse amplitude is 1.0 kV / cm, but is not limited thereto.

The quantum dot dye is preferably CdSe (cadmium selenide), CdTe (cadmium telluride), CdS (cadmium sulfide), CdTeSe (cadmium telluride selenide) alloy quantum dots, and most preferably, but not limited to CdTeSe .

Any animal other than the human may be any animal that has an immune response suppressed, but is preferably athymic nude mouse or nude rat.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, more detailed description of the present invention.

In one embodiment of the invention,

(Iii) microinjecting the quantum dot dye into cancer cells;

(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;

(Iv) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths after the incision around the cancer-generating site of the animal;

(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And

(Iii) comparing the optical color image with the normalized fluorescence image to determine whether fluorescence is present in the Bonghan duct, and providing an imaging method of metastasis of cancer cells through the Bonghan duct.

In this case, the micro-injection is preferably performed by nanoinjection (nanoinjection), lipofection, osmotic method (electrosmotic method) or electroporation (electroporation), but is not limited thereto, using an electroporation method Most preferably. In the case of using the electroporation method, the pulse width of the applied pulse is 50 to 150 μs, the pulse interval is 50 to 150 μs, the number of pulses is 15 to 25 times, and the pulse amplitude is preferably 0.5 to 2.0 kV / cm. Most preferably, the width is 100 μs, the pulse interval is 100 μs, the number of pulses is 18 and the pulse amplitude is 1.0 kV / cm, but is not limited thereto.

The quantum dot dye is preferably CdSe (cadmium selenide), CdTe (cadmium telluride), CdS (cadmium sulfide), CdTeSe (cadmium telluride selenide) alloy quantum dots, and most preferably, but not limited to CdTeSe

Any animal other than the human may be any animal that has an immune response suppressed, but is preferably athymic nude mouse or nude rat.

The present inventors have hypothesized that Bonghan duct may be related to cancer metastasis as a third coronary system, and in order to confirm that the hypothesis is correct, first, by using electroporation, a quantum dot dye is applied to cancer cells without damaging cancer cells. It was examined whether it was possible to load. The present inventors confirmed that applying a pulse width of 100 μs, a pulse interval of 100 μs, 18 pulses, and a pulse amplitude of 1.0 kV / cm, quantum dots can be loaded onto cancer cells without damaging the cancer cells (FIG. 1). Reference). This is a very encouraging result considering that conventionally, when quantum dots are loaded into cells by electroporation, they are considered undesirable because of their cytotoxicity.

Based on the above results, cancer cells labeled with these quantum dots were injected into animals to induce cancer, and then the metastasis process of these cancers was confirmed by a multispectral fluorescence imaging system.

Specifically, lung carcinoma labeled with quantum dots was injected subcutaneously in rats, and used as a model for metastasis to the periphery of the injection site and metastasis to visceral organs, and injected human ovarian cancer cells labeled with quantum dots into the abdominal cavity of mice. Used as a transition model. In vivo fluorescence imaging after inoculation of cancer cells confirmed cancer cell metastasis, and intraoperative fluorescence imaging and anatomical observation revealed that metastasis of these cancer cells occurred relatively selectively through Bonghan duct (FIGS. 2 to 2). 7). Therefore, the present inventors have confirmed that cancer metastasis may occur through the Bonghan duct, and by monitoring the cancer metastasis through the Bonghan duct, by selecting an anti-metastatic agent that can suppress the metastasis of the cancer through the Bonghan duct, It has been confirmed that it may provide clues to the development of new antitransmitters.

The screening method of cancer metastasis suppressor through Bonghan duct of the present invention is as follows:

(Iii) microinjecting the quantum dot dye into cancer cells;

(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;

(Iii) administering an anti-cancer metastasis candidate to the animal inducing cancer;

(Iii) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths by dissection around the cancer-generating site of the animal to which the candidate is administered and the control animal to which the candidate is not administered ;

(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And

(Iii) screening in vivo a method for inhibiting cancer cell metastasis through the Bonghan duct, comprising selecting a candidate substance whose fluorescence in the Bonghan duct identified on the optical color image is less than that in the Bonghan duct of the control group. to provide.

