A complex for treating tumors

Abstract

The application belongs to the technical field of biological medicine, and claims a kind of drug complex for preparing tumor treatment, which comprises BHK-21 fibroblast with final concentration of 300,000-625,000 / mL; Dulbecco's culture solution of 30-60% artificial basement membrane and 2-10% fetal bovine serum.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a compound for treating tumors. BACKGROUND

[0002] According to the latest Chinese cancer epidemiology data, kidney cancer and pelvic cancer are the second most common malignant tumors of the urogenital system in China, with about 66,800 new cases and 23,400 deaths each year. In particular, renal cell carcinoma (RCC) accounts for a large proportion of new cases of adult kidney cancer (Chen, Zheng et al. 2016). Unlike some tumors with lower malignancy, renal cell carcinoma is highly metastatic and refractory, and neither chemotherapy nor radiotherapy is effective for it (Merza and Bilusic 2017, Shingarev and Jaimes 2017).

[0003] At present, the most widely used methods in the treatment of cancer (including renal cell carcinoma) are chemotherapy, radiotherapy, surgery, hormone therapy and "targeted" therapy, and cancer immunotherapy and monoclonal antibody therapy are the two most common types of targeted therapy.

[0004] The existing anti-cancer technologies are analyzed as follows:

[0005] 1. Chemotherapy: Chemotherapy is usually accompanied by varying degrees of side effects, including nausea and vomiting, hair loss, decreased immunity and loss of appetite, etc. At the same time, chemotherapy can also damage normal cells in the human body (Munker, Gerken et al. 2018; Oun, Moussa et al. 2018; Beaver and Magnan 2016).

[0006] 2. Radiotherapy: Although its side effects are not as severe as chemotherapy, patients receiving radiotherapy often experience skin reactions such as scaling, erythema and fibrosis. In addition, this method requires expensive equipment and a professional environment (such as a hospital), and there is also a risk of inducing new cancers (Gieger and Nolan 2017).

[0007] 3. Immunotherapy: Although this method has achieved exciting results in the treatment of cancer, it is well known that immunotherapy relies on the immune system of the human body, so a series of immune response symptoms such as fever, nausea and vomiting, and general pain can occur. In severe cases, there can also be cardiopulmonary toxicity, and the therapeutic effect of immunotherapy decreases significantly with age (Hurez, Padron et al. 2018).

[0008] 4. Hormone Therapy: The most common typical side effect of hormone therapy is "Cushing's syndrome", which is also accompanied by steroid ulcer, behavioral or mental abnormalities, and muscle and bone lesions (Lin et al. 2017).

[0009] From the above, the existing methods for treating cancer have many defects and deficiencies. SUMMARY

[0010] The present application provides a complex for treating tumors.

[0011] The first aspect of the present application provides a complex for treating tumors, comprising: BHK-21 fibroblasts with a final concentration of 300,000-625,000 / mL; 30-60% artificial basement membrane and 2-10% fetal bovine serum Dulbecco's medium (DMEM).

[0012] In a preferred technical solution of the present application, the drug complex further comprises 2-10% fetal bovine serum Dulbecco's medium.

[0013] In a preferred technical solution of the present application, the final concentration of BHK-21 fibroblasts is 300,000-625,000 / mL.

[0014] In a preferred technical solution of the present application, the mass percentage of artificial basement membrane is 30%-60%.

[0015] In a preferred technical solution of the present application, the complex comprises: BHK-21 fibroblasts with a final concentration of 300,000-625,000 / mL; 30%-60% artificial basement membrane, 5% fetal bovine serum Dulbecco's medium (DMEM).

[0016] In a preferred technical solution of the present application, the artificial basement membrane is Matrigel.

[0017] The second aspect of the present application provides the use of the above-mentioned complex, i.e. for the preparation of a medicament for treating tumors.

[0018] In a preferred technical solution of the present application, the complex is used for the preparation of a medicament for treating kidney cancer.

[0019] In a preferred technical solution of the present application, the complex is used for the preparation of a medicament for treating lung cancer

[0020] In a preferred technical solution of the present application, the complex functions at a temperature of 23-37°C, preferably at a temperature of 25-37°C.

[0021] The preparation method of the complex of the present application: first, mix a certain number of BHK-21 cells with a certain volume of 30-60% ice artificial basement membrane to prepare a mixture, then add a certain number of cancer cells to make the concentration of the two kinds of cells between 300,000-625,000 / mL, and inoculate into a culture plate until the artificial basement membrane is solidified at a temperature of 24-37°C, and finally add 2-10% fetal bovine serum Dulbecco's Modified Eagle Medium (DMEM) culture solution for culture; the cancer cells are preferably labeled with red fluorescence protein (Red Fluorescence Protein; RFP).

