CN110710495A - Breast cancer PDX construction method - Google Patents
Breast cancer PDX construction method Download PDFInfo
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- CN110710495A CN110710495A CN201810760063.5A CN201810760063A CN110710495A CN 110710495 A CN110710495 A CN 110710495A CN 201810760063 A CN201810760063 A CN 201810760063A CN 110710495 A CN110710495 A CN 110710495A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0271—Chimeric animals, e.g. comprising exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Abstract
The invention discloses a breast cancer PDX construction method, which comprises the following steps: preparing a tumor tissue sample, injecting and inoculating the breast of the immunodeficient mouse into a duct, and conventionally feeding and observing the immunodeficient mouse. The invention adopts the method of breast duct injection, can inject the human breast cancer micro tissue block or the breast cancer primary cell into the breast duct of the immunodeficient mouse, constructs the human breast cancer in-situ PDX model, better accords with the actual generation and development of clinical breast cancer, and provides convenience for better research of breast cancer for human beings. The method can also be used for constructing a breast cancer cell line transplantation tumor model by injecting human or mouse derived cell lines into the breast duct.
Description
Technical Field
The invention relates to the technical field of breast cancer treatment, in particular to a breast cancer PDX construction method.
Background
A human-derived tumor xenograft model (PDX) refers to an in-vivo animal model which is constructed by inoculating a fresh tumor sample of a tumor Patient into an immunodeficiency mouse and dividing and proliferating the tumor sample by virtue of a physiological environment in the mouse. The PDX model reserves the heterogeneity, pathological characteristics, gene expression and variation of tumors of tumor patients, has high clinical relevance with the patients, has incomparable advantages of other tumor models, and has great significance for personalized treatment of tumors, research of tumor science and research and development of new drugs. However, low success rate, long cycle time and high cost are important bottlenecks that prevent the wide use of the PDX model.
Breast cancer is a common tumor in women. Modern research suggests that breast cancer is not a single disease but rather a heterogeneous collection of diseases characterized by a diversity of histologies, genomic alterations, gene expression, metastatic behavior, and therapeutic responses. In addition to tumor heterogeneity between patient populations (or between two primary tumors in a single patient), recent data also show considerable intratumoral heterogeneity (i.e., the difference between cells within a single tumor of the same patient and between the primary tumor and its metastatic tumor). This constitutes a significant impediment to effective treatment of breast cancer, and the development of a personalized therapeutic approach based on an individual's specific biology and unique tumor is an indiscriminate therapeutic strategy. The heterogeneity that exists between breast cancer internal and breast cancer, both with respect to basic and transformation studies, also presents a significant challenge to the fabrication and use of preclinical models associated with breast disease. While the PDX model is clearly an advantageous tool to address this challenge.
Efforts by researchers to establish stable, transplantable xenograft models by direct inoculation of tumor tissue from breast cancer patients into immunocompromised mice have been ongoing for decades. However, the success rate of establishing a stable transplantable model (generally defined as the ability of the mouse PDX model to be serially transplanted from P0 generation ≧ 3 generation) is only about 10%. Therefore, few stable transplantation models are available for early studies. In recent years, with the updating iteration of immunodeficient mice and the improvement of an inoculation method, the success rate of a human breast cancer PDX model is stabilized at about 20 percent.
At present, a plurality of breast cancer inoculation methods are commonly used, and breast cancer tissues or cells can be inoculated to an anterior chamber of an eyeball, a kidney subcontracting membrane, a scapular fat pad, subcutaneous (ear, face and the like) or in-situ breast fat pad of a mouse and injected into a breast catheter. However, the inoculation positions are different from the growth environment of breast cancer in clinic, and do not accord with the physiological environment for the occurrence and development of breast cancer, so the application is limited. Because of the hormone dependence of breast cancer, in order to improve the success rate of breast cancer PDX, the implantation of estradiol sustained-release tablets in mice to promote the tumor formation of the mice has also been selected. The estradiol sustained release tablet has high price and greatly increases the cost.
The breast cancer in-situ PDX model is creatively established by adopting a breast duct internal injection method, breast cancer cells or tissue blocks can be inoculated into the breast duct, a tumor microenvironment is reserved, the breast cancer in-situ PDX model is closer to clinical practical conditions, and the application value is huge.