In this case, the micro-injection is preferably performed by nanoinjection (nanoinjection), lipofection (lipfection), osmotic method (electrosmotic method) or electroporation (electroporation), but is not limited thereto, using an electroporation method Most preferably. In the case of using the electroporation method, the pulse width of the applied pulse is 50 to 150 μs, the pulse interval is 50 to 150 μs, the number of pulses is 15 to 25 times, and the pulse amplitude is preferably 0.5 to 2.0 kV / cm. Most preferably, the width is 100 μs, the pulse interval is 100 μs, the number of pulses is 18 and the pulse amplitude is 1.0 kV / cm, but is not limited thereto.

The quantum dot dye is preferably CdSe (cadmium selenide), CdTe (cadmium telluride), CdS (cadmium sulfide), CdTeSe (cadmium telluride selenide) alloy quantum dots, and most preferably, but not limited to CdTeSe

Any animal other than the human may be any animal that has an immune response suppressed, but is preferably athymic nude mouse or nude rat.

In the electroporation method used in the present invention, the quantum dots could be efficiently introduced into the cancer cells and labeled without cytotoxicity against the cancer cells, and even after several weeks, the image loss due to the diffusion of the quantum dots could be minimized, which is very useful for tracking cancer cells. It was found to be effective, and the multispectral fluorescence imaging method for the quantum dots used in the present invention was very sensitive and was able to accurately track the metastasis path of cancer through Bonghan and Lymphatic vessels.

1 is a photograph of tumor cells labeled intracellularly by QD electroporation:
A: Fluorescence and DIC micrographs (red: pseudo-coloured QD, blue: nuclei stained by DAPI) of QD electroporated human lung cancer cells (NCI-H460),
B: Transmission electron micrograph of QD delivered to NCI-H460 cells identified based on size (8-10 nm) and shape (group 4),
C: calibrated multispectral image showing efficient tumor growth 6 weeks after inoculation of QD-loaded NCI-H460, and
D: Re-visualized photograph of QD-labeled cells of tumor tissue excised from C by multiphoton microscopy.
FIG. 2 is an in vivo multispectral image of cancer cells seeded through bonghan tissue around superficially grown tumors:
A: in vivo multispectral image,
B: intra operative multispectral image, and
C: Illustration of region of interest for lymphatic and Bonghan duct sites in color image of B. FIG.
Figure 3 is a graph comparing the corrected fluorescence intensity in the region of interest of the Bonghan duct and lymphatic vessel of Figure 2C.
4 is an intraoperative multispectral image of the seeding of cancer cells through peritoneal bony tissue in a subcutaneous xenograft tumor model:
A: intrinsic image, fluorescence image and corrected fluorescence image of subcutaneous tumor,
B: intrinsic image, fluorescence image and corrected fluorescence image for viscera after open, and
C: An enlarged color image from B to liver, abdominal wall (a), small intestine (b) and bladder (c). Dotted line indicates the location of Bonghan duct.
FIG. 5 is an intraoperative multispectral image of selective implantation of cancer cells through the abdominal Bonghan duct in intraperitoneally injected ovarian cancer mice:
A: Optical color image, intrinsic image, fluorescence image and corrected fluorescence image of the incision including around the greater omentum, with arrowhead indicating Bonghan duct,
B: optical color image, intrinsic image, fluorescence image, and corrected fluorescence image of the incision containing testicular fat, with arrowhead indicating Bonghan lord, arrow indicating Bonghan duct, and
C: Optical color image, intrinsic image, fluorescence image and corrected fluorescence image of the incision surrounding the inferior vena cava, with arrowheads representing bony nodules and dashed arrows representing lymph nodes.
FIG. 6 is a graph quantitatively comparing and calculating the average number of photons in each region of interest (L: liver, S: stomach, I: small intestine, O: ovary, and GF: testicular fat).
7 is a series of photographs showing histological observations of metastasized organs and Bonghan ducts:
A: Representative fluorescence / DIC microscopy images of slices of metastasized organs (first row: small intestinal villi, second row: liver and third row: lung) in subcutaneous NCI-H460 tumor xenografts, column 1 ) Is a fluorescence image by quantum dots, the second column is an overlap image of quantum dot image and DAPI staining, and the third column is an overlap image of quantum dot image and DIC microscope image,
B: Representative fluorescence / DIC microscopy images of bony nodules (double arrows in the first row), Bonghan ducts (arrows in the second row), and abdominal Bonghan ducts (third row) around the subcutaneous tumors in a subcutaneous xenograft model of NCI-H460 cancer cells A first column is a fluorescence image by quantum dots, a second column is an image by H & E staining, a third column is an image by Verhoeff staining, and
C: Representative fluorescence / DIC microscopic images of longitudinal slices of the Bonghan ducts (first row and second row) and lymphatic vessels (third row), in particular the second and third rows are located around the same testicular fat and Fluorescence / DIC microscopy image of lymphatic vessel, description of each column is same as A.

