CN112415206B - Application of CD171 protein in exosome as tumor metastasis diagnosis marker - Google Patents

Abstract

The invention relates to the field of medical diagnostics, in particular to application of CD171 protein in exosomes as a tumor metastasis diagnosis marker. The present invention provides a method for predicting tumor metastasis and its metastatic sites. The CD171 protein in the exosomes specifically related to the invention can be used as a tumor metastasis prediction marker, and detection of the CD171 protein in the exosomes from a tissue-specific source can be used for predicting tumor metastasis sites. The method provided by the invention can be applied to the prediction of tumor metastasis, and also provides an important reference for tumor prognosis evaluation and the selection of tumor treatment schemes.

Description

Application of CD171 protein in exosomes as a diagnostic marker for tumor metastasis

technical field

The present invention relates to the field of medical diagnostics, in particular to the application of CD171 protein in exosomes as a diagnostic marker for tumor metastasis.

Background technique

Tumor occurrence is a multi-factor, multi-stage evolutionary process. In recent years, studies have found that the expression level of CD171 in cancer is positively correlated with the ability to metastasize, and CD171 protein can increase the migration of tumor cells. Metastatic tumor cells lacking CD171 can reach and drill out of capillaries through blood circulation, but lose the ability to move along the outer wall of blood vessels and grow into tumors. Normal cancer cells move along the capillaries and can squeeze out the pericytes along the way, but after knocking out CD171, the cancer cells cannot squeeze out the blocking pericytes; on the contrary, in a cancer cell with low expression of CD171, the Expression of CD171 increases the ability of cancer cells to migrate along the vascular network. The CD171 protein is key to tumor seeding.

There is an urgent need in this field for methods that can effectively predict tumor metastasis, assess tumor prognosis, and select tumor treatment options.

Contents of the invention

The present invention relates to the application of detection agent in the preparation kit;

The detection agent includes a quantitative detection agent for CD171 protein in exosomes;

The kit is used for at least one of predicting whether tumor metastasis occurs in the subject, predicting the location of metastasis, judging the prognosis of tumor treatment, and selecting a tumor treatment plan;

The exosomes are from tissue-specific exosomes in biological samples;

The tissue-specific exosomes are selected from intestinal tissue-specific exosomes, gastric tissue-specific exosomes, liver tissue-specific exosomes, lung tissue-specific exosomes, and lymphoid tissue-specific exosomes at least one of .

Optionally, as described above, the detection agent also contains at least one of the following qualitative detection agents for tissue-specific markers:

Intestinal tissue-specific marker MS4A12, gastric tissue-specific marker SLC9A4, liver tissue-specific marker SLCO1B3, lung tissue-specific marker AGER, and lymphoid tissue-specific marker CD8A.

Optionally, as mentioned above, the detection agent is used to perform any of the following methods:

Biomass Spectrometry, Electrophoresis, Chromatography, ELISA, Immunofluorescence, Immunochemiluminescence, Immunoturbidimetry, Western Blotting, and Dot Blot.

Optionally, in the above-mentioned application, the detection agent is a specific antibody against the substance to be detected.

Optionally, as mentioned above, the biological sample is selected from cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspiration fluid, bronchoalveolar washing fluid, urine, semen, vaginal secretion , mucus, saliva, sputum, and cleared lysates obtained from biological tissue samples.

The present invention also designs a kit, which contains exosome enrichment and purification reagents and detection reagents as defined above;

The kit is used for at least one of predicting whether tumor metastasis occurs in the subject, predicting the location of metastasis, judging the prognosis of tumor treatment, and selecting a tumor treatment plan.

Optionally, the above-mentioned kit also contains at least one of a solid phase carrier, a protease inhibitor, a blocking solution, a chromogenic reagent and a washing buffer.

Optionally, in the above-mentioned kit, the solid phase carrier is a chemiluminescence plate.