Disclosure of Invention
The invention provides a method for constructing breast cancer PDX, aiming at solving the defects in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for constructing breast cancer PDX comprises the following steps:
1) tumor sample collection: tumor tissues which are vigorous in growth at the edge of a tumor and have no ulcer and necrosis are cut in an operation and are quickly put into a centrifuge tube containing tissue preservation solution, and the centrifuge tube is put into an ice bag and transported to a laboratory at low temperature.
2) Tumor sample treatment: tumor tissue viability was detected in a laboratory biosafety cabinet with 0.4% trypan blue staining solution and non-viable tissue was removed. The tissue was then sterilized by soaking in 0.2% iodophor solution for 30 seconds and rinsed with 3% double antibody in PBS. Put into new culture dish with the tumor tissue that washes totally, stab the tissue with No. 7 syringe needle on one side, wash with a small amount of PBS solution on one side, stab the tissue into the slice after, reuse two tweezers twist, extrude the tissue to rinse in the PBS solution, then collect liquid, filter through 300 mesh filter screens. And collecting the filtered microtissue block suspension into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 rpm, discarding most of liquid in the centrifuge tube, and reserving a small amount of culture medium to suspend the microtissue block for later use.
Collagenase may be added to the microtissue mass suspension as necessary to digest the microtissue mass into single cells for use. The tumor sample can also be prepared into single cell suspension by adopting a GentleMACS full-automatic mild tissue processor which is gentle and gentle in the America.
3) Mouse inoculation: in SPF-class animal houses, adult female nude mice (BALB/C-nu/nu) or other highly immunodeficient female mice are selected, and after anesthesia, the mice are fixed on an operation plate in a supine position, and the haired mice need to have their abdominal hairs shaved off to completely expose the 4 th pair of nipples. Disinfecting local skin with 0.5% iodophor, cutting off the papilla tip with a pair of 4 th pair of ophthalmology scissors by means of a dissecting microscope, exposing the mammary duct, sucking 20 mul of the uniformly mixed micro tissue block suspension or cell suspension (about 10 ten thousand cells/papilla) by using a 50ul plain Hamilton micro-injection needle (30G), slowly injecting into the mammary duct, and disinfecting the wound by using a 0.5% iodophor cotton swab.
4) Mouse feeding and observation: mice were housed in IVC cages in SPF-grade animal rooms and were kept routinely. Observing the tumor-bearing mice twice a week, touching the texture of the mammary gland with a hand, measuring the size of the tumor with a vernier caliper, and recording a tumor growth curve;
5) pathology and genetic testing: when the tumor volume grows to 1500mm3And when the left and right parts or the surface of the body has ulcer, the tumor body is removed by operation, one part of the tumor body is taken out for pathological detection and gene sequencing, one part of the rest tissues is put into the frozen stock solution for freezing and the other part is continuously passed.
Further, in the step 1), tumor tissues which are vigorous in tumor edge growth and free of ulcer and necrosis are cut out in the operation, the tumor tissues are quickly put into a centrifuge tube containing tissue preservation solution, and the centrifuge tube is put into an ice bag for low-temperature transportation to a laboratory.
Further, in step 2), the breast cancer tissue block is also required to be processed in a blocking manner, and the operation method is as follows: tumor tissue viability was detected in a laboratory biosafety cabinet with 0.4% trypan blue staining solution and non-viable tissue was removed. The tissue was then sterilized by soaking in 0.2% iodophor solution for 30 seconds and rinsed with 3% double antibody in PBS. Put into new culture dish with the tumor tissue that washes totally, stab the tissue with No. 7 syringe needle on one side, wash with a small amount of PBS solution on one side, stab the tissue into the slice after, reuse two tweezers twist, extrude the tissue to rinse in the PBS solution, then collect liquid, filter through 300 mesh filter screens. And collecting the filtered microtissue block suspension into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 rpm, discarding most of liquid in the centrifuge tube, and reserving a small amount of culture medium to suspend the microtissue block for later use.
Further, in step 3), in injecting the mouse, the operator fixes the nipple with the ophthalmological forceps and holds the micro-syringe with hand, and the needle is aligned with the cut nipple, pierces into the mammary duct, and slowly injects the suspension. After injection, the mice need not to be sutured, are bred conventionally after being wiped by iodophor, and can be injected with multiple nipples (the number of the nipples of the mice is 5).
Further, in step 3), said small tissue mass suspension may be replaced withThe breast cancer cell suspension can be prepared by human breast cancer cell strain or cell line, or human breast cancer primary cell, collecting breast cancer cells, and preparing into 1 × 10 cells with culture medium6Cell suspension per ml.