Hereinafter, the present invention will be described by way of specific examples. The present invention is not intended to be limited to the scope of the present invention.

General Experiment Method

1. Loading of quantum dots using electroporation

CdTeSe alloy quantum dots (QD) were synthesized under cadmium-rich conditions using methods similar to those previously reported (Bailey et al ., J. Am. Chem. Soc. , 125 (23): 7100-7106, 2003). . The emission peak of the quantum dot was 800 nm and had a full width at hat maximum of 50 nm. Hydrodynamic diameter was 8-10 nm. 500 nm CdTeSe alloy quantum dots were loaded with an electroporator after loading 2 × 10 ⁷ NCI-H460 human lung cancer cells and SK-OV-3 human ovarian cancer cells, which are cancer cells purchased from Korea Cell Line Bank (Seoul, Korea). And suspended in 2 ml of RPMI-1640 medium and allowed to stand for 5 minutes, followed by application of pulse width of 100 μs, pulse interval of 100 μs, pulse number 18 and pulse amplitude of 1.0 kV / cm without cell death. Optimal intracellular quantum dot delivery was performed. Then, the cells were washed several times with cell culture medium to remove free quantum dot dye.

2. Fabrication of Tumor Model Animals

Tumor model animals were 5-7 week old female nude mice (Balb-c-nu / nd, Japan SLC, Inc., Japan), which were bred in a filter-laminar flow cabinet and using a sterilization process. It was manipulated. Animal-related experiments were performed in accordance with the guidelines of Seoul National University. Two sets of tumor models were used, the first being a standard subcutaneous (sc) xenograft mouse model (Welch et al ., Clin. Exp. Metastasis , 15 (3): 272-306, 1997). In this case, five mice were anesthetized by intraperitoneally (ip) injection of zoletil / rompun and then 1 × 10 ⁷ NCI-H460 human lung cancer cells suspended in 1 ml RPMI-1640 medium for subcutaneous cancer formation. Inoculated by subcutaneous injection to the back. Another tumor model is an intraperitoneal experimental metastasis model. 1 ml of RPMI-1640 medium in which SK-OV-3 human ovarian cancer cells were suspended at 1 × 10 ⁷ cells / ml was injected into the abdominal cavity of five nude mice.

3. Confirmation of tumor formation in vivo in Bonghan tissue

To identify cancer cell migration through the Bonghan duct around subcutaneous tumors and visceral organs in a subcutaneous xenograft tumor model, we have developed a self-made multiplex with dual fluorescence and color detectors for mice 8 weeks after subcutaneous inoculation of tumor cells. Images were obtained using a spectral imaging system. All intraoperative imaging procedures were performed under general anesthesia via intraperitoneal injection of zoletil / romfun. First, the side of the tumor skin was excised and the skin of the tumor was carefully removed to expose the tumor with an intact membrane. Bonghan ducts and blood vessels were then confirmed by visualizing the surgical anatomy directly through the color channels of the imaging setup. At the same time, near infra-red (NIR) fluorescence was recorded from cancer cells inside Bonghan ducts and tumor tissues, and further image processing was achieved by correcting tissue attenuation variation and background autofluorescence. The Bonghan tissue on the surface of the iso organ was irradiated abdomen to confirm the escape of cancer cells from the superficial tumor through the Bonghan duct. To investigate the selective transplantation of cancer cells in the intraperitoneal bony tissue, the inventors observed an experimental metastatic mouse model 1 to 4 weeks after intraperitoneal inoculation in the same imaging method.