The present invention also relates to a system, which includes an information acquisition module and a data analysis report module;

The function of the information acquisition module includes: obtaining the concentration of CD171 protein in exosomes, and providing the result to the data analysis module;

The function of the data analysis reporting module includes: judging whether the concentration is ≥ 150 pg/mL, if yes, output high risk of tumor metastasis; if not, output low risk of tumor metastasis.

The beneficial effects of the present invention are:

The present invention provides a method for predicting tumor metastasis and its metastasis site. The CD171 protein in exosomes specifically involved in the present invention can be used as a marker for tumor metastasis prediction, and the detection of CD171 protein in tissue-specific exosomes can be used to predict tumor metastasis sites. The method provided by the invention can not only be applied to the prediction of tumor metastasis, but also provide an important reference for tumor prognosis assessment and selection of tumor treatment schemes.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is the detection result of the total exosomal CD171 protein of metastatic and non-metastatic cancer samples in one embodiment of the present invention;

Figure 2 is the detection results of exosome CD171 protein from tissue-specific source of samples from patients with gastric cancer liver metastases in one embodiment of the present invention;

Fig. 3 is the detection result of exosome CD171 protein from tissue-specific source of intestinal cancer liver metastases in one embodiment of the present invention;

Fig. 4 shows the detection results of CD171 protein in exosomes from tissue-specific sources of samples from patients with lung metastasis of liver cancer in one embodiment of the present invention.

Detailed ways

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment.

Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the invention are disclosed in or are apparent from the following detailed description. It is to be understood by those of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended to limit the broader aspects of the invention.

The present invention relates to the application of detection agent in the preparation kit;

The detection agent includes a quantitative detection agent for CD171 protein in exosomes;

The kit is used for at least one of predicting whether tumor metastasis occurs in the subject, predicting the location of metastasis, judging the prognosis of tumor treatment, and selecting a tumor treatment plan;

The exosomes are from tissue-specific exosomes in biological samples;

The tissue-specific exosomes are selected from intestinal tissue-specific exosomes, gastric tissue-specific exosomes, liver tissue-specific exosomes, lung tissue-specific exosomes, and lymphoid tissue-specific exosomes at least one of .

The present invention provides a new marker for diagnosis: CD171 protein in exosomes.

Wherein, compared with the control level from a subject without said tumor metastasis, an increase in the level of CD171 protein in the exosomes of the subject indicates that there may be tumor metastasis in the subject, and in the subject Elevated levels of CD171 in tissue-specific derived exosomes indicated corresponding tumor metastatic sites.

The term "marker" as used herein refers to a molecule to be used as a target for analysis of a patient's experimental sample. Examples of such molecular targets are proteins or polypeptides. Proteins or polypeptides used as markers in the present invention are intended to include naturally occurring variants of said proteins as well as fragments of said proteins or said variants, particularly immunologically detectable fragments. An immunologically detectable fragment preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 contiguous amino acids of said marker polypeptide. Those skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (eg, during inflammation) and may be degraded or cleaved into such fragments. Certain markers are synthesized in an inactive form that can be subsequently activated by proteolysis. As will be apparent to the skilled artisan, proteins or fragments thereof may also be present as part of a complex. Such complexes can also be used as markers in the sense of the present invention. Additionally, or in the alternative, the marker polypeptide or variant thereof may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation.

In some embodiments, the tumor is a solid tumor.

In some embodiments, the quantitative detection agent also contains at least one of the detection agents selected from the following tissue-specific markers:

Intestinal tissue-specific marker MS4A12, gastric tissue-specific marker SLC9A4, liver tissue-specific marker SLCO1B3, lung tissue-specific marker AGER, and lymphoid tissue-specific marker CD8A.

In some embodiments, the quantitative detection agent is used to perform any of the following methods:

Biomass Spectrometry, Electrophoresis, Chromatography, ELISA, Immunofluorescence, Immunochemiluminescence, Immunoturbidimetry, Western Blotting, and Dot Blot.