Compared with the prior art, the method for in-situ PDX model construction of the human breast cancer by adopting the method of in-vitro injection of the breast duct can better meet the actual clinical breast cancer occurrence and development, save expensive reagent consumables and save a large amount of medical cost.
Detailed Description
The breast cancer PDX construction method provided by the invention mainly comprises the following steps:
1) tumor sample collection: tumor tissues which grow vigorously and have no ulcer necrosis are cut in the operation, and the tumor tissues are quickly put into a centrifuge tube containing tissue preservation solution, and the centrifuge tube is put into an ice bag for low-temperature transportation.
2) Tumor sample treatment: tumor tissue viability was detected in a laboratory biosafety cabinet with 0.4% trypan blue staining solution and non-viable tissue was removed. The tissue was then sterilized by soaking in 0.2% iodophor solution for 30 seconds and rinsed with 3% double antibody in PBS. Put into new culture dish with the tumor tissue that washes totally, stab the tissue with No. 7 syringe needle on one side, wash with a small amount of PBS solution on one side, stab the tissue into the slice after, reuse two tweezers twist, extrude the tissue to rinse in the PBS solution, then collect liquid, filter through 300 mesh filter screens. And collecting the filtered microtissue block suspension into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 rpm, discarding most of liquid in the centrifuge tube, and reserving a small amount of culture medium to suspend the microtissue block for later use.
Collagenase may be added to the microtissue mass suspension as necessary to digest the microtissue mass into single cells for use. The tumor sample can also be prepared into single cell suspension by adopting a GentleMACS full-automatic mild tissue processor which is gentle and gentle in the America.
3) Mouse inoculation: in SPF-class animal houses, adult female nude mice (BALB/C-nu/nu) or other highly immunodeficient female mice are selected, and after anesthesia, the mice are fixed on an operation plate in a supine position, and the haired mice need to have their abdominal hairs shaved off to completely expose the 4 th pair of nipples. Disinfecting local skin with 0.5% iodophor, cutting off the papilla tip with a pair of 4 th pair of ophthalmology scissors by means of a dissecting microscope, exposing the mammary duct, sucking 20 mul of the uniformly mixed micro tissue block suspension or cell suspension (about 10 ten thousand cells/papilla) by using a 50ul plain Hamilton micro-injection needle (30G), slowly injecting into the mammary duct, and disinfecting the wound by using a 0.5% iodophor cotton swab.
4) Mouse feeding and observation: mice were housed in IVC cages in SPF-grade animal rooms and were kept routinely. Observing the tumor-bearing mice twice a week, touching the texture of the mammary gland with a hand, measuring the size of the tumor with a vernier caliper, and recording a tumor growth curve;
5) pathology and genetic testing: when the tumor volume grows to 1500mm3And when the left and right parts or the surface of the body has ulcer, the tumor body is removed by operation, one part of the tumor body is taken out for pathological detection and gene sequencing, one part of the rest tissues is put into the frozen stock solution for freezing and the other part is continuously passed.
The construction method of the breast cancer cell suspension comprises the following steps:
preparation of breast cancer cells: the adenocarcinoma cells can be human breast cancer cell strains or cell lines, and can also be human breast cancer primary cells. Collecting cells, preparing the cells into 1 × 10 cells by using the culture medium6Cell suspension per ml.
The invention adopts a method of mammary duct internal injection to construct a human breast cancer in-situ PDX model. Compared with the existing method, the method is more suitable for the reality of clinical breast cancer occurrence and development, and saves expensive reagent consumables. Using this approach we have successfully constructed 3 breast cancer PDX models (micro tissue mass inoculation).
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the patent and protection scope of the present invention should be subject to the appended claims.
