WO2007107038A1

WO2007107038A1 - Constructing tumor model in vitro and its application

Info

Publication number: WO2007107038A1
Application number: PCT/CN2006/000432
Authority: WO
Inventors: Hua Liu; Wei Yang
Original assignee: Hua Liu; Wei Yang
Priority date: 2006-03-20
Filing date: 2006-03-20
Publication date: 2007-09-27

Abstract

A culture equipment that constructs the tumor in vitro, the application of this equipment and a method for culturing tumors in vitro with this equipment. The culture equipment comprises three-dimensional cell culture unit, in which includes the cavity for culturing the tumor cells or tumors and the cavity wall for defining said cavity, said cavity wall contains biodegradable materials and can allow the permeation of nutriment and metabolize, and tumor cells or tumors in the said cavity.

Description

Creation and application of in vitro tumor model

Technical field

The invention belongs to the field of biotechnology and medicine, and relates to the creation and application of an in vitro tumor model. Background technique

Tumors account for the second highest rate of mortality in the world and in China. In order to overcome the tumors, many developed countries have spent huge sums of money and a large amount of manpower to conduct research, but no breakthrough has been made. At least 7 million people worldwide die from cancer every year, including about 1.3 million in China. Therefore, the prevention and research of cancer is of great significance.

Tumor development is a multi-factor, multi-stage, complex and gradual process. The transformation of normal cells into malignant tumor cells requires a multi-step and multi-stage process, including the initiation phase, the promotion phase and the evolution phase. This makes cancer research more complicated.

The biological research of cancer cells includes a series of contents, such as cancer cell membrane, glycoprotein sugar chain structure, cell transmembrane signaling, apoptosis and regulation, differentiation induction and malignant transformation of cells, information transmission of malignant growth, and therapeutic molecules. Target, the role of oncogenes and tumor suppressor genes, and mechanisms of cancer metastasis.

In the past 30 years, great progress has been made in the treatment of tumors. In addition to the improvement of traditional tumor treatment methods such as tumor surgery, radiation therapy, and chemotherapy, with the development of basic disciplines such as tumor molecular biology and cell biology, new treatment methods and methods are constantly emerging, and some explorations Gradually from laboratory research to clinical research. However, to truly evaluate its clinical efficacy remains to be further studied and clinical observation.

In vitro studies of tumors are mainly directed to various tumor cell lines or strains, or primary cultured tumor cells, which have a mechanism for exploring tumorigenesis at the cellular and molecular levels, as well as for clinical diagnosis, drug screening, drug susceptibility testing, etc. Important theoretical significance and practical application value. Such as the identification of malignant transformation of normal cells in cell culture. In vitro cell experiments have the advantages of rapid, stable, quantitative and economical, and are important experimental methods for conducting research, but the results cannot be directly transferred to the clinic. Therefore, the overall experiment of animal solid tumors is an important part of the research. In order to further observe the relationship between tumor and host, it is sometimes necessary to inoculate tumor cell lines into nude mice and observe the shape of their three-dimensional tissue structure. Nude mice have higher cost, longer time-consuming, more affected factors, and are not conducive to continuous dynamic observation. This has limited its application value.

Tumor infiltration and metastasis is one of the biological characteristics of malignant tumors. The incidence of clinical metastasis is quite rampant. Some people have reported that more than 60% of patients with malignant tumors have found metastasis at the time of initial diagnosis. Tumor invasion and metastasis are the most dangerous stages in the development and progression of tumors. According to statistics, more than 80% of cancer patients die from invasion and metastasis. Therefore, tumor metastasis is a very interesting clinical treatment problem. There have been many studies on tumor invasion, invasion and metastasis, but this issue has not yet been fully understood and solved.

At present, it is difficult to observe the whole process of infiltration and metastasis of malignant tumors directly from mammals. There are not many spontaneous tumors in animals that have metastasized in the short term. Therefore, when studying the mechanism of tumor invasion and metastasis, it is often necessary to use a method of replicating animal models. The experimental tumor metastasis model requires repeated injections of more than 10,000 tumor cells in the animal, or 10 consecutive screenings to isolate subpopulations with high metastatic potential.

Therefore, there is an urgent need to develop a feature that has the same characteristics as a tumor in a mammal, and can be conveniently used. An in vitro solid tumor model for tumor research and drug screening. Summary of the invention

It is an object of the present invention to provide a method for preparing an in vitro solid tumor model, and an in vitro solid tumor model prepared by the method.

The object of the present invention is also to provide a tumor culture device constructed in vitro, by which the growth, differentiation, migration, infiltration, expansion, metastasis, or apoptosis of tumor cells or tumors can be conveniently observed, and screening is used for inhibition. Or substances that kill tumor cells or tumors, as well as screening for chemical, physical, biological, immunological, radiological, or Chinese medicine therapies that can be used to inhibit or kill tumor cells or tumors.

In a first aspect of the invention, an in vitro construction of a tumor culture device is provided, the device comprising:

(a) a three-dimensional cell culture unit comprising a cavity for culturing a tumor cell or tumor and a cavity wall defining the cavity, the cavity wall containing a biodegradable material, and the cavity The wall is permeable to: nutrients, metabolites;

(b) a tumor cell or tumor located within the cavity of the three-dimensional cell culture unit.

In another preferred embodiment of the invention, the device is located in a liquid medium.

In another preferred embodiment of the present invention, the tumor is selected from the group consisting of: nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, breast cancer, colon cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral cancer, salivary gland tumor, Nasal and paranasal sinus malignant tumors, laryngeal cancer, ear tumors, ocular tumors, thyroid tumors, mediastinal tumors, chest wall, pleural tumors, small intestine tumors, biliary tract tumors, pancreatic and periampullary tumors, mesenteric and retroperitoneal tumors, kidneys Tumor, adrenal tumor, bladder tumor, prostate cancer, testicular tumor, penile cancer, endometrial cancer, ovarian malignancy, malignant trophoblastic tumor, vulvar and vaginal cancer, malignant lymphoma, multiple myeloma, soft tissue tumor, bone Tumor, skin and accessory tumors, malignant melanoma, nervous system tumors, or pediatric tumors.

In another preferred embodiment of the invention, the tumor is a solid tumor having a diameter of 50 w m to 100 mm.

In another preferred embodiment, the solid tumor comprises: a microscopic tumor composed of a plurality of cells to a macroscopic tumor (e.g., a tumor of l-10 mm diameter), or larger.

In another preferred embodiment, the solid tumor has a diameter of l () 0 y m - 80 mm, and more preferably, the solid tumor has a diameter of 150 m - 60 mm _; further preferably, the entity The diameter of the tumor is 200 μ ni - 50 mm, such as 100 μ πι, 200 μπι, 500 μ ηι, lmm, 5 mm, 10 mm, or 20 mm.

In another preferred embodiment, a three-dimensional cell culture unit containing a larger cavity (eg, 2 mm, 5 mm, 10 mm, 15 mm, or larger in diameter) can be prepared, and the in vitro constructed tumor culture device is placed in a bioreactor. Larger tumors are cultured.

In a second aspect of the invention, there is provided the use of the device for preparing an in vitro solid tumor model.

In a third aspect of the invention, the use of the device is provided, the device being used for:

(ί) Observing the growth, differentiation, migration, invasion, expansion, metastasis, or apoptosis of tumor cells or tumors;

(ii) screening for substances that inhibit tumors;

(iii) screening for substances that promote tumors (eg, carcinogens);

(iv) Screening for chemical, physical, biological, immunological, radiological, or TCM therapies that can be used to inhibit or kill tumor cells or tumors. In a fourth aspect of the invention, a method of constructing a tumor in vitro is provided, the method comprising:

(1) placing a tumor cell or tumor in a cavity of a three-dimensional cell culture unit, the three-dimensional cell culture unit comprising a cavity for culturing the tumor cell or tumor and a cavity wall defining the cavity, The cavity wall contains a biodegradable material, and the cavity wall is permeable to: nutrients, metabolites;

(2) A three-dimensional cell culture unit containing tumor cells or tumors is placed in a medium to grow tumor cells or tumors, thereby constructing a solid tumor in vitro.

In a fifth aspect of the invention, a tumor model is provided, the tumor model being a tumor obtained by the method described above. More preferably, the tumor has a diameter of from 50 μm to 100 mm.

In a sixth aspect of the invention, there is provided the use of the tumor model, the tumor model for:

(i) observing the growth, differentiation, migration, invasion, expansion, metastasis, or apoptosis of tumor cells or tumors;

(ii) screening for substances that inhibit tumors;

(iii) screening for substances that promote tumors (eg, carcinogens);

(iv) Screening for chemical, physical, biological, immunological, radiological, or TCM therapies that can be used to inhibit or kill tumor cells or tumors.