4. Multispectral Imaging and Analysis

For in vivo and intraoperative imaging of cancer cells in Bonghan tissues, we used a self-made multispectral imaging system. The developed system can generate a fluorescent image that corrects tissue optical attenuation and self-fluorescence while simultaneously generating an optical color image. Typically, 6 images were obtained for each experiment, 650 ± 5 nm, 750 ± 12.5 nm, 800 ± 12.5 nm, dark current image and optical color image for the same emission wavelength as above. . Then, the present inventors have found that non-were obtained to normalize the fluorescent image having a wavelength of λ _x is 750 ± 12.5 nm one image of I _x and the wavelength λ _y is 800 ± 12.5 nm image of when the time I _y, respectively, the I _x And I _y can be obtained by the following formulas I and II, respectively:

I _x = I _fx -I _dx (Ⅰ)

I _y = I _fy -I _dy (II).

_Wherein I _fx and I _fy are the initial fluorescence images and I _dx and I _dy are the I _fx and I _fy Each is a corresponding dark current image obtained without irradiation of light for the same exposure time. The inventors also obtained I _z , which is an attenuation (intrinsic) image at the irradiation wavelength of λ _z = 650 ± 5 nm, by the following formula III:

I _z = I _iz -I _dz (III).

Where I _iz is an intrinsic image and I _dz is a corresponding noise image. We then describe U _x , which is the corresponding normalized fluorescence image And U _y were calculated using the following equations IV and V in a similar manner as previously reported (Ntziachristos et al ., J. Biomed. Opt ., 10 (6): 064007, 2005):

(Ⅳ),

(Ⅳ),

(Ⅴ).

(Ⅴ).

In the above formula, the threshold T _i was set to 3% of the maximum of the denominator value to prevent low signal-to-noise ratio signals from contributing to the final images U _x and U _y .

After the attenuation measurement, the spectral correction of the quantum dots and the self-fluorescent signal was performed using a linear unmixing algorithm (Zimmermann et al ., Adv. Biochem. Eng. Biotechnol . 95: 245-265, 2005). The inventors obtained the emission spectra of pure quantum dots by measurement through the above-developed setup, and obtained the emission spectra of background self-fluorescence by identifying areas where the signal is clearly pure self-fluorescence. The non-shared image was obtained by solving equations VI and VII for the following shared system:

U _x = S _Ax C _A + S _Qx C _Q (Ⅵ)

U _y = S _Ay C _A + S _Qy C _Q (Ⅶ).

In the above formula, U _x and U _y are detected normalized fluorescence images, and S _Ax , S _Ay , S _Qx and S _Qy are known from the standard emission spectra obtained in advance, channel λ _x = 750 ± 12.5 nm and λ _y = 800 ± 12.5 nm relative contribution of background self-fluorescence and quantum dots, C _A and C _Q are unknown unmixed images for the self-fluorescence and quantum dots, respectively to be.

5. Microscopy and Histological Analysis

To investigate the ability of electroporation to provide effective delivery of quantum dots to cancer cells, we performed multiphoton microscopy and transmission electron microscopy analysis of the electroporated cells. Understanding multiphoton microscopy, the electroporated cells were fixed in 4% paraformaldehyde for 20 minutes, washed several times with phosphate buffered saline (PBS), embedded in anti-smearing agents, and then multiphoton microscopy (Zeiss LSM 510). META, Germany) to obtain an image. Nuclei were labeled with DAPI (Molecular Probes, USA). For transmission electron microscopy, the cells were fixed for 10 hours at 4 ° C. with 2.5% glutaraldehyde solution in 0.1 M cacodylate acid buffer at pH 7.4, followed by post-fixation with osmium tetraide and dehydration. The immobilized cells were embedded in Epon812 and then sliced to 50 nm thickness, stained with uranil acetate and lead citrate, and images were taken by operating a transmission electron microscope (JEOL JEM-1010, Japan) at 80 kV. It was.

After obtaining in vivo or surgical images of cancer cells in Bonghan tissues, ex vivo retests were performed with a multi-photon microscope (Zeiss LSM 510 META, Germany) or an optical microscope (Olympus BX51, Japan). For this purpose, samples were sliced to 5 μm and then fixed with 4% formaldehyde. The samples were left overnight in the fixative, washed several times with PBS the next day and counterstained with DAPI. Several consecutive tissue slices were stained with hematoxylin and eosin stains Verhoeff staining to confirm histological properties of Bonghan ducts.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the following examples.

