CN110747168A - Prostate cancer in-situ PDX model construction method - Google Patents
Prostate cancer in-situ PDX model construction method Download PDFInfo
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Abstract
The invention discloses a prostate cancer in-situ PDX model construction method, which comprises the following steps: pretreatment of tissue blocks, inoculation of ventral leaves of the prostate of a mouse and conventional breeding and observation of the mouse, wherein the ventral leaves of the prostate of the mouse are close to the lower abdomen, so that the tumorigenesis condition can be observed by naked eyes of a human. The invention has simple operation, does not need to buy expensive reagent consumables to separate tissues into single cells, still maintains the original microenvironment of tumor tissues due to the tissue blocks, grows by adhering to the reproductive system of mice, better simulates the clinical practical situation, and is close to the body surface to facilitate the observation and operation of naked eyes of people.
Description
Technical Field
The invention relates to the technical field of cancer research and treatment, in particular to a prostate cancer in-situ PDX model construction method.
Background
Prostate cancer (PCa) is the most common malignancy among men in the united states. In the statistics of 2012 tumor patients in China, the incidence rate of prostate cancer is also listed in the 6 th incidence rate of malignant tumors in men, and in recent years, the incidence rate also tends to increase year by year. According to statistics, the incidence of prostate cancer in China has increased by more than 10 times in recent 20 years.
Prostate cancer is a hormone-dependent cancer and despite many new therapies for advanced patients, the overall survival rate remains relatively short due to the development of resistance to endocrine therapy in some patients. These mechanisms include interference of the Androgen Receptor (AR) axis and inhibition of androgen biosynthesis. Malignant tumor cells derived from the epithelial layer of the prostate, including the secretory cavity, basal cells and rare neuroendocrine cells, contribute to a high degree of heterogeneity in PCa. It is not clear which type of epithelial cells represent the origin of prostate cancer, either luminal or basal stem cells or both, but it is conceivable that the mechanisms therein are very complex.
To overcome the well-known limitations and difficulties in PCa research and therapy, a prostate cancer human-derived tumor xenograft model (PDX) has emerged.
The PDX model is a model of a transplant tumor formed by implanting tissue blocks, circulating tumor cells, and primary cells derived from tumor patients into the body of an immunodeficient mouse. The PDX model relies on the environmental growth provided by animals, preserving the human primary tumor microenvironment and basic biological properties. One PDX model corresponds to one patient, and can well reflect the difference of tumors among different individuals. PDX is more responsive to clinical patient conditions than traditional models.
A human prostate cancer PDX model is established by inoculating human prostate cancer tissues or primary cells into immunodeficient mice (such as Balb/C-nu/nu, NOD-SCID, NOG \ NSG, NPG and the like) in situ or ectopically. The tumor formation period after inoculation needs 2-12 months, the average tumor formation rate is 23-75%, and the tumor formation rate is closely related to the tumor invasiveness. The construction method of the prostate cancer PDX model commonly comprises subcutaneous inoculation, in-situ inoculation and kidney envelope inoculation, wherein the kidney envelope is inoculated to have the highest tumor formation rate due to the enrichment of blood vessels (the transplantation success rate of high-immunity-deficient mice such as NOG and NPG can reach about 90%). However, the kidney envelope inoculation operation is difficult to operate and difficult to observe. In addition, since prostate cancer is an androgen-dependent tumor, embedding androgen tablets to promote neoplasia and better mimic clinical conditions are often considered in constructing PDX models. The conventional prostate cancer in-situ PDX model is characterized in that a relatively large tumor block (2 multiplied by 2mm 3) is inoculated on the prostate ventral part of an immunodeficient mouse through an operation, and because the tumor block is large and prostate tissues are thin, the operation difficulty of inoculating the tumor block is relatively high, and cells in the tumor block are relatively difficult to obtain nutrition. The invention improves the operation method of the in-situ PDX model of human prostate cancer, inoculates the tumor micro-tissue block into the prostate side lobe of a male immunodeficiency mouse at multiple points through percutaneous injection, maintains the microenvironment and heterogeneity of the tumor tissue, has good survival rate of the tumor micro-tissue block and simple operation, and is expected to be widely applied to personalized and accurate medical treatment of prostate cancer, clinical pre-drug screening of new anti-cancer drugs and tumor research of prostate cancer.