In a seventh aspect of the invention, there is provided a method of screening for a substance which inhibits tumor growth or promotes tumor growth, comprising the following steps:

(1) in the test group, placing the in vitro constructed tumor culture device in a system containing a candidate substance; and, placing the in vitro constructed tumor culture device in a system containing no candidate substance and other conditions being the same , as a control group;

(2) Observing the growth, differentiation, migration, infiltration, expansion, or metastasis of the tumor in the tumor culture device in vitro in the test group and the control group, if the tumor grows, migrates, infiltrates, expands, or metastasizes in the test group. Slower than the control group (preferably significantly slower than the control group, for example, the growth, migration, infiltration, expansion, or metastasis rate of the tumor in the test group is 20% slower than the control group; preferably 50% slower, more preferably 80% slower, Or tumor cell apoptosis or death occurs, the candidate substance is a substance that inhibits tumor growth; if the growth, migration, infiltration, expansion, or metastasis of the tumor in the test group is faster than the control group (preferably significantly faster than the control) For example, in the test group, the tumor growth, migration, infiltration, expansion, or metastasis rate is 20% faster than the control group; preferably 50% faster, more preferably 80% faster, and the candidate substance is to promote tumor growth. Substance.

In another preferred embodiment, the system containing the candidate substance is: a solution or a medium containing the candidate substance.

In another preferred embodiment, a method for screening a carcinogen is provided, wherein a normal tissue cell is substituted for a tumor cell and placed in a cavity of the three-dimensional cell culture unit; a candidate substance is added to observe growth and differentiation of normal tissue cells. Condition, if normal tissue cells are cancerous, the candidate substance is a carcinogen.

In another preferred embodiment, the cancer suppressing substance is an anti-metastatic drug.

In another preferred embodiment, the in vitro constructed tumor culture device or tumor model has a use selected from the group consisting of:

(1) viral etiology of tumors; (2) studies of chemical and physical carcinogenic factors; (3) genetic and tumor correlation studies; (4) tumor cell biology studies; (5) oncogenes and tumor suppressor genes (6) study the relationship between cell growth, apoptosis and tumor; (7) tumor enzymology research; (8) tumor differentiation and reversal research; (9) study the relationship between cell signal transduction and tumor; Study on the mechanism of tumor formation; (11) Study on invasion, invasion and metastasis of tumor; (12) Immunology of tumors; (13) study of tumor markers; (14) study of tumor stroma; (15) pathological diagnosis of tumors; (16) imaging studies of tumors; (17) radiotherapy studies of tumors (18) Chemotherapy studies of tumors; (19) Biotherapeutic research of tumors; (20) Traditional Chinese medicine research on tumors; (21) Research on tumor hyperthermia and cryotherapy; (22) Laser treatment research of tumors; 23) Microwave treatment of tumors; (24) Electrochemical therapy of tumors, radiofrequency therapy; (25) Interventional treatment of tumors; (26) Tumor-directed and induced differentiation treatment studies; (27) Research on tumor prevention Or (28) a study of the prognosis of tumors.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

Figure 1A shows the growth of a liver cancer cell line SMMC 7721 using a general two-dimensional culture system;

1B-Fig. 1H shows the growth of tumors, tumor cells and tumors in the lumen of a three-dimensional culture unit in vitro using a liver cancer cell line SMMC 7721 and an in vitro culture device. The magnifications of Figures 1A-1H are: 20x, 20x, 10x, 10x, 20x, 20x, lOx, 10x.

Fig. 2A shows the growth state of the liver cancer cell line ATCC HB-8065 cultured in a two-dimensional culture system; Fig. 2B - Fig. 2D shows the tumor tissue, tumor in vitro, using the liver cancer cell line ATCC HB-8065 and an in vitro culture device. The growth of cells and tumors in the lumen of a three-dimensional culture unit. The magnifications of Figures 2A-2D are: 20x, 10x, 10x, 10x.

Figures 3A-3D show the growth of ATCC CRL 2254 cells in a three-dimensional culture unit in the lumen of a three-dimensional culture unit. The magnifications of Figures 3A-3D are: 2 (bc, 20x, 20x, 20x).

Figure 4A shows the growth of colon cancer cell line ATCC CCL-209 cultured in a general two-dimensional culture system; Figure 4B - Figure 4H shows the in vitro construction of tumors using colon cancer cell line ATCC CCL-209 and an in vitro culture device, The growth status of tumor cells and tumors in the lumen of a three-dimensional culture unit. The magnifications of Figures 4A-4H are: 10x, 10x, 10x, 4x, 10x, lOx, 20x, 20x.

5A and 5C show the growth state of the tumor in the lumen of the three-dimensional culture unit before the application of the chemotherapeutic drug oxazadine in the colon cancer model; FIGS. 5B and 5D show the tumor after the application of the esazadine treatment Growth condition in the lumen of the three-dimensional culture unit. The magnifications of Figures 5A-5D are: 20x, 20x, 20x, 20x.

Fig. 6A shows the growth state of ovarian cancer cell line ATCC HTB-161 cultured in a general two-dimensional culture system; Fig. 6B - Fig. 6D shows that ovarian cancer cell line ATCC HTB-161 and an in vitro culture device were used to construct a tumor in vitro. , the growth of tumor cells and tumors in the lumen of a three-dimensional culture unit. The magnifications of Figures 6A-6D are: 10x, 20x, 20x, 40x.

Figure 7A shows the growth of breast cancer cell line ATCC HTB-22 in a general two-dimensional culture system; Figure 7B - Figure 7D shows the in vitro construction of tumors using a breast cancer cell line ATCC HTB-22 and an in vitro culture device. The growth of cells and tumors in the lumen of a three-dimensional culture unit. The magnifications of Figures 2A-2D are: 10x, 10x, 20x, 10x.

Fig. 8A shows the growth state of the small cell lung cancer cell line ATCC HTB-171 cultured in a general two-dimensional culture system; Fig. 8B - Fig. 8H shows that the tumor was constructed in vitro using the small cell lung cancer cell line ATCCHTB-171 and an in vitro culture device. , the growth of tumor cells and tumors in the lumen of a three-dimensional culture unit. Figure 2A-2D magnification The times are: 10x, 20x, 20x, lOx, lOx, lOx, lOx, 10x. detailed description

Through extensive and intensive research and experimentation, the present inventors have created a method for constructing a tumor in vitro and a tumor model obtained by the method, and an in vitro solid tumor model can be prepared by simulating an in vivo environment using a culture device for constructing a tumor in vitro. The constructed tumor has cell biological characteristics similar to malignant tumors in vivo and can secrete tumor-associated antigens. The method can observe the growth, differentiation, migration, movement, infiltration, metastasis, or apoptosis of tumor cells and tumors at any time. The in vitro tumor model provides a unique environment for cancer research and can perform a variety of basic and clinical studies for specific tumors. It will provide valuable insights into the research of guiding prevention, assisting diagnosis, improving treatment and evaluating prognosis of tumors. The present invention has been completed based on this.

As used herein, "tumor" may also refer to a tumor tissue formed by a plurality of tumor cells. Tumor culture device constructed in vitro

In the present invention, there is provided an in vitro construction of a tumor culture device, the device comprising:

In a preferred form of the invention, the device is located in a culture medium. The medium may be any medium suitable for growth, differentiation, migration, and the like of a desired cultured tumor cell or tumor; preferably, the medium is a liquid medium.

In a preferred mode of the invention, the tumor is a solid tumor having a diameter of 50 y m-100 mm.

In another preferred embodiment, a three-dimensional cell culture unit containing a larger cavity (e.g., 2 mm, 5 mm, 10 mm in diameter) can be prepared.

15 mm, or larger, the in vitro constructed tumor culture device is placed in a bioreactor to culture a larger tumor.

In a preferred form of the invention, the nutritional ingredients include, but are not limited to, oxygen, protein, sugar, fat, vitamins, hormones.

In a preferred embodiment of the invention, the metabolites include, but are not limited to, carbon dioxide, cellular metabolites. In a preferred mode of the invention, the cavity wall contains 80-100% by weight of biodegradable material.

The size of the cavity is not particularly limited in the present invention, depending on the actual tumor cell or tumor culture needs. In a preferred mode of the invention, the cavity has a cross-sectional area of 0.1-100 mm ² and a length of 1-1000 mm, and the cavity wall has a thickness of 0.1-10 mm.

In a preferred mode of the present invention, the cavity wall has a thickness of 0.1 to ₆ mm _; more preferably, the cavity wall has a thickness of 0.1 to 2 mm.

In a preferred form of the invention, the cavity wall is permeable to a liquid medium.

In a preferred form of the invention, the cavity wall is substantially free of holes visible to the naked eye (eg, having a diameter greater than 2 mm) hole).

In a preferred mode of the present invention, the biodegradable material is a material which is melted at a relatively high temperature (for example, 50-100 ° C) and solidified at a normal temperature (such as 25-37 'C); or A material that is liquid at low temperatures (eg, 4 ° C) and solidifies at room temperature (eg, 25-37 Torr).

In a preferred mode of the present invention, the biodegradable material is a gel formed by a biodegradable substance, wherein the biodegradable substance is selected from the group consisting of: agar, agarose, hydrogel, collagen, Matrigel, or a combination thereof.

In a preferred form of the invention, the biodegradable material is a permeable, transparent or translucent biodegradable material.

In a preferred mode of the invention, the biodegradable material has a concentration of the biodegradable material of 0.1-10 g/100 ml of biodegradable material (SP 0.1-10%).