The quantitative detection agent is usually a reagent that specifically detects the CD171 protein and optional tissue-specific markers in exosomes, for example, a lectin that specifically binds to one of them, a suitable protein that specifically binds to one of them Ligands or antibodies and antibody fragments that specifically bind to one of them. A specific binding agent has an affinity for its corresponding target molecule of at least 10 ⁷ l/mol. A specific binding agent preferably has an affinity for its target molecule of 10 ⁸ 1/mol, or more preferably 10 ⁹ 1/mol.

In some embodiments, the quantitative detection agent is a specific antibody against the substance to be detected.

In some embodiments, the specific antibody is a monoclonal antibody or a polyclonal antibody.

In some embodiments, the biological sample is selected from cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspiration fluid, bronchoalveolar wash, urine, semen, vaginal secretion, mucus, saliva , sputum, and cleared lysate obtained from a biological tissue sample.

The present invention also provides a kit, which contains an exosome enrichment and purification reagent and a quantitative detection reagent as defined above;

The kit is used for at least one of predicting whether tumor metastasis occurs in the subject, predicting the location of metastasis, judging the prognosis of tumor treatment, and selecting a tumor treatment plan.

In some embodiments, the kit further contains at least one of a solid phase carrier, a protease inhibitor, a blocking solution, a chromogenic reagent, and a washing buffer.

The protease inhibitor can be one or more of Indinavir, Saquinavir, Ritonavir, Nelfinavir, Amprenavir and other ingredients.

The blocking solution can be one or more of BSA, bovine serum, skim milk, TBST and other components.

The chromogenic solution can be determined according to the labeled substance on the antibody, for example, when the labeled substance is horseradish peroxidase, the chromogenic reagent can be luminol.

The washing buffer can be composed of PBS, TBS and the like.

Wherein, the protease inhibitor, blocking solution, chromogenic solution, and washing buffer can be packaged in the kit in the form of working concentration, or can be packaged in the form of their concentrated mother solution (such as 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50 times concentrated mother liquor).

The solid phase carrier is usually used to coat the antibody, and the solid phase carrier material used for coating the antibody can be polystyrene, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agar Sugar gel and other materials, the carrier can be in the form of test tubes, EP tubes, porous plates (especially chemiluminescent plates), micro-reaction plate wells, small beads (especially magnetic beads), small discs, etc.

A preferred solid support is a chemiluminescent plate. It contains 16, 32, 48, 64, 96 or more pores.

According to yet another aspect of the present invention, it also relates to a system, the system comprising an information acquisition module and a data analysis report module;

The function of the information acquisition module includes: obtaining the concentration of CD171 protein in exosomes, and providing the result to the data analysis module;

The function of the data analysis reporting module includes: judging whether the concentration is ≥ 150 pg/mL, if yes, output high risk of tumor metastasis; if not, output low risk of tumor metastasis.

In some embodiments, the exosomes come from diagnosed cancer tissue (denoted as A).

In some embodiments, the exosomes can also come from a tissue that may be transferred (denoted as B); further, if the concentration of CD171 protein in B is ≥ 150pg/mL, it means that the tumor cells of A are transferred to the tissue of B. high risk.

The present invention also relates to a method for diagnosing whether tumor metastasis occurs, said method comprising:

a) Enrichment and purification of exosomes;

b) detecting the concentration of CD171 protein in the exosomes;

c) If the concentration is ≥ 150 pg/mL, the probability of metastasis is high.

In some embodiments, in step a), the total exosomes are separated and enriched from the subject's biological sample according to the principle of molecular sieve chromatography (for example, using CL-6B filler).

In some embodiments, step b) simultaneously detects intestinal tissue-specific exosomes, gastric tissue-specific exosomes, liver tissue-specific exosomes, lung tissue-specific exosomes, and lymphoid tissue-specific exosomes at least one of the markers.

In some embodiments, the marker is selected from at least one of the following: intestinal tissue-specific marker MS4A12, gastric tissue-specific marker SLC9A4, liver tissue-specific marker SLCO1B3, lung tissue-specific marker AGER, and Lymphoid tissue-specific marker CD8A.

The subject is usually a mammal, preferably a primate, more preferably a human.