Disclosure of Invention
The invention provides a prostate cancer in-situ PDX model construction method for 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 an in-situ PDX model of prostate cancer 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 housing, adult male nude mice are selected (BALB/C-nu/nu) Or other male mice with high immunodeficiency, the mice are fixed on an operation board in a supine position after anesthesia, and the haired mice need to shave the lower abdomen hair off and expose the position of the prostate ventral lobe. Sterilizing topical skin with 0.5% iodophor, making an incision of about 5mm length on lower abdomen with ophthalmic scissors, and lifting with ophthalmic scissorsThe skin was incised at the margins, exposing the ventral lobes of the prostate. Sucking 20-50 μ l of the mixed micro tissue block suspension or cell suspension with a 100ul microsyringe, injecting into the ventral lobe of prostate gland at multiple points, suturing the incision, sterilizing the sutured part with 0.5% iodophor cotton swab, and finishing inoculation. The prostate cancer micro tissue blocks or primary cell suspensions can be inoculated directly into the skin after the operation is skilled, and the skin does not need to be cut.
4) Mouse feeding and observation: mice were routinely housed in SPF grade animal house IVC cages, tumor bearing mice were observed twice a week, the prostate texture was felt by hand, and tumor size was measured with a vernier caliper and tumor growth curves were recorded.
5) Pathology and genetic testing: when the tumor volume reaches 1500mm3Or when the surface has ulcer, stripping off the tumor body, taking a part of the tumor body for pathological detection and gene sequencing, putting a part of the rest tissues into the frozen stock solution for freezing and preserving, and continuously passaging a part of the rest tissues.
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), a blocking process of the tissue block is also required, and the specific 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), when the mice are inoculated, the mice are fixed on an operation plate in a supine position after anesthesia, and the haired mice need to remove the lower abdominal hair and expose the position of the prostate ventral lobe bulge. After disinfecting the topical skin with 0.5% iodophor, an incision of about 5mm length was made in the lower abdomen with ophthalmic scissors, and then the skin at the edge of the incision was lifted with ophthalmic scissors to expose the ventral lobe of the prostate. Sucking 20-50 μ l of the mixed micro tissue block suspension or cell suspension with a 100ul microsyringe, injecting into the ventral lobe of prostate gland at multiple points, suturing the incision, sterilizing the sutured part with 0.5% iodophor cotton swab, and finishing inoculation. The prostate cancer micro tissue blocks or primary cell suspensions can be inoculated directly into the skin after the operation is skilled, and the skin does not need to be cut.
The invention adopts the syringe needle to repeatedly puncture tissues to produce a large number of tiny tissue blocks, and then the tiny tissue blocks are injected into the ventral lobe of the prostate of a mouse to construct a prostate cancer PDX model.
Compared with the traditional common method, the method has the following beneficial effects: 1. the mouse can grow depending on the reproductive system of the mouse, and the reality of the occurrence and development of clinical prostate cancer is better met; 2. no need of additional androgen supplementation, high tumor formation rate; 3. because of the tissue block, the original microenvironment of the tumor tissue is still reserved; 4. is close to the body surface, and is convenient for the naked eye observation and operation.
Detailed Description
The present invention will be described in detail with reference to examples.
The prostate cancer in-situ PDX model construction method provided by the invention mainly comprises the following steps:
pretreatment of tissue blocks:
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. 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.
Mouse inoculation:
in SPF-class animal housing, adult male nude mice are selected (BALB/C-nu/nu) Or other male mice with high immunodeficiency, the mice are fixed on an operation board in a supine position after anesthesia, and the haired mice need to shave the lower abdomen hair off and expose the position of the prostate ventral lobe. After disinfecting the topical skin with 0.5% iodophor, an incision of about 5mm length was made in the lower abdomen with ophthalmic scissors, and then the skin at the edge of the incision was lifted with ophthalmic scissors to expose the ventral lobe of the prostate. Sucking 20-50 μ l of the mixed micro tissue block suspension or cell suspension with a 100ul microsyringe, injecting into the ventral lobe of prostate gland at multiple points, suturing the incision, sterilizing the sutured part with 0.5% iodophor cotton swab, and finishing inoculation. The prostate cancer micro tissue blocks or primary cell suspensions can be inoculated directly into the skin after the operation is skilled, and the skin does not need to be cut.