Preferably, the biodegradable substance is dissolved in 50-99.99% or less: water, physiological saline, PBS buffer, or a cell culture medium containing an extracellular matrix, a growth factor, a hormone, and a vitamin. More preferably, the biodegradable material contains 80-99.5% of the following: water, physiological saline, PBS buffer, or a cell culture medium containing extracellular matrix, growth factor, hormone, vitamin.

In a preferred mode of the invention, the solvent of the biodegradable substance includes, but is not limited to, water, physiological saline, PBS buffer, or culture solution.

In a preferred mode of the present invention, the biodegradable substance has a concentration of 0.1-5 g/100 ml of biodegradable material (0.1%-5%); more preferably, the biodegradable substance The concentration is from 0.5 g/100 ml to 2 g/100 ml (0.5% to 2%).

In a preferred mode of the present invention, the biodegradable substance is agar or agarose, and the concentration is 0.1-

10 g of 100 ml biodegradable material (0.1-10%); more preferably, the concentration of the agar or agarose is 0.1-5 g/100 ml biodegradable material (0.1%-5%). In a preferred embodiment of the invention, agar is used as a biodegradable material at a concentration of lg/100 ml of biodegradable material (1%, biodegradable material using PBS buffer as solvent).

In a preferred mode of the present invention, the cavity is elongated and the interface shape is selected from the group consisting of: rectangular, square, circular, elliptical, oval, pentagonal, hexagonal, or spiral;

Or the shape of the cavity corresponds to the size and shape of the tumor.

In a preferred mode of the invention, the cavity is in the shape of a cylinder (circular or approximately circular in cross section). The prototype mold has an outer diameter of 6 mm, an inner diameter of 5 mni, a wall thickness of lmm, and a length of 200 mm. The circular wire has a diameter of 0.2 mm and a length of 300 mm.

Some preferred cavities are cuboids (ie, the inner cavity is square or rectangular in cross section, or approximately square or rectangular), and the cube (ie, the inner cavity is square or rectangular in cross section, or approximate to a square or rectangular shape). Cylinder (ie, the lumen is circular or elliptical in cross section, or approximately circular or elliptical).

In a preferred mode of the present invention, the cavity has a cross-sectional area of 0.2-60 mm ² and a length of 3-600 mm _; more preferably, the cavity has a cross-sectional area of 0.5-30 mm ² . The length is 5-300mn! .

In a preferred mode of the invention, the cavity has a rectangular parallelepiped shape (rectangular or approximately rectangular in cross section).

In a preferred mode of the present invention, the ends of the cavity are closed; or, optionally, the cavity Both ends are open.

In a preferred embodiment of the invention, the three-dimensional cell culture unit contains 1-100 cavities.

In a preferred embodiment of the present invention, the three-dimensional cell culture unit contains 2 to 50 of the cavities; more preferably, the three-dimensional cell culture unit contains 2 to 10 of the cavities.

In a preferred mode of the invention, an extracellular matrix, or a nutrient component, may be added to the biodegradable material of the inner cavity wall.

In a preferred embodiment of the invention, the extracellular matrix promotes cell adhesion.

In a preferred embodiment of the present invention, the nutrient component includes, but is not limited to: a medium, a protein, a fat, a sugar, a vitamin, an extracellular matrix, a hormone, a growth, which is necessary or preferred for cell growth, differentiation, or proliferation. Factors, pharmacologically active factors and/or trace elements.

The present invention can construct various kinds of tumors. In a preferred mode of the present invention, the tumor is selected from the group consisting of: nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, breast cancer, colon cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral cancer, salivary gland tumor, Nasal and paranasal sinus malignant tumors, laryngeal cancer, ear tumors, ocular tumors, thyroid tumors, mediastinal tumors, chest wall, pleural tumors, small intestine tumors, biliary tract tumors, pancreatic and periampullary tumors, mesenteric and retroperitoneal tumors, kidneys Tumor, adrenal tumor, bladder tumor, prostate cancer, testicular tumor, penile cancer, endometrial cancer, ovarian malignancy, malignant trophoblastic tumor, vulvar and vaginal cancer, malignant lymphoma, multiple myeloma, soft tissue tumor, bone Tumor, skin and accessory tumors, malignant melanoma, nervous system tumors, pediatric tumors.

In the device of the present invention, tumor cells can form tumors at an early stage (48 to 72 hours), and can also be grown for a long period of time in the conditions of in vitro culture.

Tumor cells can be extensively expanded in the device of the present invention, and proliferating cells can be recovered without damage. Method for constructing tumor in vitro

In the present invention, there is provided a method of constructing a tumor in vitro using the in vitro constructed tumor culture apparatus of the present invention, the method comprising:

(a) placing a tumor cell or tumor in a cavity of a three-dimensional cell culture unit, the three-dimensional cell culture unit comprising a cavity for culturing the tumor cell or tumor and a cavity wall defining the cavity, The cavity wall contains a biodegradable material, and the cavity wall is permeable to: nutrients, metabolites;

(b) A three-dimensional cell culture unit containing tumor cells or tumors is placed in a liquid medium to grow tumor cells or tumors.

The use of the method of the present invention for tumor cell or tumor culture is characterized in that the cells can grow, proliferate, differentiate, and mature in a three-dimensional space, which is different from the conventional two-dimensional culture.

The three-dimensional spatial growth of tumor cells or tumors located in a three-dimensional cell culture unit is achieved by providing a space and structure similar to in vivo growth for the growth and movement of tumor cells or tumors through a three-dimensional cell culture unit, as well as Extracellular matrices and growth factors that facilitate cell adhesion and growth. In addition, co-culture of two or more tumor cells or tumors can be used to promote interaction between cells and cell substrates as well as cells and cells. The combination of one or more of the foregoing provides a desirable growth environment for tumor cells or tumors. N2006/000432 The method of the present invention can be used to culture various kinds of tumor cells or tumors, and the nutrients required for cell growth, proliferation, differentiation, and migration can be first added to the three-dimensional cells when the three-dimensional cell culture unit is fabricated. In the culture unit, the tumor cells are introduced therein and cultured directly; or alternatively, the cells and the extracellular matrix and the growth factor may be introduced together in a three-dimensional cell culture unit; or alternatively, in a three-dimensional cell culture unit The tumor cells are first introduced, and the three-dimensional cell culture unit with the tumor cells is immersed in a medium containing nutrients required for cell culture. The medium is generally a liquid medium.

In a preferred embodiment of the present invention, before the tumor cells are cultured by the apparatus of the present invention, the tumor cells are cultured according to a usual method until the cells are over 80% to 90%, and the digested tumor cells are subjected to capillary action. Introducing a cavity of a three-dimensional cell culture unit. Depending on the application, the tumor cells can also be introduced into the cavity together with various extracellular matrices, such as various types of collagen, or injected into the cavity by other conventional methods.

After injecting the tumor cells, the three-dimensional cell culture unit containing the cells in the cavity is placed in a desired culture medium, cultured in a desired conventional carbon dioxide incubator, or placed in a bioreactor.

Two or more tumor cells or tumors are cultured together or separately according to various needs and specific experimental conditions. This allows observation of the interaction between cells at the cellular and molecular levels. For example, observe the interaction and influence between cells and cells, or between cells and extracellular matrices, cell contact, activation or inhibition of cell contact. Observe hormones, growth factors, receptors, immunoglobulins, and cytokines secreted by different cells. It can also study the effects of hormones such as paracrine, autocrine, cytokines, neuropeptides, neurotransmitters and other complex intercellular signaling molecules on tumor cells in the development and progression of malignant tumors.

The combined culture of tumor cells or tumors with other cells or cells can also be used to study the response and effects between immune cells and tumor cells to aid in immunodiagnosis and to guide immunotherapy. Tumor model

The in vitro constructed tumor culture device of the present invention provides an ideal three dimensional environment for tumor cells to approximate growth and movement in vivo. Therefore, an active solid tumor or microtumor having tumor characteristics such as growth, differentiation, migration, infiltration, expansion, or metastasis can be prepared by using the in vitro constructed tumor culture apparatus of the present invention, and the solid tumor or microtumor has a patient Similar features of tumors in vivo, and thus can be used as tumor models for various studies and experiments.

In a preferred mode of the present invention, the tumor model may be present in a cavity of a three-dimensional cell culture unit for constructing a tumor culture device in vitro; or, the tumor model may be separated from the cavity and used directly. For research and testing, such as drug screening.

Compared with monolayer cell culture in a two-dimensional environment, the tissue structure of the three-dimensional tumor model obtained by the present invention more realistically reflects the cell differentiation function of the structural scaffold of the tumor. It can therefore be used to study the etiology of tumors, the formation and development of tumors, tumor invasion, metastasis, tumor angiogenesis, tissue construction, expression of tumor genes, and allow sensitivity studies for radiotherapy, chemotherapy, or immunotherapy. Treatment of individualized research.