Claims (5)
1. A breast cancer PDX construction method is characterized by comprising the following steps:
1) tumor sample collection: cutting tumor tissues with vigorous growth and no ulcer necrosis at the edge of the tumor in the operation, quickly filling the tumor tissues into a centrifugal tube containing tissue preservation solution, filling the centrifugal tube into an ice bag, and transporting the ice bag to a laboratory at a low temperature;
2) tumor sample treatment: detecting the activity of tumor tissues by using 0.4 percent trypan blue dye solution in a biological safety cabinet in a laboratory, and removing tissues without activity; then placing the tissue into 0.2% iodophor solution to be soaked for 30 seconds for disinfection, and then washing the tissue by using PBS solution containing 3% double antibody; putting the washed tumor tissue into a new culture dish, poking the tissue by using a No. 7 needle while washing by using a small amount of PBS solution, poking the tissue into a sheet shape, screwing and extruding the tissue by using two forceps, rinsing in the PBS solution, collecting liquid, and filtering by using a 300-mesh filter screen; collecting the filtered micro-tissue block suspension into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 rpm, discarding most of liquid in the centrifuge tube, and reserving a small amount of culture medium to suspend the micro-tissue block for later use;
3) mouse inoculation: in an SPF-level animal room, selecting an adult female nude mouse or other high-immune-deficiency female mice, fixing the mice on an operation plate in a supine position after anesthesia, and shaving off the abdominal hair of the haired mice to completely expose the 4 th pair of nipples; sterilizing local skin with 0.5% iodophor, cutting off the papilla tip with a pair of 4 micro-ophthalmic scissors by means of a dissecting microscope, exposing a mammary duct, sucking 20 μ l of uniformly mixed micro tissue block suspension or cell suspension (about 10 ten thousand cells/papilla) by using a 50ul plain Hamilton micro-injection needle (30G), slowly injecting into the mammary duct, and sterilizing a wound by using a 0.5% iodophor cotton swab;
4) mouse feeding and observation: feeding mice in IVC cages of SPF-level animal rooms, and conventionally feeding; observing the tumor-bearing mice twice a week, touching the texture of the mammary gland with a hand, measuring the size of the tumor with a vernier caliper, and recording a tumor growth curve;
5) pathology and geneAnd (3) detection: when the tumor volume grows to 1500mm3And when the left and right parts or the surface of the body has ulcer, the tumor body is removed by operation, one part of the tumor body is taken out for pathological detection and gene sequencing, one part of the rest tissues is put into the frozen stock solution for freezing and the other part is continuously passed.
2. The method of constructing PDX for breast cancer according to claim 1, wherein: in the step 1), tumor tissues which are vigorous in growth and have no ulcer necrosis are cut in the operation and are quickly put into a centrifuge tube containing tissue preservation solution, and the centrifuge tube is put into an ice bag for low-temperature transportation.
3. The method of constructing PDX for breast cancer according to claim 1, wherein: in step 2), the breast cancer tissue block is also required to be processed in a blocking manner, and the specific operation method comprises the following steps: detecting the activity of tumor tissues by using 0.4 percent trypan blue dye solution in a biological safety cabinet in a laboratory, and removing tissues without activity; then placing the tissue into 0.2% iodophor solution to be soaked for 30 seconds for disinfection, and then washing the tissue by using PBS solution containing 3% double antibody; putting the washed tumor tissue into a new culture dish, poking the tissue by using a No. 7 needle while washing by using a small amount of PBS solution, poking the tissue into a sheet shape, screwing and extruding the tissue by using two forceps, rinsing in the PBS solution, collecting liquid, and filtering by using a 300-mesh filter screen; and collecting the filtered microtissue block suspension into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 rpm, discarding most of liquid in the centrifuge tube, and reserving a small amount of culture medium to suspend the microtissue block for later use.
4. The method of constructing PDX for breast cancer according to claim 1, wherein: in step 3), when injecting the mouse, the operator uses the micro-ophthalmic scissors to cut off the 4 th pair of papilla tips, exposes the mammary duct, uses a 50ul plain Hamilton micro-injection needle to suck 20 mul of the uniformly mixed micro tissue block suspension or cell suspension (about 10 ten thousand cells/papilla), slowly injects the micro tissue block suspension or cell suspension into the mammary duct, sterilizes the wound with a 0.5% iodophor cotton swab, and conventionally feeds the mouse.
5. The method of constructing PDX for breast cancer according to claim 4, wherein: in step 3), the microtissue block suspension can be replaced by breast cancer cell suspension, the breast cancer cell suspension can be prepared by human or mouse breast cancer cell strain or cell line, or human breast cancer primary cell, after collecting breast cancer cells, the cells are prepared into 1 × 10 cells by using culture medium6Cell suspension per ml.
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Cited By (1)
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| CN114223614A (en) * | 2021-12-10 | 2022-03-25 | 广东省实验动物监测所 | Construction method and application of breast cancer cell strain in-situ transplantation model |
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