To clarify, the ideal scenario for diagnosis is a situation where a single event or process causes a variety of diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as is the case with many cancer types. As the skilled artisan will appreciate, a diagnosis without a biochemical marker is 100% specific and as 100% sensitive for a given multifactorial disease. Conversely, biochemical markers (such as the CD171 protein concentration in exosomes demonstrated herein) can be used to assess, for example, the presence or severity of a disease with a certain probability or predictive value. Therefore, in routine clinical diagnosis, various clinical symptoms and biological markers are usually considered comprehensively to diagnose, treat and control the underlying disease.

Embodiments of the present invention will be described in detail below in conjunction with examples.

Example 1 Isolation of exosomes from serum tissue-specific sources

Plasma was taken out from the -80°C refrigerator, 2 mL of each plasma sample was thawed quickly, and then 0.6 mL of thromboplastin-D was added to incubate at room temperature for 60 minutes. Add 1.4 mL of phosphate buffered saline containing protease inhibitor cocktail. Centrifuge at 3000g for 20 minutes at 4°C. After centrifugation, the supernatant solution was collected using CL-6B filler to separate and enrich the total exosomes, and concentrated to a volume of 2 mL. Take 200uL of exosome solution, add 2uL of biotinylated antibody of tissue-specific markers, and then add 50uL of 3% BSA, and place in a refrigerator at 4°C for 1 hour. After that, 25uL of streptavidin agarose was added, and then 50uL of 3% BSA was added, and left at 4°C for 30 minutes. Under the condition of 4°C, centrifuge at 400g for 10 minutes, fully remove the supernatant and keep the precipitate. Add 50uL of 0.05M glycine-hydrochloric acid (pH=3) to the precipitate, and vortex for 10 seconds. The resuspended tissue-specific exosome particles were stored in a -80°C refrigerator.

Example 2 Determination of CD171 protein level expressed in exosomes

Take the extracted exosome sample out of the -80°C refrigerator, thaw it quickly, add 0.5mL of M-PER, and adjust the pH value of the solution to 8.0 with 1M Tris-HCl at pH 8.6. Incubate in a constant temperature water bath at 37°C for 10 minutes, vortex for 10 seconds to completely lyse the exosomes. Take the CD171 detection kit out of the refrigerator and equilibrate to room temperature, add 100uL of the calibrator and the sample to be tested respectively, and set up duplicate holes. Incubate for 1 hour in a 37°C incubator. After washing the plate 3 times, 100uL of detection antibody was added, and incubated in a 37°C incubator for 1 hour. After washing the plate 3 times, 100uL of chemiluminescence substrate was added, and the luminescence value was read by a chemiluminescence instrument. Draw a calibration curve based on the luminescence value of the calibrator, and calculate the concentration of the sample from the calibration curve.

Example 3 Detection of total exosomal CD171 protein in metastatic and non-metastatic cancer samples

Collect 50 plasma samples of cancer metastasis, including 10 gastric cancer samples, 10 intestinal cancer samples, 10 lung cancer samples and 10 liver cancer samples; collect cancer non-metastasis plasma samples, including 10 gastric cancer samples, 10 intestinal cancer samples, 10 lung cancer samples and 10 liver cancer samples. After isolation and enrichment, the total exosomes were lysed and the expression level of the surface marker CD171 protein was detected. It can be seen from Figure 1 that most of the CD171 expression levels of non-metastatic cancer samples are lower than 150 pg/mL, and most of the CD171 expression levels of metastatic cancer samples are higher than 150 pg/mL.

Table 1 Detection of total exosomal CD171 protein in metastatic and non-metastatic cancer samples

Example 4 Detection of CD171 protein in tissue-specific source exosomes and prediction of tumor metastatic sites

Plasma samples were collected from 30 patients with metastatic cancer, including 10 patients with liver metastases from gastric cancer, 10 patients with liver metastases from intestinal cancer, and 10 patients with lung metastases from liver cancer. Exosomes from tissue-specific sources were isolated and captured, and the expression level of the surface marker CD171 protein was detected. Figure 2 Detection results of tissue-specific exosomal CD171 protein from patients with gastric cancer liver metastasis. It can be seen from the figure that the expression level of exosomal CD171 protein from liver tissue is significantly higher than that of other tissues. Figure 3 The detection results of tissue-specific exosomal CD171 protein from patients with intestinal cancer liver metastases. It can be seen from the figure that the expression level of exosomal CD171 protein from liver tissue is significantly higher than that of other tissues. Figure 4 The detection results of tissue-specific exosomal CD171 protein from patients with liver cancer and lung metastasis. It can be seen from the figure that the expression level of exosomal CD171 protein from lung tissue is significantly higher than that of other tissues.