The in vitro constructed tumor culture device of the present invention provides a space and structure for tumor cell growth similar to growth in vivo, and also provides extracellular matrix and growth factors for cell adhesion and growth, and promotes tumor cells and cell substrates, and The interaction between cells and cells. In a preferred embodiment of the invention, it is found that the malignant cells in the culture device can form a microcancer similar to carcinoma in situ. Malignant tumors formed and in vivo Similar cell biological properties of malignant tumors. It is characterized by the rapid growth of tumor cells; central necrosis occurs early. Under the microscope, the cells can be observed to form microvilli and foot processes, including leafy and filopodia. The resulting tumor has a variety of irregularities, including lobulated, globular, nodular, and cauliflower-like tumors. These malignant tumors have no envelope, and sometimes short, thick pedicles, or broad-base tumors, are observed, similar to the various shapes of tumors in the body. Method for screening tumor-inhibiting substances or carcinogens

The in vitro constructed tumor culture device of the present invention or a tumor model obtained by the device can be used to screen for tumor suppressing substances.

At the time of screening, the in vitro constructed tumor culture device of the present invention or the tumor model obtained by the device may be exposed to a candidate substance to observe growth, differentiation, migration, infiltration, expansion, or metastasis of the tumor cell or tumor, Thereby, it can be judged which candidate substance is a substance effective for suppressing the tumor, or the effectiveness of various drugs for a specific tumor can be compared. In addition, various candidate substances can also be tested in combination, or a preferred dosage of a particular drug for a particular tumor can be tested.

In a preferred embodiment of the present invention, the following method can be used to screen for a tumor-inhibiting drug or carcinogen- (a) in the test group, the in vitro constructed tumor culture device is placed in a system containing a candidate substance; And, the in vitro constructed tumor culture device is placed in a system containing no candidate substance and other conditions are the same as the control group; (b) observing the growth and migration of the tumor in the in vitro constructed tumor culture device of the test group and the control group; Infiltration, expansion, or metastasis, if the tumor grows, migrates, infiltrates, expands, or metastasizes in the test group slower than the control group (preferably significantly slower than the control group, eg, tumor growth, migration, The rate of infiltration, expansion, or metastasis is 20% slower than the control group; preferably 50% slower, more preferably 80% slower, or tumor cell apoptosis or death occurs, then the candidate substance is a tumor suppressor; Tumor growth, migration, infiltration, expansion, or metastasis in the group was faster than the control group (preferably significantly faster than the control group, eg, swollen in the test group) Growth, migration, invasion, expansion extension, or 20% faster than the transfer speed control; faster preferably 50%, more preferably 80% or more faster), then the candidate substance is a carcinogen.

The system containing the candidate substance is: a solution or a medium containing the candidate substance.

The invention also provides a method for screening a carcinogen, wherein the normal tissue cells are substituted for the tumor cells and placed in the cavity of the three-dimensional cell culture unit; the candidate substance is added, and the growth and differentiation of the normal tissue cells are observed, if normal When the tissue cells are cancerous, the candidate substance is a carcinogen.

In general, the candidate drug can be directly administered directly to the tumor model obtained by the in vitro constructed tumor culture device of the present invention, or the drug can be added to the medium and infiltrated into the in vitro constructed tumor culture device of the present invention via the biodegradable material.

Alternatively, these initially screened materials may constitute a screening library so that one can ultimately screen for drugs that are useful for inhibiting tumors or identify carcinogens.

Further, the in vitro constructed tumor culture apparatus or tumor model of the present invention can also be used as a metabolic conversion system and a tester to screen various carcinogens and physiological cell active molecules, growth regulators. Application of in vitro construction of tumor culture device or in vitro tumor model

Applications of the in vitro constructed tumor culture devices or in vitro tumor models of the present invention include, but are not limited to: 6 000432 Observing the growth, differentiation, migration, invasion, expansion, metastasis, or apoptosis of tumor cells or tumors

In a preferred mode of the present invention, the three-dimensional cell culture unit in the in vitro constructed tumor culture device is permeable, and the entire process of tumor cell growth and even the developed tumor can be observed and dynamically tracked at any time. Tumor cell growth similar to the in vivo environment can be observed in the device, forming tumors, in situ and ectopic movement of tumor cells, dissemination and implantation of tumor cells, penetrating infiltration of tumor cells, secondary The formation and shedding of sexual tumors and their dissemination in the pipeline, tumor cells parked in the distance, clonal growth. Metastasis of metastatic tumors can also be observed, and multiple tumors can be formed.

At present, the prior art has not fully understood the many details of the tumor metastasis process, so further clarification of the molecular mechanism of tumor invasion and transformation is the basis of tumor metastasis prevention research and anti-metastatic drug development. The in vitro constructed tumor culture device of the present invention can study the in situ movement and ectopic movement of tumor cells in vitro, the separation of tumor cells and the loss of cell contact inhibition, the effect of interstitial on tumor infiltration, and tumor cell products and other related components. Its role in clarifying the molecular mechanisms of tumor cell-specific motility, adhesion, invasion, metastasis, and regulation of organ-derived soluble factors.

The in vitro construction of a tumor culture device or tumor model of the present invention can also observe the difference between malignant tumors and benign tumors as well as the degree of tumor malignancy. The biological characteristics of tumor cells with different degrees of malignancy in this culture system are consistent with clinical observations. For example, the malignant properties of liver cancer cells and small cell lung cancer cells are greater than those of colon cancer.

The in vitro construction of a tumor culture device or tumor model of the present invention can also study the mechanism of drug metabolism changes and drug resistance formation of anticancer drugs. The device or tumor model of the present invention can observe the morphology of tumor cells and tumors under various environmental conditions which can be arbitrarily regulated, and can be used to observe the effects of hormones, growth factors, nutrients, metabolites and pharmacologically active factors on their growth.

The device or tumor model of the present invention can also isolate and identify highly metastatic cell sublines in a population of tumor cells, and select cells with different levels of metastatic potential. And the biological characteristics of these cells related to invasion and metastasis such as genetic code, cell surface structure, receptor type and distribution, antigenic characteristics, metabolic properties, invasiveness, adhesion to vascular endothelial cells, etc., and production of local blood coagulation factors The ability of tumor angiogenic factors, as well as the response to immune responses, etc.

It is often encountered in clinical practice that some tumors become more aggressive and have a higher degree of malignancy over time. In this case, the tumor is qualitatively changed again, which is different from the increase in the number of tumor cells and the increase in volume. The biological basis of increased tumor malignancy (including accelerated growth, invasion, and distant metastasis) is the emergence of subpopulations with different phenotypes within the tumor. Some subpopulations have faster growth rates, and some have stronger Invasive, some have higher metastatic potential, and some have weaker antigenicity to escape the body's immune mechanisms. Some subpopulations have altered susceptibility to hormones and drugs and dependence on growth factors. It can be seen that although most tumors are of monoclonal (single-center) origin, when the tumors are clinically discovered, their descendants have apparent heterogeneity. The device or tumor model of the invention allows for the observation, isolation and identification of these subpopulations of tumor cells with different phenotypes.

The device or tumor model of the invention can also be used for the isolation and identification of tumor stem cells. Another aspect of solid tumor heterogeneity is that only a few cells exhibit "clonal advantage" growth in culture or in vivo. These tumor cells may be cancer stem cells. Because most cancer cells with tumors of clonal origin produce tumors, cells must have cells with different gene phenotypes, including tumor cells with limited or no regenerative capacity, or have an infinite increase. The ability of cancer cells, suggesting that the growth process experienced by cancer cells that produce tumor disease is similar to the process of normal stem cell self-proliferation and differentiation. If the growth of solid tumors is driven by cancer stem cells, it will have a profound impact on the treatment of cancer. All current phenotypic cancer cells are considered to have unlimited proliferative capacity and can be metastasized. However, over the years, it has been found that cancer cells can be detected at locations remote from the primary tumor, but never exhibit metastatic disease. One of the possible reasons is that most cancer cells lack the ability to form new tumors, only a small number of cancer stem cells have the ability to cause metastasis, so the fundamental purpose of treatment should be to find and kill these cancer stem cells, the device of the present invention Or a tumor model can observe the "clonal advantage" growth phenomenon of tumor cells and the formation of secondary tumors in vitro. This will help the isolation and identification of cancer stem cells, and the therapeutic drugs and methods for cancer stem cells will achieve more lasting effects, even to cure metastatic tumors. Screening for drugs that prevent or treat tumors, as well as chemistry, physics, biology, immunology, radiology, and traditional Chinese medicine for the prevention or treatment of tumors

(a) Application of tumor radiotherapy

The in vitro constructed tumor culture device or tumor model of the present invention can be used to screen for radiation therapy methods useful for inhibiting tumors. Including but not limited to: Studying the effectiveness of various radiation therapies to inhibit tumors; radiosensitivity; dose rate effects, radiation resistance, radiosensitization, etc.

For example, the in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of conformal radiation therapy (protoplast irradiation), that is, radiotherapy according to the shape of the tumor, which minimizes the normal tissue surrounding the tumor, thereby improving the tumor. The dose of radiation increases the local control rate of the tumor, thereby improving the survival rate.

(b) Application of tumor chemotherapy

The in vitro constructed tumor culture device or tumor model of the present invention can be used for research: tumor drug resistance, action mechanism of anticancer drug and relationship with cell cycle, adverse reaction of anticancer drug, metabolism of anticancer drug, anticancer Distribution of drugs, metabolism of anticancer drugs, adjuvant chemotherapy for surgery or radiotherapy.