Example 1: Validation of the utility of electroporation for in vivo metastasis studies

The method we used to establish a model for imaging cancer cells inside the Bonghan duct in living animals is to label cancer cells with near-infrared quantum dots (NIR QD) by electroporation. By using electroporation with optimal parameters for electrical pulses, DHLA-packed CdTeSe quantum dots are effectively delivered to cells, as confirmed by multi-photon microscopy (FIG. 1A) and transmission electron microscopy (FIG. 1B). It became.

Loading of electroporation-based quantum dots resulted in agreement with other findings of high performance parallel delivery and intermittent formation of intracellular quantum dot aggregation (FIG. 1A) (Chen et al ., Nano Lett ., 4 (10)). : 1827-1832, 2004; Derfus et al ., Adv. Mater ., 16 (12): 961-966, 2004). Some quantum dots entered the nucleus, but most remained cytoplasmic. To investigate the behavior of quantum dot-labeled cancer cells under in vivo conditions, we subcutaneously injected NCI-H460 human lung cells labeled with quantum dots to the back of immunodeficient mice. From the sixth week of surgery, the present inventors confirmed that quantum dot-labeled cancer cells developed efficiently into tumors as well as unlabeled cancer cells (FIG. 1C).

In vivo multispectral imaging was able to record tumor growth with high contrast and high sensitivity, thanks to tissue optical attenuation correction and self-fluorescent non-interflow (FIG. 1C). Histological examination of the exfoliated tumors revealed that most cancer cells still retain quantum dots after 6 weeks after in vivo inoculation, minimizing the spread of quantum dots (FIG. 1D). It is understood that the tumorigenic capacity of quantum dot-labeled cancer cells may provide an ideal tool for identifying metastatic processes in Bonghan ducts in living animals, as identified herein.

Example 2: Formation of metastatic colonies and migration of cancer cells via Bonghan duct

We investigated the tumor growth and metastasis process of quantum dot-labeled cells using in vivo multispectral imaging of NIR fluorescence signals in NCI-H460 cell inoculation regions of athymic immunodeficient mice (n = 5). At this time, the fluorescence measurement value was corrected as described above in consideration of tissue attenuation and self-fluorescence in order to more clearly show the tumor boundary, the intrinsic image and the fluorescence image were excited at 650 ± 5 nm and 800 ± 12.5 Photographed at emission wavelength of nm. Three weeks after transplantation, we found that the quantum dot-labeled cells spread and clustered in the vicinity of the injection site (FIG. 2A). As shown in FIG. 2A above, tumor growth at the QD-labeled cell injection site, denoted a, was imaged with a strong NIR signal, and new tumors were observed at adjacent sites labeled b. The successful colony-forming ability of these quantum dot-labeled cancer cells shows that the cytotoxicity caused by the electroporation process is negligible. Anatomical confirmation of the proliferation pathway and detailed investigation of cancer cell behavior were performed through careful skin resection (FIG. 2B). NIR fluorescence imaging examined quantum dot-labeled cancer cells that migrated from the original tumor to the newly grown tumor via a Bonghan duct that is distinct from the lymphatic pathway (FIG. 2B). The distinction between lymphatic and Bonghan ducts is their anatomical features, for example, Bonghan ducts are white and have a bumpy surface of bundle structure, whereas lymphatic vessels have a very transparent and smooth surface of a single lumen structure, which is a large number of prior art. It is a simple but key criterion learned from the morphological analysis of the study (Soh et al ., J. Acupunct. Meridian Stud . 2 (2): 93-106, 2009). The path of migration of the tumor cells was compared quantitatively by calculating the average photon count for similar regions of interest shown as boxes in FIG. 2C (FIG. 3). The tumor cells preferred to migrate through the Bonghan duct (dashed arrow) relative to the lymphatic vessel (solid arrow), the intensity of the signal in the Bonghan duct was about twice as high as that of the lymphatic vessel. Average signal intensity from blood vessels was little significant in terms of signal to noise ratio.