Pathology and genetic testing: when the tumor volume reaches 1500mm3Or when the surface has ulcer, stripping off the tumor body, taking a part of the tumor body for pathological detection and gene sequencing, putting a part of the rest tissues into the frozen stock solution for freezing and preserving, and continuously passaging a part of the rest tissues.
The invention creatively adopts the syringe needle to repeatedly puncture tissues to produce a large number of micro tissue blocks, and then the micro tissue blocks are injected into the ventral lobe of the prostate of a mouse to construct a prostate cancer PDX model. Compared with the traditional common method, the method has the following advantages: 1. the mouse can grow depending on the reproductive system of the mouse, and the reality of the occurrence and development of clinical prostate cancer is better met; 2. no need of additional androgen supplementation, high tumor formation rate; 3. because of the tissue block, the original microenvironment of the tumor tissue is still reserved; 4. is close to the body surface, and is convenient for the naked eye observation and operation. By using the method, 4 PDX models are successfully constructed, pathological analysis shows that the pathological structures of the tumor-bearing of a PDX model mouse are consistent with those of a patient tumor, and the identification of the PDX model mouse by a PCR method is humanized tissue.
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 (3)
1. A method for constructing an in-situ PDX model of prostate cancer is characterized by comprising the following steps:
1) tumor sample collection: cutting tumor tissue with vigorous tumor edge growth and no ulcer necrosis during operation, rapidly placing into a centrifuge tube containing tissue preservation solution, placing the centrifuge tube into an ice bag, and transporting to a laboratory at 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: selecting adult male nude mice or other male mice with high immunodeficiency in an SPF (specific pathogen free) animal room, fixing the mice on an operation board in a supine position after anesthesia, shaving the hair of the lower abdomen of the haired mice, exposing the position where the ventral lobe of the prostate is raised, disinfecting local skin by 0.5% iodophor, making an incision with the length of about 5mm on the lower abdomen by using an ophthalmic scissors, and then lifting the skin at the edge of the incision by using the ophthalmic scissors to expose the ventral lobe of the prostate; sucking 20-50 μ l of the mixed micro tissue block suspension or cell suspension with a microinjector 100ul, injecting into the ventral lobe of prostate gland at multiple points, suturing the incision, sterilizing the sutured part with 0.5% iodophor cotton swab, and finishing inoculation;
4) mouse feeding and observation: conventionally feeding the mice in an IVC cage of an SPF-level animal room, observing the tumor-bearing mice twice every week, touching the texture of the prostate by hands, measuring the size of the tumor by a vernier caliper, and recording the growth curve of the tumor;
5) pathology and genetic testing: when the tumor volume reaches 1500mm3Or when the surface has ulcer, stripping off the tumor body, taking a part of the tumor body for pathological detection and gene sequencing, putting a part of the rest tissues into the frozen stock solution for freezing and preserving, and continuously passaging a part of the rest tissues.
2. The method for constructing an in situ PDX model of prostate cancer according to claim 1, wherein: in step 1), the tissue block is further processed in a blocking manner, and the specific operation method is as follows: 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.
3. The method for constructing an in situ PDX model of prostate cancer according to claim 1, wherein: in the step 3), when the mice are inoculated, the mice are fixed on an operation plate in a supine position, and the lower abdomen hairs of the haired mice need to be shaved off to expose the position of the prostate ventral lobe; sterilizing local skin with 0.5% iodophor, making an incision with length of about 5mm on lower abdomen with ophthalmic scissors, and lifting skin at edge of the incision with ophthalmic scissors to expose abdominal side of prostate; sucking 20-50 μ l of the mixed micro tissue block suspension or cell suspension with a microinjector 100ul, injecting into the ventral lobe of prostate gland at multiple points, suturing the incision, sterilizing the sutured part with 0.5% iodophor cotton swab, and finishing inoculation; the prostate cancer micro tissue blocks or primary cell suspensions can be inoculated directly into the skin after the operation is skilled, and the skin does not need to be cut.
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