In vitro susceptibility testing of chemotherapeutic drugs is also a method to guide clinical medication. Commonly used detection methods are: MTT colorimetric assay, tumor stem cell colony formation assay, radionuclide incorporation metabolism, human cancer mouse kidney subcapsular transplantation, nude mouse tumor transplantation, flow cytometry DNA analysis Wait. However, there are still some problems in clinical use. For example, due to the heterogeneity of tumor cells, there are still differences in the response of tumor cells to chemotherapeutic drugs in vivo, and not all tumor cell responses are consistent. The biological behavior and drug sensitivity of tumor cells in vitro have changed, and there are still no effective quality control standards. The results vary widely. Therefore, after using this test method, the survival rate of tumor patients is not much improved. In addition, it is difficult to detect tumors with slow growth and division. The in vitro solid tumor model created by the present invention can be used to improve this aspect of the study.

The in vitro constructed tumor culture device or tumor model of the present invention can be used to find new targets for anticancer drug action. Most of the commonly used anticancer drugs currently target the DNA or protein of tumor cells. In order to develop new anticancer drugs with different mechanisms of action, it is necessary to find new targets for anticancer drugs and to change the target of anticancer drugs. This is also a solution to the problem of drug resistance.

The in vitro constructed tumor culture device or tumor model of the invention can be used to study therapeutic doses of tumor drugs.

The in vitro constructed tumor culture device or tumor model of the present invention can be used for in vitro susceptibility testing of chemotherapeutic drugs. The in vitro constructed tumor culture device or tumor model of the present invention can be used in multidrug resistance studies to find unique methods or drugs to reverse multidrug resistance.

(C) Tumor virus etiology application

Leukemias, sarcomas, breast cancers, skin cancers, and kidney cancers of amphibians, poultry, caries, mammals, and primates have been shown to be associated with the etiology of the virus. Moreover, some human lymphoma, nasopharyngeal cancer, cervical cancer, liver cancer, and adult T-cell leukemia have also been associated with the virus. The in vitro constructed tumor culture device or tumor model of the present invention can be used for research on the etiology of tumor virus.

(d) Application of chemical and physical carcinogenic factors

The in vitro constructed tumor culture device or tumor model of the present invention can be used to study chemical and physical carcinogenic factors. It can also be used for the detection and identification of chemical carcinogens, the study of the principles and processes of chemical carcinogenesis, the study of the role of ionizing radiation and tumors, as well as oncogenes and tumor suppressor genes in radiation carcinogenesis, and the factors affecting radiation carcinogenesis. Further, the in vitro constructed tumor culture apparatus or tumor model of the present invention contributes to the diagnosis of radiation-induced tumors, ultraviolet-induced tumors, and electromagnetic wave-induced tumors.

(e) Application of genetic and tumor related research

The in vitro constructed tumor culture device or tumor model of the present invention can be used for genetic and tumor related research, including but not limited to research: tumor formation and inheritance, cell signaling system and tumor, cell backup defense system and tumorigenesis, tumor cause Inter-genetic interaction with the environment, oncogenes and tumor suppressor genes in tumor inheritance, tumor immunogenetic markers, cytogenetic markers, biochemical genetic markers, chromosomal diseases and somatic-specific chromosomal abnormalities, or genetic polymorphisms of drug-metabolizing enzymes Sex and so on.

(f) Application of tumor cell biology

体外Using the in vitro constructed tumor culture device or tumor model of the present invention, it is convenient to observe tumor cells, such as nucleus, chromosome, nucleolus; cytoplasm, microfilament, middle filament; cell membrane, cell membrane surface glycan structure change, cell membrane surface other Changes in adhesion molecules; tumor growth characteristics, tumor cell proliferation kinetics, tumor angiogenesis on tumor growth, tumor progression and heterogeneity on tumor growth.

The in vitro constructed tumor culture device or tumor model of the present invention can be used for research: 1 tumor cell proliferation kinetics; 2 tumor blood vessel formation; 3 tumor progression to form a new, more malignant subclone.

(g) Study on oncogenes and tumor suppressor genes

In the process of proliferation, differentiation and apoptosis, cells in the body are regulated by both positive and negative regulatory signals in vivo. Positive signals cause cells to enter the proliferative cycle and inhibit their differentiation; while negative signals inhibit cell proliferation and promote their maturation. The oncogene regulates a positive signal, while the tumor suppressor gene regulates a negative signal. Tumors originate from the abnormal regulation of cell proliferation and differentiation, allowing cells to proliferate continuously and fail to differentiate and apoptosis in time. The in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of oncogenes and tumor suppressor genes.

(h) Study the relationship between cell growth, apoptosis and tumor

Apoptosis is closely related to tumors, that is, tumors are not only diseases with abnormal proliferation and differentiation, but also diseases with abnormal apoptosis. If apoptosis is inhibited, cell survival is prolonged, and mortality is reduced; cells that are normally apoptotic continue to survive, and the number of cells increases, showing the advantage of growth, which may be converted into tumor cells. The in vitro constructed tumor culture device or tumor model of the present invention can be used in this aspect of the study. Such as analysis and tumor cell apoptosis Changes in the most important genes such as p53 can be used to track the occurrence and development of tumors, and even the apoptotic index can be determined as an indicator of tumor grade.

Apoptosis is inhibited, breaking the balance of cell proliferation and apoptosis in normal tissues, resulting in a decrease in cell death. If the body cannot restore the regulation of proliferation and apoptosis, it will lead to an increase in the number of cells, which shows a growth advantage, which is an important basis for tumor formation. The in vitro constructed tumor culture device or tumor model of the present invention can be used for this. Research on the one hand.

In addition, the in vitro constructed tumor culture device or tumor model of the present invention can be used for: research on apoptosis and cell cycle, research on apoptosis and tumor growth, study on relationship between apoptosis and tumor-induced differentiation, apoptosis and Tumor treatment, prognostic relationship studies, or apoptosis and tumor infiltration and metastasis studies.

(i) Application of tumor enzymology

Malignant behaviors of the tumor, such as regulation of uncontrolled growth, morphological and functional dedifferentiation, and even invasion, invasion and metastasis, are closely related to the specific metabolism of the tumor. Almost all metabolic pathway enzymes have more or less viability changes in malignant tumors, and enzyme system aberrations are the result of gene regulation and expression changes. Therefore, tumor enzymology research is very important for understanding the development of tumors. It is also very valuable to find the enzymatic markers of tumors as clinical diagnostic indicators or targets for drug treatment.

The in vitro constructed tumor culture device or tumor model of the present invention can be used for tumor enzymology research, including but not limited to - general characteristics of malignant tumor metabolism and enzyme activity change; anabolism and catabolism; proliferation-related enzymes and differentiation-related enzymes; Related enzymes and evolution-related enzymes; important enzymatic changes in malignant tumors; enzymatic changes in glucose metabolism; enzymatic changes in lipid metabolism; enzymatic changes in amino acid metabolism; enzymatic changes in nucleotide metabolism Important enzymology in nucleic acid metabolism; changes in biotransformation and detoxification enzymes; changes in protein kinases and protein phosphatases; enzymatic changes in glycolipid metabolism; glycoprotein sugar chain processing enzyme changes; isozymes in malignant tumors The type of change.

Differentiation and reversal of ω tumors

The in vitro constructed tumor culture device or tumor model of the present invention can be used for tumor differentiation and reversal studies. Such as the endogenous and exogenous differentiation inducer of malignant tumor; the structure and function of differentiation inducer; the specificity of differentiation inducer; tissue and cell specificity; the specificity of induced differentiation; the differentiation of solid tumor Induction; differentiation of human hepatocellular carcinoma; signal transduction of differentiation inducer; CAMP signaling pathway; phospholipid signaling pathway; tyrosine residue dephosphorylation signal; differentiation agent-nuclear receptor complex signal.

(k) Cell Signal Transduction and Tumor

The in vitro constructed tumor culture device or tumor model of the present invention can be used for cell signal transduction and tumor research. Such as cell signal transduction disorders and tumorigenesis, direct signal transduction; indirect signal transduction; transmembrane signal transduction; cell membrane receptors; signal amplification and cellular effects; signal regulation and suspension; oncogenes and growth factors; Genes and growth factor receptors; oncogenes and intracellular signal transduction molecules; oncogenes and nuclear transcription factors; cell adhesion molecules and tumor metastasis; several types of cell adhesion molecules involved in tumor metastasis; tumor metastasis processes and cell adhesion Attached to the role of molecules.

(1) Study on the mechanism of tumor formation

The in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of the mechanism of tumor occurrence. Such as biological problems related to tumorigenesis, hyperplasia; differentiation; anaplastic; dysplasia and transformation; malignant transformation. Tumor tissue, the source of tumor cells; single or multicenter occurrence of tumors; tumorigenesis process. Gene regulation and tumorigenesis, Changes in DNA during cell carcinogenesis; regulation of histones; regulation of non-histones; transcription factors; repressor proteins that regulate transcription; relationship between chromatin structure and gene activity; post-transcriptional regulation;

(m) Tumor invasion, invasion and metastasis

Tumor invasion refers to the attack of malignant tumor cells from the primary tumor to surrounding tissues, which is marked by the tumor cells breaking through the basement membrane. Tumor metastasis refers to the process in which malignant tumor cells are detached from their primary sites, transported through various channels, and continue to proliferate and grow in discrete tissues to form tumors of the same nature. Tumor invasion and metastasis is an extremely complex process involving multiple physiological and biochemical changes.