Example 3: Abdominal metastasis of cancer cells through Bonghan duct in subcutaneous xenograft model

We next examined abdominal metastases in a subcutaneous xenograft model (n = 5) using a midline incision to the abdomen. Intraoperative multispectral imaging confirmed spontaneous metastasis from subcutaneous tumors (a, b and c in FIG. 4B), with confirmation of superficial growth fluorescent tumors 8 weeks after subcutaneous injection of quantum dot-labeled NCI-H460 cells (FIG. 4A). A strong fluorescence signal emitted by the metastatic cells was detected in the Bonghan duct, which was located from the liver towards the abdominal wall and was also present on the small intestine and bladder (a, b and c in FIG. 4B). An enlarged anatomical observation confirmed that the Bonghan duct is a translucent and freely moving coronary structure (indicated by dotted lines in FIG. 4C) with unique characteristics that distinguish it from those of blood vessels and lymphatic vessels. In such cases, the data presented show significantly higher capacity of Bonghan ducts in metastasis of cancer.

Example 4: Abdominal metastasis of cancer cells through Bonghan duct in intraperitoneal ovarian cancer xenograft model

The results of intraperitoneal injection of quantum dot-labeled cancer cells also showed a significant relationship between Bonghan tube and cancer metastasis. Specifically, the present inventors were able to demonstrate quantum dot-labeled ovarian cancer cell infiltration in Bonghan tissue using intraperitoneal (i.p.) inoculation method in athymic immunodeficient mice (n = 5). Two or three weeks after inoculation, site-specific transplantation of quantum dot-labeled SK-OV-3 in peritoneal tissue following intra-operative guidance from the NIR multispectral imaging system was investigated. Numerous quantum dot-labeled ovarian cancer cells selectively infiltrated into Bonghan ganglia, frequently identified as translucent point structures in the greater omentum (FIG. 5A). A complex network of Bonghan tissues A complex network of Bonghan tissues transplanted with a number of distinctly transplanted quantum dot-labeled ovarian cancer cells was observed in testicular fat (FIG. 5B). Visualization of seeded cancer cells in Bonghan tissue without interference of light scattered from adipose tissue could be obtained by sophisticated multispectral imaging. We quantitatively compared the accumulation of quantum dot-labeled cancer cells in bony and lymph nodes in the analyzed cases (FIG. 5C). The mean fluorescence intensity value is similar to the interest in Bonghan nodes near the inferior vena cava (eg, freely moving structures connected to freely moving Bonghan ducts, arrowheads) and two lymph nodes (eg, enclosed structures connected to lymphatic vessels, dotted arrows). Obtained from the area (FIG. 5C). The Bonghan ganglia were differentiated from lymph nodes by higher infiltration of quantum dot-labeled cancer cells (FIG. 6).

Example 5: Histopathological Analysis

In order to confirm the presence of the NIR fluorescence signal from the non-injured cancer cells and the distinctive morphology of the bony tissues, the present inventors further confirmed the metastasis process in the bony tissues as a result of subsequent histopathological analysis (FIG. 7). ). As shown in FIG. 7A, metastasized organs were observed in a subcutaneous NCI-H460 mouse xenograft model in which superficial tumors were successfully produced. As shown by the characteristic NIR fluorescence generated from quantum dots (overlapping with red), after escaping from superficial tumors, a population of quantum dot-labeled cancer cells developed intestinal villi (first row), liver (second row) and lung ( Last row). The development of metastases in other organs means that labeling cells with quantum dots has no detectable side effects on the viability and function of cancer cells, and the subcutaneous NCI-H460 xenograft model is suitable for the study of metastasis. FIG. 7B shows representative images of quantum dot-labeled cancer cells stagnated in Bonghan ganglia (first row), Bonghan duct (second row) and peritoneal Bonghan duct (last row) near superficial tumors. Microscopic confirmation of NIR fluorescence confirmed that a number of quantum dot-labeled cancer cells were located in the Bonghan duct and Bonghan duct samples consistent with in vivo data (FIGS. 2 and 4). Through H & E staining and Berghofe staining, which is consistent with previous reports (Soh et al ., J. Acupunct. Meridian Stud ., 2 (2): 93-106, 2009; Ogay et al ., J. Acupunct. Meridian Stud , 2 (2): 107-117, 2009) A typical alignment of the rod sheath nucleus can be observed in the multi-steel structure and the longitudinally cut Bonghan duct in the transversely cut Bonghan pelvis. FIG. 7C shows the identification of intraperitoneally inoculated quantum dot-labeled cancer cells penetrating the Bonghan duct, as already confirmed by in vivo multispectral imaging in FIG. 5. NIR quantum dots located in the cytoplasm surrounding the nucleus of cancer cells are clearly observed inside the Bonghan duct (first row). Furthermore, microscopic observations revealed a number of quantum dot-labeled cancer cells infiltrating multiple cavities in the Bonghan duct, surrounded by rod-shaped nuclei in adipose tissue (second row), but in lymphoid tissues of the same adipose tissue. Fewer cancer cells were observed in a single clean lumen (last row). In this regard, it should be noted that no frequent spots were released into the extracellular matrix and the quantum dots were all located within the cancer cells through all histopathological observations, which further confirms the reliability of the results in vivo.