[0022] BHK-21: a cell strain derived from fibroblasts of newborn hamster kidney, which was first established by Stoker and Macpherson in 1961 (Stoker and Macpherson, 1961). Because BHK-21 is very sensitive to viral infection, this cell has been widely used as a host for virus propagation and biopharmaceutical production for many years.

[0023] Artificial basement membrane is a reconstituted basement membrane extract, which is usually mainly used to simulate the in vivo microenvironment for 3D culture of cells in vitro. Matrigel (BD Bioscience) is a soluble 3D culture medium with rich gel network structure, which is originally extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma rich in extracellular matrix proteins. Because Matrigel contains a large amount of macromolecular proteins in the extracellular matrix, such as laminin (60%), collagen (30%), actin (8%) and heparan sulfate proteoglycans, as well as a large number of growth factors necessary for cell stimulation and signal transduction, such as FGF, EGF, TGF-β, Matrigel can largely simulate the structure, composition, physical properties and functions of the basement membrane of cells in vivo, thereby providing favorable growth conditions for in vitro culture of cells (Hughes, Postovit et al. 2010, Benton, Kleinman et al. 2011, Benton, Arnaoutova et al. 2014).

[0024] The present application aims to protect a new complex for treating cancer: (i) the formula of BHK-21 cell and Matrigel complex as an anticancer agent in cancer treatment and (ii) the application thereof. The complex has been proven to be able to efficiently inhibit the proliferation and spread of tumors. Compared with the prior art, the complex of the present application has the following advantages:

[0025] 1. Unlike chemotherapy which usually requires expensive reagents and radiotherapy which requires expensive instruments, the compound of the present invention has better cost-effectiveness, estimated to be no more than 2000 RMB in general.

[0026] 2. Traditional chemotherapy is to kill cancer cells with cytotoxic compounds. In contrast, the therapeutic effect of the compound of the present invention is to control and inhibit the growth of cancer cells by relying on the inherent affinity of BHK-21 vasculature to cancer cells (shown in Figure 2).

[0027] 3. In Matrigel, CaKi-1 cells have a tendency to adhere to BHK-21 vasculature. The actual effect is that the BHK-21 cells in the invention can act like a "dust collector" to remove any cancer cells scattered nearby (Figures 3 and Figure 4 ).

[0028] The present invention is a compound composed of two main components, namely (i) a basement membrane extract named Matrigel , which is a commercially available product on the market; and (ii) baby hamster kidney cells (BHK-21), which are a type of adherent cells commonly used for protein expression.

[0029] In this patent application, it is shown that the compound has a dual role of capturing and inhibiting the proliferation and spread of kidney cancer cells (CaKi-1) and lung cancer cells (Lung Adenocarcinoma, A549). The invention not only serves as a novel and feasible method for the prevention and treatment of cancer, but also has great potential in the development and commercialization of intellectual property rights. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a schematic diagram of an embodiment of the present invention, Figure 1A 1A is the control group, and 1B is the experimental group.

[0031] Figure 2A CaKi-1 cells were inoculated and cultured with Matrigel alone, Figure 2B showing CaKi-1 cells on the vasculature of BHK-21 cells in the compound under a regular microscope.

[0032] Figure 3 shows CaKi-1 cells adhering to the vasculature of BHK-21 cells under a fluorescence microscope, Figure 3A as the bright field background, Figure 3B RFP-labeled CaKi-1 cells observed under a fluorescence microscope, Figure 3C a composite picture of the first two, showing RFP-labeled CaKi-1 cells adhering to the outside of BHK-21 vasculature.

[0033] Figure 4 Process diagram of the spheroid formation of CaKi-1 cells wrapped by the complex of the present application.

[0034] Figure 5A Control diagram of CaKi-1 cells cultured alone and CaKi-1 cells mixed with BHK-21 under the same conditions; Figure 5B Bar chart of 5A.

[0035] Figure 6A Control diagram of RFP-labeled A549 cells mixed with 50% Matrigel alone and RFP-labeled A549 cells mixed with the complex under the same conditions; Figure 6B The number of surviving A549 cells at the end of the experiment in Example 4 is shown. The complex wrapped A549 cells and inhibited their proliferative growth. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application are described below with reference to the accompanying drawings.