The in vitro constructed tumor culture device or tumor model of the present invention can be used to study the metastasis of different tumors, including the frequency of transfer, morning and evening, pathways, sites, etc., as well as mechanisms for studying the organ metastasis tendency of tumors.

(n) Tumor and immunity

The in vitro constructed tumor culture device or tumor model of the present invention can be used for tumor and immune research, including but not limited to: tumorigenesis and immune status, immunodeficiency and tumorigenesis, localization of tumor antigens, and mechanism of tumor cell antigen production.

In addition, the in vitro constructed tumor culture device or tumor model of the present invention can be used for the following tumor immunological studies: 1 antigenic characteristics of transformed cells; 2 host immune response to tumor cells; 3 host immune effect during malignant cell growth; Regulates the immune system's ability to recognize tumor cells and promote tumor regression. Tumor cells differ from normal cells in that their common characteristics include heterogeneity, self-discipline, growth-free, invasive, invasive, and metastatic, which are somewhat related to immune regulation.

To date, purified tumor-specific antigens that are lacking in normal tissues have not been extracted from human tumors. The in vitro solid tumor model created by the present invention can be used for the study of tumor specific antigens.

(o) Study of tumor markers

The in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of tumor markers. Such as protein tumor markers: alpha-fetoprotein, carcinoembryonic antigen, tissue polypeptide antigen, prostate specific antigen, cytokeratin 19 fragment, squamous cell-associated antigen, acid ferritin, alpha-antitrypsin; carbohydrate tumor marker: CA -125, CA19-9, CA50, CA242; Enzyme tumor markers: acid phosphatase, alkaline phosphatase, Y-glutamyl transferase, placental-type glutathione S-transferase, α-L-rock Alginase, proteolytic enzyme, polyamine; or hormonal tumor marker.

(P) Imaging diagnosis of tumors

The in vitro constructed tumor culture device or tumor model of the present invention can be used for imaging diagnosis of tumors of tumors. Tumor imaging plays an important role in the early detection of tumors, staging of tumors, estimation of preoperative surgical resection, development of treatment plans, and post-treatment follow-up.

(q) Pathological diagnosis of tumor

The in vitro constructed tumor culture device or tumor model of the present invention can be used for pathological diagnosis of tumors. To determine whether it is a tumor, a benign or malignant degree of tumor, it is currently mainly dependent on pathological diagnosis. Pathological diagnosis is recognized as the final diagnosis and is the gold standard. Although "metastatic" is an indicator for judging the benign and malignant tumors, it is necessary to make a prospective diagnosis and treatment before the tumor metastasizes in clinical and pathological practice. Tumor growth characteristics, gross morphology, tissue structure, cell morphology, mitotic figures, ultrastructure and many other factors are the basis for judging good and malignant. In addition to the qualitative changes in "transfer", the above factors are only changes in quantity. In vitro created by the present invention Solid tumor models can be used for the qualitative aspects of tumor "metastatic". ,

(r) TCM treatment of tumors

The in vitro constructed tumor culture device or tumor model of the present invention can be used to find a traditional Chinese medicine having an anticancer effect and an effective ingredient thereof, and to prepare an anticancer preparation.

(s) Tumor hyperthermia and cryotherapy studies

At present, both in vitro and clinical practice have demonstrated that heating can enhance the therapeutic effects of radiotherapy and chemotherapy. The in vitro constructed tumor culture device or tumor model of the present invention can be used for research in tumor hyperthermia.

The combination of cryoablation, radiotherapy, chemotherapy, etc. has attracted the attention of people in the field, and some have achieved encouraging results. The in vitro constructed tumor culture device or tumor model of the present invention can be used in the study of tumor hyperthermia.

(t) Laser treatment of tumors

The in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of laser treatment of tumors. Lasers can cut bones, stop bleeding, and vaporize tumor tissue because of their effects on heat, pressure, light, and electromagnetic fields. The experiment found that after the laser partially destroyed the tumor, the residual cancer could resolve by itself. After the melanoma is completely destroyed by the laser, the tumor cells can not be regrown, suggesting that the role of the laser is also related to immunity.

(u) Microwave treatment of tumors

The in vitro constructed tumor culture device or tumor model of the present invention can be used for microwave treatment of tumors. Microwave treatment of tumors mainly uses its thermal effects. The water content in tumor tissue can be as high as 89%, while the water content of normal soft tissue is generally below 65%. Since water is a kind of coupling molecule, it has a large dielectric constant and can strongly absorb microwave energy and convert it into heat energy. Therefore, microwave radiation can selectively destroy tumors.

(V) Electrochemical therapy, radiofrequency therapy research

The in vitro constructed tumor culture device or tumor model of the present invention can be used for the study of electrochemical therapy, radiofrequency therapy. Electrochemical therapy (ECT), also known as direct current therapy, is based on the ionization of direct current, which changes the living environment of tumor tissue and causes disorder in the metabolism of tumor cells.

(w) Interventional treatment of tumor

The in vitro constructed tumor culture device or tumor model of the present invention can be used for interventional treatment of tumors. Interventional treatment of tumors is mainly based on the supply of blood vessels in most malignant tumors. The arterial selective intubation chemotherapy (TAI) and embolization (TAE) tumors support the blood vessels, which can greatly increase the concentration of drugs in the tumor and cut off the tumor. The source of nutrition promotes ischemic necrosis of the tumor.

(X) Tumor biotherapy

The in vitro constructed tumor culture device or tumor model of the present invention can be used in biotherapeutic studies of tumors. Tumor biotherapy includes two aspects: tumor immunotherapy and gene therapy. The former is the basis of tumor biotherapy, and the latter is the direction of tumor biotherapy. The biological treatment of tumor refers to a treatment method for inhibiting or eliminating tumor growth by regulating the biological reaction of the body itself by the action mechanism of the tumor host or the action of the biological preparation, and is characterized in that not only a large amount of biological preparation is obtained by genetic recombination, but also Biological effects include the entire regulatory system of immunity, nerves and endocrine.

At present, the scope of tumor biotherapy mainly includes cytokines, adoptive cellular immunotherapy, monoclonal antibodies, tumor vaccines and gene therapy. Due to the variety and number of cancer patients, the lack of effective treatment methods, and poor prognosis, the clinical urgency of new therapeutic methods such as gene therapy is strong, patients and their families are easy to accept, and ethical issues are less, so tumor gene therapy The research is the most popular, and the most attention, the current gene therapy clinical projects are mostly tumor gene therapy research. Although the current research on gene therapy is developing rapidly, it has to be widely used in clinical practice, especially in the field of tumors. There are still many problems worthy of further study. Need to find and select more effective target genes; construct specific and highly targeted expression vectors; select recipient cells for more ideal gene therapy and gene transfection methods; also consider the fusion of multiple gene combination therapy The effect of protein on the body. Although the gene therapy research of tumors has achieved some gratifying results, there are still few breakthroughs. The in vitro solid tumor model created by the present invention can contribute to this aspect of the study.

(y) Tumor targeting and differentiation therapy

The in vitro constructed tumor culture device or tumor model of the present invention can be used for tumor targeting and differentiation induction treatment research. The basic principle of tumor-directed therapy is to use the anti-tumor antibody specificity for tumor antigens. With highly specific pro-tumor substances as carriers, cytotoxic substances such as radionuclides, chemotherapeutics, toxins, etc. can be carried as warheads. A variety of cytotoxic substances that specifically kill tumors, but are less toxic to the host.

Many tumor cells are similar in morphology and metabolism to undifferentiated or poorly differentiated embryonic cells, and their malignant behavior is often negatively correlated with their degree of differentiation. That is, poorly differentiated people have a high degree of malignancy, while highly differentiated people have a low degree of malignancy. . Therefore, it is conceivable to change the degree of malignancy by inducing poorly differentiated tumor cells to become more mature cells, and finally to alleviate or even cure the tumor. At present, research and observation of differentiation therapy have involved a variety of human tumors, such as colon cancer, gastric cancer, bladder cancer, liver cancer and the like. There are a variety of differentiation inducers for different tumor cells, and they have relative specificity. However, most of the current researches are experimental studies, and there are not many people who actually enter clinical research. The in vitro constructed tumor culture device or tumor model of the present invention can be used in this aspect of the study.

(z) Cancer prevention and prognosis

The in vitro constructed tumor culture device or tumor model of the present invention can be used for research in tumor prevention. Including but not limited to: detection of suspected carcinogens; search for carcinogens; early detection; early diagnosis; early treatment.

The in vitro constructed tumor culture device or tumor model of the present invention can be used for research on tumor prognosis, and how to detect high-risk recurrence and metastasis cases. Obtaining tumor cells or tumors from a three-dimensional cell culture unit

Since the three-dimensional cell culture unit uses a biodegradable material in the tumor in vitro culture device of the present invention, the material not only facilitates material exchange inside and outside the cavity wall of the three-dimensional cell culture unit, but also facilitates acquisition of a tumor. Cells or tumors.