Table 2 Detection of CD171 protein in tissue-specific exosomes from patients with gastric cancer liver metastases

Table 3 Detection of CD171 protein in exosomes from tissue-specific sources of colorectal cancer liver metastases

Table 4 Detection of CD171 protein in tissue-specific exosomes from patients with liver cancer and lung metastasis

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. The application of the detection agent in the preparation of the kit,

the detection agent comprises a quantitative detection agent of CD171 protein in exosomes;

the kit is used for predicting at least one of tumor metastasis sites, prognosis of tumor treatment based on the tumor metastasis sites, and selecting a tumor treatment regimen based on the tumor metastasis sites;

the exosomes are derived from tissue-specific exosomes in a biological sample;

the tissue-specific exosome is selected from at least one of intestinal tissue-specific exosome, gastric tissue-specific exosome, liver tissue-specific exosome, lung tissue-specific exosome, and lymphoid tissue-specific exosome;

the tumor is at least one selected from intestinal cancer, liver cancer, gastric cancer and lung cancer;

the tumor transfer part is selected from at least one of intestinal cancer tissue, liver cancer tissue, gastric cancer tissue, lung cancer tissue and lymph tissue;

the biological sample includes a plasma sample.

2. The use according to claim 1, wherein the detection agent further comprises at least one qualitative detection agent selected from the group consisting of tissue-specific markers:

intestinal tissue-specific markers, gastric tissue-specific markers, liver tissue-specific markers, lung tissue-specific markers, and lymphoid tissue-specific markers.

3. The use according to claim 1 or 2, wherein the detection agent further comprises at least one qualitative detection agent selected from the group consisting of tissue-specific markers:

intestinal tissue specific marker MS4a12, gastric tissue specific marker SLC9A4, liver tissue specific marker SLCO1B3, lung tissue specific marker agrer, and lymphoid tissue specific marker CD8A.

4. The use according to any one of claims 1 to 3, wherein the detection agent is used to perform any one of the following methods:

biological mass spectrometry, electrophoresis, immunofluorescence, immunonephelometry, and dot blotting.

5. The use according to claim 4, wherein the detection agent is a specific antibody against the substance to be detected.

6. The use according to claim 1, wherein the tumour is a solid tumour.

7. The use according to any one of claims 1 to 6, wherein the kit further comprises a total exosome enrichment reagent.

8. The use according to claim 1, wherein the kit further comprises at least one of a solid support, a protease inhibitor, a blocking solution, a chromogenic agent and a wash buffer.

9. The use according to claim 8, wherein the solid support is a chemiluminescent plate.

10. A system comprising an information acquisition module and a data analysis reporting module;

the information acquisition module has the functions of: obtaining the concentration of CD171 protein in an exosome from a tissue-specific exosome in a biological sample, the biological sample comprising a plasma sample, and providing the result to the data analysis reporting module;

the tissue-specific exosome is selected from at least one of intestinal tissue-specific exosome, gastric tissue-specific exosome, liver tissue-specific exosome, lung tissue-specific exosome, and lymphoid tissue-specific exosome;

the functions of the data analysis reporting module include: and predicting a tumor metastasis site according to the concentration and the tissue corresponding to the exosome, wherein the tumor is selected from at least one of intestinal cancer, liver cancer, gastric cancer and lung cancer, and the tumor metastasis site is selected from at least one of intestinal cancer tissue, liver cancer tissue, gastric cancer tissue, lung cancer tissue and lymph tissue.