[0037] I. Drugs and reagents

[0038] 2.1 Drugs: (BD Bioscience); BHK-21 cell strain (Shanghai Cell Bank GNHa10); A549 cell strain (Shanghai Cell Bank TCHu150); CaKi-1 cells (Shanghai Cell Bank TCHu135)

[0039] 2.2: LS-55 fluorescence spectrometer (PerkinElmer Life Sciences), C1-Si laser confocal microscope (Nikon), upright fluorescence microscope (Nikon), inverted fluorescence microscope (Nikon), multifunctional enzyme label instrument (Thermo Scientific), FACSAria II flow cytometry sorter (BD Bioscience), automatic cell counter (Invitrogen).

[0040] II. Examples

[0041] (I) Figure 1 is a mode diagram of the present application, which simply summarizes the research content and method of the present application.

[0042] To establish the experimental model, cancer cells and BHK-21 cells were suspended in culture medium at a certain concentration ratio with solid phase support for 3D culture. Taking the CaKi-1 cells as an example: first, the red fluorescent protein was introduced into and stably expressed in the kidney cancer cells CaKi-1, and then mixed at a concentration ratio of CaKi-1 625,000 / mL:BHK-21 625,000 / mL:Matrigel 50% in DMEM. The control group was the same number of CaKi-1 added to 50% Matrigel alone, and then the two groups of cells were respectively planted into the culture hole and placed in 37°C until it became solid, and finally the complete culture medium containing 5% fetal bovine serum was added, and placed in a 5% CO2 / 37°C incubator for 37 days.

[0043] Figure 1A In the control group of Example 1B, CaKi-1 cells were inoculated into 50% Matrigel and a complex of 625,000 / mL:BHK-21 cells.

[0044] (II) Example 1

[0045] CaKi-1 cells were adsorbed to the vessel wall of BHK-21 cells in a matrix gel

[0046] Method:

[0047] 1. Purchase CMV-mCherry-T2A-Luc lentivirus and related antibiotics (Jinman Biotechnology Co., Ltd.).

[0048] 2. Inoculate ordinary CaKi-1 cells in a 10cm culture dish and culture with complete culture medium. The complete culture medium is: 5% fetal bovine serum, penicillin-streptomycin solution (1X) and DMEM culture medium.

[0049] 3. When the density of CaKi-1 cells reaches 60%-80%, infect the cells with the above lentivirus, and then screen with Blasticidin until RFP-labeled CaKi-1 cells are obtained.

[0050] 4. Screen the infected CaKi-1 cells again with a flow cytometer to obtain RFP-high-expressing cells.

[0051] 5. Culture BHK-21 cells with complete culture medium until enough cells are obtained.

[0052] 6. Culture high-RFP-expressing CaKi-1 cells with complete culture medium containing 5ug / ml Blasticidin until enough cells are obtained for use.

[0053] 7. The cells were collected by trypsinization and counted using an automated cell counter.

[0054] 8. 50,000 CaKi-1 cells were resuspended in 40 ul of culture medium and mixed with 40 ul of cold Matrigel. The mixture was immediately pipetted into a 24 well plate, 5 ul per well, and repeated 6 times.

[0055] 9. The mixture was pipetted into a 24 well plate and incubated at 37°C until it formed a semi-sphere and solidified on the plate. Finally, complete culture medium was added and the cells were incubated.

[0056] 10. 50,000 BHK-21 cells were resuspended in 40 ul of cold Matrigel to form a complex, and mixed with 50,000 CaKi-1 cells resuspended in 40 ul of culture medium. The mixture was pipetted into a 24 well plate and incubated as described above.

[0057] 11. The cells were observed and photographed using an inverted fluorescent microscope.

[0058] Results: As shown in Figures Figure 2A 2B, Figure 3A 3B, 3C

[0059] Figure 2A shows CaKi-1 cells at a concentration of 625,000 / ml seeded with 50% Matrigel alone;

[0060] Figure 2B shows CaKi-1 cells seeded with a complex of BHK-21 cells and Matrigel at the same concentration;

[0061] Figure 3A shows the morphology of BHK-21 cells in the complex under green fluorescent light in a fluorescent microscope;

[0062] Figure 3B shows RFP labeled CaKi-1 cells observed under a fluorescent microscope;

[0063] Figure 3C shows the red fluorescent protein labeled CaKi-1 cells adhering to the BHK-21 vessels.

[0064] In the absence of BHK-21 cells, the CaKi-1 cells formed independent cell masses of various sizes after 5 days of incubation at 37°C. In the presence of BHK-21 cells, most of the CaKi-1 cells adhered to the vessels between the BHK-21 cells and did not form independent cell masses

[0065] Conclusion: This example effectively demonstrates the inhibitory effect of the complex on the diffusion of CaKi-1 kidney cancer cells. The attraction and adhesion of BHK-21 vasculature to CaKi-1 cells in the matrix gel, and the adsorption of CaKi-1 cells to the vessel wall of BHK-21 cells in the matrix gel.