Those skilled in the art will appreciate that tumor cells or tumors can be obtained from the three-dimensional cell culture unit using a variety of chemical and physical methods. For example, the cavity wall of the three-dimensional cell culture unit can be cut by a cutting tool to obtain the tumor cell or tumor.

Further, the tumor cells or tumors may be obtained by biochemical degradation, for example, collagen may be digested with collagenase to obtain the culture. The main advantages of the present invention are:

(1) For the first time, tumors can be constructed under in vitro conditions, and growth, differentiation, migration, invasion, expansion, metastasis, or apoptosis of tumor cells or tumors can be observed in the three-dimensional cell culture unit. Biological characteristics, and can secrete tumor-associated antigens.

(2) Using the in vitro constructed tumor culture apparatus or tumor model of the present invention, various tumor-specific studies such as screening of cancer suppressing drugs, screening of carcinogenic substances, and effectiveness studies on various existing tumor therapies can be performed.

(3) The in vitro tumor model provides a unique environment between the in vitro two-dimensional tumor cell culture system and the in vivo animal experiment tumor model for cancer research, and can be widely used in various basic and clinical studies of tumors. .

(4) Tumor cells In this system, tumors can be formed at an early stage (48 to 72 hours), and can also be grown for a long period of time in vitro under conditions of in vitro culture. In addition to the above-described other features of the invention, such as transparency and dynamic observation of tumor cells at any time. These advantages make the three-dimensional cell culture system of the present invention very suitable for the observation of the multi-stage, multi-step complex process of tumor development. Therefore, this in vitro solid tumor model similar to the cell growth, proliferation and development process of the in vivo environment will It is invaluable in the research of guiding prevention, assisting diagnosis, improving treatment and evaluating prognosis of tumors. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1 Liver cancer in vitro culture device and preparation of liver cancer model (hepatoma cell line SMMC 7721)

In this example, a liver cancer cell model SMMC 7721 and an in vitro culture device were used to construct a tumor in vitro to prepare a solid liver cancer model.

In the culture, the components of the medium used were as follows: RPMI 1640 (Sigma) 1000 ml, 100-fold penicillin + streptomycin (GIBCO 15140-122) 10 ml, fetal bovine serum 100 ml. The culture conditions were 5% carbon dioxide and the temperature was 37 Torr.

The three-dimensional culture unit used is made of 1% agarose, and the cavity has a circular or nearly circular cross section. The circular cavity has an inner diameter of 2-3 mm and a cavity wall thickness of l mm.

The liver cancer cell line SMMC 7721 was cultured under the above conditions, and the growth state of the tumor was shown in Fig. 1B - Fig. 1H. Tumor cells In the three-dimensional cell culture unit, the growth rate is fast, and central necrosis occurs early. After about 48-72 hours, it can be observed that the hepatoma cell line SMMC 7721 cells accumulate to form a plurality of tumors, and then the tumor tissue cells protrude from the external environment, and the metastatic cells exhibit various forms including micro-tumors. Tumor cell movement and/or detachment to the periphery of the tumor can also be observed. At the same time, it can be seen that a large number of cells are separated from the maternal tumor and transferred to the periphery and the distant place, and the primary tumor is centered on the radial type to expand and expand the tumor. In about a week, tumor cells proliferated in a large amount, forming multiple tumors, including secondary tumors. Sometimes short and thick pedicles can be observed.

After the first week of culture, a tumor with a diameter of about 1.5 is formed, which can be used as a liver cancer model for screening. 2 drugs or a variety of basic and clinical research and trials.

Further, the inventors also cultured a liver cancer cell line SMMC 7721 as a control in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like. The growth of the cells in a two-dimensional culture system is shown in Fig. 1A. It can be observed that the liver cancer cell line SMMC 7721 is in a planar growth state in a two-dimensional space and cannot form a tumor. Example 2 In vitro culture apparatus for liver cancer and preparation of liver cancer model (hepatoma cell line ATCC HB-8065) In this example, a liver cancer cell line ATCC HB-8065 and an in vitro culture device were used to construct a tumor in vitro, thereby preparing another solid liver cancer. model.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the medium components were as follows: Minimum essential medium (Eagle) with Earle's BSS (Gibco 11700-077), 100 times glutamine 10 ml (25030-081), 0.1 mM non-essential amino acids (11140-050), 1.0 mM sodium pyruvate (11360-070), 100 times penicillin + streptomycin (GIBCO 15140-122) 10 ml, fetal bovine serum 100 ml. The cells were introduced into a cavity of a three-dimensional culture unit and placed in the medium under the conditions of 5% carbon dioxide and a temperature of 37 °C.

The liver cancer cell line ATCC HB-8065 was cultured under the above conditions, and the growth state of the tumor was shown in Fig. IB - Fig. 1D. The liver cancer cell line ATCC HB-8065 is in a three-dimensional cell culture unit, and the tumor tissue cells protrude from the outside of the cell pseudopod, mainly showing the formation of a substantial tumor. However, the movement of the cells was significantly less than that of the liver cancer cell line SMMC 7721. The tumor metastasis was dominated by cell shedding. Early central necrosis of the cells can be observed, and secondary tumors are detached and metastasized from the primary tumor. And the formation of a single secondary tumor can be seen.

By the first week of culture, tumors with a diameter of about 2.0 mm can be formed, which can be used as a model of liver cancer, 'for screening drugs or for various basic and clinical studies and trials.

Further, the present inventors also cultured a liver cancer cell line ATCC HB-8065 in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like as a control. The growth of the cells in a two-dimensional culture system is shown in Fig. 2A, and it was observed that the liver cancer cell line ATCC HB-8065 could not form a tumor in a two-dimensional space. Example 3 In vitro culture of hepatocytes ATCC CRL 2254

In order to study the difference between tumor cells and normal tissue cells, the present inventors cultured normal hepatocytes and observed their growth characteristics for comparison.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the composition of the medium is as follows: DMEM/F12

(GIBC012400-016) 1000ml, 100 times penicillin + streptomycin (GIBCO 15140- 122) 10ml, 100 times 10 ITS (Sigma I 1884) 10ml, dexamethasone (Sigma D 8893) lml, fetal bovine serum 100ml. The cells were introduced into a cavity of a three-dimensional culture unit and placed in the medium under the conditions of 5% carbon dioxide, temperature 37. C.

Hepatocyte ATCC CRL 2254 was cultured under the above conditions, and the growth state of the cells is shown in Figs. 3A to 3D. Hepatocyte ATCC CRL 2254 grows in a three-dimensional cell culture unit in a completely different manner than malignant cells. Hepatocyte ATCC CRL 2254 grows relatively slowly, without early central necrosis, and the formation of microscopic organ morphology Comparative rules, there is obvious envelope, no invasion and transfer phenomenon.

Moreover, normal tissue cell culture can grow, differentiate, mature, or even form new microscopic organs in a three-dimensional cell culture unit, including tissue-specific structures. For example, hepatocyte ATCC CRL 2254 can be cultured in a three-dimensional cell culture unit to form a micro-shaped liver organ, and envelope formation can be observed, as well as a clearly visible duct-like structure and a cystic structure. Example 4 Colon cancer cell line ATCC CCL-209 in vitro culture apparatus and preparation of colon cancer model In this example, a colon cancer cell line ATCC CCL-209 and the in vitro culture apparatus of the present invention were used to construct a tumor in vitro to prepare a solid colon. Cancer model.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the composition of the medium is as follows: 2 mM

L-Glutamine Ham's F12K medium (Gibco 21700-026) 1000 ml, 100-fold penicillin + streptomycin (GIBCO 15140-122) 10 ml, 100X glutamine 10 ml, fetal bovine serum 100 ml. The cells were introduced into a cavity of a three-dimensional culture unit and placed in the medium under the conditions of 5% carbon dioxide at a temperature of 37 °C.

The colon cancer cell line ATCC CCL-209 was cultured under the above conditions, and the growth of the tumor was shown in Fig. 4B-4H. Tumor cell growth and formation of tumors, in situ and ectopic movement of tumor cells, dissemination and implantation of tumor cells, penetrating infiltration of tumor cells, formation of malignant tumors and in vivo can be observed in a three-dimensional cell culture unit. Similar cell biological properties of malignant tumors. It is characterized by the rapid growth of tumor cells and the early stage of central necrosis. The formation of cell foot processes, including leaf and vesicular pseudopods, can be observed under a microscope. The resulting tumor has a variety of irregularities, including lobulated, globular, nodular, and cauliflower-like tumors. These malignant tumors have no envelope and are broad-based tumors.

By the second week of culture, a tumor with a diameter of about 2.0 mm can be formed, which can be used as a colon cancer model for screening drugs or for various basic and clinical studies and trials.

Further, the inventors also cultured colon cancer cell line ATCC CCL-209 in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like as a control. The growth of the cells in a two-dimensional culture system is shown in Fig. 4A, and it can be observed that the colon cancer cell line ATCC CCL-209 is in a planar growth state in a two-dimensional space, and no tumor can be formed. Example 5: Drug Screening with Colon Cancer Model

In the present example, the colon cancer model obtained in Example 4 was used for drug screening test.