As described above, the present inventors were able to confirm the presence of additional pathways of cancer cells from the vicinity of the primary tumor to the abdominal cavity through in vivo imaging of cancer cell migration and transplantation in the Bonghan tissue, and the Bonghan tissue was found in various stages of cancer development. It was found that during the period it can operate independently of the vascular and lymphatic pathways. Thus, the findings by the inventors may provide new clues for the development of therapeutic strategies targeting these new structures associated with tumor metastasis. On the other hand, the high contrast and high sensitivity in vivo imaging system provided by the present invention will be of great help in understanding the mechanism of cancer cell migration and infiltration in the bony tissue.

Since the method of the present invention is efficient and highly sensitive, it can be usefully used for screening metastasis suppressor and metastasis mechanism of cancer through Bonghan duct.

Claims (10)

(Iii) microinjecting the quantum dot dye into cancer cells;
(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;
(Iv) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths after the incision around the cancer-generating site of the animal;
(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And
(Iii) comparing the optical color image with the normalized fluorescence image to determine whether fluorescence is present in the Bonghan duct, the method of measuring metastasis of cancer cells through the Bonghan duct. The method of claim 1, wherein the microinjection is performed by electroporation. The method of claim 2, wherein the electroporation is performed through a Bonghan duct, characterized in that the pulse width 50 to 150 μs, pulse interval 50 to 150 μs, pulse number 15 to 25 times and pulse amplitude 0.5 to 2.0 kV / cm Method for measuring metastasis of cancer cells. The method of claim 1, wherein the quantum dot dye is a CdTeSe alloy quantum dot. The method of claim 1, wherein the animal other than the human is a nude mouse. (Iii) microinjecting the quantum dot dye into cancer cells;
(Ii) injecting cancer cells injected with the quantum dot dye into an animal other than a human to induce cancer development;
(Iii) administering an anti-cancer metastasis candidate to the animal inducing cancer;
(Iii) obtaining a fluorescence image and an optical color image according to a plurality of emission wavelengths by dissection around the cancer-generating site of the animal to which the candidate is administered and the control animal to which the candidate is not administered ;
(Iii) obtaining a normalized fluorescence image by considering a dark current image and an intrinsic fluorescence image in the fluorescence image; And
(Iii) screening a candidate substance for inhibiting cancer cell metastasis through the Bonghan duct, comprising selecting a candidate substance whose fluorescence shown in the Bonghan duct identified on the optical color image is less than that shown in the Bonghan duct of the control group. The method of claim 6, wherein the micro-injection is performed by electroporation method in vivo screening of a substance for inhibiting cancer cell metastasis through Bonghan duct. The method of claim 7, wherein the electroporation is performed through a Bonghan duct, characterized in that the pulse width 50 to 150 μs, pulse interval 50 to 150 μs, pulse number 15 to 25 times and pulse amplitude 0.5 to 2.0 kV / cm In vivo screening methods of substances that inhibit cancer cell metastasis. The method of claim 6, wherein the quantum dot dye is a CdTeSe alloy quantum dot in vivo screening method of a substance for inhibiting cancer cell metastasis through Bonghan duct. The in vivo screening method of a substance for inhibiting cancer cell metastasis through Bonghan ducts according to claim 6, wherein the animal other than human is a nude mouse.

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