[0066] Example 2

[0067] Method:

[0068] 1. Same as steps 1-7 and 9 in Example 1.

[0069] 2. After the RFP-labeled CaKi-1 cells and the complex are both solidified onto the plate, add complete culture medium for culture.

[0070] 3. Continuous observation and photographic recording are performed using an inverted microscope.

[0071] Results: As Figure 4 The pictures of continuous microscopic observation for 0-6 days are shown, and the cells form BHK-21 / CaKi-1 spheroids in the matrix gel within 6 days.

[0072] Conclusion: Within one week, CaKi-1 and the complex can jointly form a cocoon-like spheroid.

[0073] Example 3

[0074] Method:

[0075] 1. Same as steps 1-11 in Example 1.

[0076] 2. After all the cells are cultured in 50% matrix gel for 37 days, they are treated with trypsin / EDTA until the BHK-21 / CaKi-1 spheroids are dissolved and the CaKi-1 cells are suspended.

[0077] 3. All the cells in the two groups are collected separately, and the red fluorescent protein-labeled CaKi-1 cells are counted.

[0078] 4. For the CaKi-1 cells cultured alone, after collection, direct counting is performed using an automatic cell counter.

[0079] 5. For the CaKi-1 cells cultured with the complex, all the collected cells are resuspended in 100 ul of culture medium, 10 ul is taken out, and the CaKi-1 cells are counted under a fluorescence microscope, and then the total number of CaKi-1 cells is calculated.

[0080] 6. Each sample is subjected to 4 repeated experiments.

[0081] Results: As Figure 5ARFP-labeled CaKi-1 cells were cultured alone in 50% Matrigel for 37 days; and RFP-labeled CaKi-1 cells were mixed with the complex and cultured for 37 days under the same conditions. In the absence of BHK-21 cells, CaKi-1 cells proliferated rapidly and almost covered the entire surface of the culture well; while in the mixed culture of the two cells, CaKi-1 cells were trapped and enclosed in the spheroids formed by BHK-21 and could not spread to the surrounding area.

[0082] Figure 5B It was shown that the number of CaKi-1 cells cultured alone for 37 days reached about 495,000, while the number of CaKi-1 cells mixed with BHK-21 under the same conditions finally survived only about 850. In summary, the number of CaKi-1 cells cultured with the complex was much lower than that of the cells cultured alone (p<0.0001).

[0083]

[0084] Conclusion: The spheroids formed by the complex trapped CaKi-1 cells, which were enclosed in the spheroids and prevented the proliferation and spread of CaKi-1 cells:

[0085] Example 4

[0086] Method:

[0087] As in Example 3

[0088] Results: As Figure 6A RFP-labeled A549 cells were mixed with 50% Matrigel alone and cultured for 25 days; and RFP-labeled A549 cells were mixed with the complex and cultured for 25 days under the same conditions. In the absence of BHK-21 cells in the complex, A549 cells proliferated rapidly and spread to the surrounding area; while in the mixed culture of A549 cells and the complete complex, A549 cells were wrapped in the spheroids formed by the complex and could not spread to the surrounding area.

[0089] Figure 6B To count the number of surviving A549 cells at the end of the experiment, the number of A549 cells cultured alone proliferated to about 97,200; while the number of A549 cells mixed with the complex survived only about 152. Therefore, the number of A549 cells mixed with the complex was much less than that of the cells cultured alone (p<0.0001).

[0090]

[0091] Conclusion: The spheroids formed by the complex can also trap A549, wrap A549 cells in the spheroids, and inhibit proliferation and spread of the A549 cells.

[0092] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and practice described. Accordingly, all such variations are intended to be included within the scope of the present application as defined in the following claims, along with full acknowledgment of such equivalents herein.

Claims

1. A complex for treating tumors, characterized in that, The complex comprises: BHK-21 fibroblasts at a final concentration of 300,000–625,000 cells / mL; 30%–60% artificial basement membrane; and Dulbecco's culture medium containing 5% fetal bovine serum; the tumor is renal cell carcinoma or lung cancer. The artificial basement membrane is Matrigel. ® Matrix adhesive.

2. Use of the complex of claim 1 in the preparation of a medicament for treating kidney cancer or lung cancer.

3. The use according to claim 2, characterized in that, The complex functions at temperatures between 23 and 37°C.

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