The colon cancer model and the chemotherapeutic drug eroxadine were directly placed in the culture medium of the colon cancer model at a concentration of 5 (Fig. 5A-5D) showing that the colon cancer model was treated with the chemotherapeutic drug oxazadine ( Comparison of Figures 5A, 5C) and after processing (Figure 5B, Figure 5D). The results showed that tumor cells showed significant apoptosis and death after the use of chemotherapeutic drugs.

At the same time, measuring the concentration of carcinoembryonic antigen in the cell culture solution showed that the concentration of carcinoembryonic antigen in the cell culture solution was significantly lowered after the use of the chemotherapeutic drug. In the colon cancer model without esazadine, the carcinoembryonic antigen was 65.43 ng/mL, and in the colon cancer model with esazadine, the carcinoembryonic antigen concentration was reduced to 10.34 ng/mL. Example 6 Ovarian cancer cell line ATCC HTB-161 in vitro culture and preparation of ovarian cancer model In this example, ovarian cancer cell line ATCC HTB-161 and the in vitro culture device of the present invention were used to construct a tumor in vitro to prepare an entity. Ovarian cancer model.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the medium components were as follows: RPMI 1640 medium (Gibco 23400-013) 800 ml, 1.0 mM sodium pyruvate, 0.01 mg/ml bovine insulin, 100 times penicillin + streptomycin (GIBCO 15140) -122) 10 ml, fetal bovine serum 200 ml. The cells were introduced into a cavity of a three-dimensional culture unit and placed in the medium under the conditions of 5% carbon dioxide and a temperature of 37 Torr.

The ovarian cancer cell line ATCC HTB-161 was cultured under the above conditions, and the growth state of the tumor is shown in Fig. 6B-6D. It can be observed in the three-dimensional cell culture unit that ovarian cancer cells can accumulate and grow to form tumors, and the formed tumors are vesicular and have a cell structure similar to that of papillae. The malignant tumor formed has cell biological characteristics similar to those of malignant tumors in the body, which is characterized by rapid growth of tumor cells and early necrosis. Under the microscope, it is observed that the cells have the formation of foot processes, including vesicular and filopodia. The metastatic cells present in a variety of different forms including minimasses. Tumor cell movement and/or shedding can also be observed around the tumor. At the same time, it can be seen that the cells are separated from the maternal tumor and transferred to the periphery and the distant place, and the formed tumor has various irregular shapes. These malignant tumors have no envelope.

The tumor can be used as an ovarian cancer model for screening drugs or for various basic and clinical studies and trials.

Further, the present inventors also cultured an ovarian cancer cell line ATCC HTB-161 in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like as a control. The growth of the cells in a two-dimensional culture system is shown in Fig. 6A, and it was observed that the ovarian cancer cell line ATCC HTB-161 could not form a tumor in a two-dimensional space. Example 7 Breast cancer cell line ATCC HTB-22 in vitro culture apparatus and preparation of breast cancer model In this example, a breast cancer strain ATCC HTB-22 and an in vitro culture device of the present invention were used to construct a tumor in vitro, thereby preparing a solid breast. Cancer model.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the medium components were as follows: Minimum essential medium (Eagle) containing 2 mM glutamine and Earle's BSS (Gibco 11700-077), sodium bicarbonate 1 · 5 g / L, non-essential amino acids 0.1 mM, pyruvic acid Sodium l mM, bovine insulin 0.01 mg/ml, 100 times penicillin + streptomycin (GIBCO 15140-122) 10 ml, fetal bovine serum 100 ml. The cells were introduced into a cavity of a three-dimensional culture unit and placed in the medium under the conditions of 5% carbon dioxide at a temperature of 37 °C.

The breast cancer cell line ATCC HTB-22 was cultured under the above conditions, and the growth state of the tumor is shown in Fig. 7B-7D. Breast cancer cell lines can accumulate in three-dimensional cell culture units, and tumor formation can be observed in about one week. The tumor was observed under the microscope. The malignant tumor formed has cell biological properties similar to malignant tumors in the body. It shows that the growth rate of tumor cells is fast. The resulting tumor has a variety of irregularities. The metastatic cells present in a variety of different forms including minimasses. Tumor cell movement and/or shedding can also be observed around the tumor. At the same time, the cells can be separated from the maternal tumor and transferred to the periphery and the distant. In about one week, the tumor cells proliferate in a large amount, forming multiple tumors, including secondary tumors.

The tumor can be used as a breast cancer model for screening drugs or for various basic and clinical studies and trials. T N2006/000432 In addition, the present inventors also cultured a breast cancer cell line ATCC HTB-22 in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like as a control. The growth of the cells in a two-dimensional culture system is shown in Fig. 7A, and it can be observed that the breast cancer cell line ATCC HTB-22 is in a planar growth state in a two-dimensional space, and no tumor can be formed. Example 8 Small cell lung cancer cell line ATCC HTB-171 in vitro culture apparatus and preparation of lung cancer model In this example, a small cell lung cancer cell line ATCC HTB-171 and the in vitro culture device of the present invention were used to construct a tumor in vitro, thereby preparing a small entity. Cell lung cancer model.

The three-dimensional culture unit used in this embodiment is the same as that in the first embodiment. When cultured, the medium components were as follows: RPMI 1640 (Sigma) 1000 ml, 100-fold penicillin + streptomycin (GIBCO 15140-122) 10 ml, fetal bovine serum 100 ml. The culture conditions were 5% carbon dioxide and the temperature was 37 Torr.

The small cell lung cancer cell line ATCC HTB-171 was cultured under the above conditions, and the growth of the tumor was shown in Fig. 8B-8H. Small cell lung cancer cell line ATCC HTB-171 grows in a three-dimensional cell culture unit, showing a rapid growth rate. After about 48-72 hours, small cell lung cancer cells can be observed to accumulate and form multiple tumors, and then the tumor tissue cells are oriented. The external environment protrudes from the cell pseudopod, and the metastatic cells present in various forms including microscopic tumors. Tumor cell movement and/or shedding can also be observed around the tumor. At the same time, a large number of cells are separated from the maternal tumor and transferred to the periphery and distant places.

After the first week of culture, a tumor with a diameter of about 0. 5-1. 0 can be formed, which can be used as a lung cancer model for screening drugs or for various basic and clinical studies and experiments.

Further, the inventors also cultured a small cell lung cancer cell line HTB-171 in a general two-dimensional culture system (ordinary cell culture dish) under the same medium, culture temperature and the like as a control. The growth of the cells in a two-dimensional culture system is shown in Fig. 8A, and it can be observed that the small cell lung cancer cell line HTB-171 is in a planar growth state in a two-dimensional space, and no tumor can be formed. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

Rights request

A culture apparatus for constructing a tumor in vitro, characterized in that said apparatus comprises -

2. Apparatus according to claim 1 wherein said apparatus is located in a liquid medium.

3. The device according to claim 1, wherein the tumor is selected from the group consisting of: nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, breast cancer, colon cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral cavity. Cancer, salivary gland tumor, nasal cavity and paranasal sinus malignancy, laryngeal cancer, ear tumor, ocular tumor, thyroid tumor, mediastinal tumor, chest wall, pleural tumor, small intestine tumor, biliary tract tumor, pancreatic and periampullary tumor, mesenteric Retroperitoneal tumor, kidney tumor, adrenal tumor, bladder tumor, prostate cancer, testicular tumor, penile cancer, endometrial cancer, ovarian malignant tumor, malignant trophoblastic tumor, vulvar cancer and vaginal cancer, malignant lymphoma, multiple myeloma , soft tissue tumors, bone tumors, skin and accessory tumors, malignant melanoma, nervous system tumors, or pediatric tumors.

4. The device according to claim 1, wherein the tumor is a solid tumor having a diameter of 50 μπι to 100 mm.

5. Use of the device of claim 1 , wherein the device is for: preparing an in vitro solid tumor model.

6. Use of the device of claim 1 wherein said device is for use -

(ii) screening for substances that inhibit tumors;

(iii) screening for substances that promote tumors;

7. A method of constructing a tumor in vitro, the method comprising:

A tumor model, characterized in that the tumor model is a tumor obtained by the method according to claim 7, having a diameter of 50 y m - 100 mm.

9. Use of a tumor model according to claim 8 wherein said tumor model is used to:

(i) observing the growth, differentiation, migration, invasion, expansion, metastasis, or apoptosis of tumor cells or tumors; (ii) screening for substances that inhibit tumors;

(iii) screening for substances that promote tumors;

(IV) Screening for chemical, physical, biological, immunological, radiological, or TCM therapy that can be used to inhibit or kill tumor cells or tumors.

10. A method of screening for a substance that inhibits or promotes tumor growth, comprising the steps of:

(1) In the test group, the in vitro constructed tumor culture apparatus according to claim 1 is placed in a system containing a candidate substance; and the in vitro constructed tumor culture apparatus according to claim 1 is placed without a candidate substance In other systems with the same conditions, as a control group;

(2) Observing the growth, differentiation, migration, infiltration, expansion, or metastasis of the tumor in the tumor culture device in vitro in the test group and the control group, if the tumor grows, migrates, infiltrates, expands, or metastasizes in the test group. Slower than the control group, the candidate substance is a substance that inhibits tumor growth; if the growth, migration, infiltration, expansion, or metastasis of the tumor in the test group is faster than the control group, the candidate substance is a substance that promotes tumor growth. .