Serum-free exclusive culture medium for cancer stem cells, method for four-stage screening of cancer stem cell lines, and isolated cancer stem cell lines

Abstract

The present invention relates to a serum-free exclusive cancer stem cell culture medium for screening and culturing cancer stem cell lines, characterized by comprising a basic culture medium and a supplement composition added to the basic culture medium, the supplement composition mainly comprising a serum substitute, glutamine dipeptide, a ROCK inhibitor and TGF-beta. The present invention also provides a multi-stage screening method for establishing a stable and large-scale proliferation of cancer stem cell lines using the serum-free dedicated medium, and cancer stem cell lines obtained by such methods. The cancer stem cell strain obtained by the invention has high purity, can be greatly amplified, can reach millions of times of cells compared with those obtained by a traditional screening method, and can stably maintain the dryness of the cancer stem cell strain in vitro for a long time.

Description

Technical Field

[0001] The present invention relates to the screening and culturing method of cancer stem cells. More particularly, the present invention relates to an exclusive culture medium applied to the screening and culturing of cancer stem cells, and a four-stage screening technology platform established by using the exclusive culture medium for cancer stem cells to isolate and obtain stable cancer stem cell lines. Background Art

[0002] The vast majority of cancer cells (more than about 98%) do not have the ability to form tumors, drug resistance and metastatic ability, while a very small part (0.01 - 2%) of cancer cells have the potential of self-renewal, proliferation and multi-directional differentiation. This group of cancer cells is defined as cancer stem cells (CSCs), which are the main causes of tumor drug resistance, recurrence and metastasis. For researchers of cancer stem cells, if they can isolate and culture cancer stem cells from general cancer cells, establish a reliable tumor model, and further reveal the functional characteristics of cancer stem cells and the mechanisms of tumorigenesis and metastasis, so as to target and eliminate the stem cell-like cancer cell population, it will be expected to become one of the reliable strategies for treating malignant tumors.

[0003] There are three common methods for isolating CSCs in the prior art, including: establishment culture method, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Among them, the establishment culture method is based on the growth characteristics of cancer stem cells. Under serum-free and non-adherent culture conditions, differentiated tumor cells will die, while cancer cells with stemness will survive and proliferate to form floating tumor spheres.

[0004] Some specific markers will be expressed on the surface of CSCs, such as CD44, CD133, etc. Generally, they are identified by methods such as flow cytometry, immunocytochemistry, immunofluorescence, Western blot (WB), etc. The traditional surface antigen screening method uses stem cell antigens such as CD44, CD24, CD133, etc., and uses fluorescent antibodies against these antigens to sort cancer stem cells. Although its advantage is that cancer stem cells can be quickly sorted out, because the surface antigens of cancer stem cells in different cancers are different, it is impossible to screen cancer stem cells for cancers whose specific surface antigens are unknown, and it is also difficult to effectively sort and distinguish tumorigenic cancer stem cells from metastatic cancer stem cells; in addition, the antibody screening cost used is high and screening needs to be repeated, and only a very small amount of cancer stem cells (only hundreds or thousands) can be sorted out, and they will differentiate in a short time (about five days), and the characteristics of cancer stem cells cannot be maintained continuously and stably amplified. As a result, the traits of CSCs are difficult to maintain, and their proliferation ability is unstable. Generally speaking, the CSCs obtained by using the traditional antibody screening method are easy to differentiate and do not have stability, because they lose their original stem cell characteristics after being cultured for several days.

[0005] To solve the above problems, the present invention takes the lead in developing a specific medium for cancer stem cell screening and culture, and further based on the behavior screening mode of cancer stem cells, establishes a screening technology platform that can be rapidly tested according to different needs, or used for unlimited culture and maintenance of highly pure and stable cancer stem cells. Summary of the Invention

[0006] On the one hand, the present invention relates to a serum-free specific medium for cancer stem cells, comprising a basal medium and a supplement composition added to the basal medium, comprising: 10-30 vol% (volume percentage) serum substitute, 1-10 mM alanyl-glutamine, 2-20 μM ROCK inhibitor, and 0.2-10 ng / ml TGF-β1. In some specific embodiments of the present invention, the basal medium supplement composition further comprises: 5-20 ng / ml fibroblast growth factor, 0.1-5 mM MEM non-essential amino acid solution, 20-100 μg / ml L-ascorbic acid, and 5-20 mM lactate. In a preferred embodiment of the present invention, the basal medium supplement composition comprises: 15-25 vol% KnockOut serum substitute, 2-5 mM alanyl-glutamine, 0.5-2 mM MEM non-essential amino acid solution, 5-15 ng / ml fibroblast growth factor, 20-60 μg / ml L-ascorbic acid, 5-15 mM lactate, 5-10 μM ROCK inhibitor, and 1-5 ng / ml TGF-β1.

[0007] In specific embodiments of the present invention, the basal medium can be any known basal medium for cell culture, including (but not limited to): DMEM, DMEM / F12, RPMI 1640, MEM, etc. In specific embodiments of the present invention, the basal medium supplement composition further comprises 1000X (diluted 1000 times) chemically defined lipid concentrate and 100X (diluted 100 times) SPITE medium supplement.

[0008] On the other hand, the present invention provides a method for establishing a cancer stem cell line, comprising: a cancer stem cell screening step of screening cancer stem cells with cancer stem cell characteristics from a parental cancer cell population based on behavior screening; and culturing the selected cancer stem cells in the serum-free specific medium for cancer stem cells of the present invention to expand into a cancer stem cell line.

[0009] In some specific embodiments of the present invention, the cancer stem cell line is a stable tumorigenic cancer stem cell line for long-term culture, and the cancer stem cell screening step includes a four-stage screening step: the first stage, tumorigenicity screening; the second stage, self-renewal ability screening; the third stage, treatment resistance screening; and the fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present invention, the tumorigenicity screening is to culture the parental cancer cell suspension in a 1-1.5 vol% agarose medium to simulate the environment of tumor formation in vitro, and screen out cancer cells with high tumorigenicity. In a preferred embodiment of the present invention, the self-renewal ability screening is to use 3D amplification technology to screen out cancer stem cells that can survive in suspension and form tumor spheres. In a preferred embodiment of the present invention, the treatment resistance screening is to use a channel blocker to block the drug efflux channels specific to drug-resistant cancer stem cells, and use a fluorescent dye to simulate the drug, and then sort out the cancer stem cells that produce fluorescence. In a preferred embodiment of the present invention, the single-cell recurrence ability screening is to prepare a single-cell suspension of the selected cancer cells and culture them in the serum-free exclusive medium for cancer stem cells of the present invention, and screen out the cancer stem cell line that can form the largest tumor from a single cell in the shortest time.

[0010] In some specific embodiments of the present invention, the cancer stem cell line is a stable metastatic cancer stem cell line for long-term culture, and the cancer stem cell screening step includes a four-stage screening step: the first stage, metastatic cancer stem cell screening; the second stage, self-renewal ability screening; the third stage, treatment resistance screening; and the fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present invention, the metastatic cancer stem cell screening is to use an invasion chamber to simulate the environment of tumor metastasis in vitro, and screen out cancer cells with the ability of epithelial-mesenchymal transition (EMT) and high invasion ability. In a preferred embodiment of the present invention, the self-renewal ability screening is to use 3D amplification technology to screen out cancer stem cells that can survive in suspension and form tumor spheres. In a preferred embodiment of the present invention, the treatment resistance screening is to use a channel blocker to block the drug efflux channels specific to drug-resistant cancer stem cells, and use a fluorescent dye to simulate the drug, and then sort out the cancer stem cells that produce fluorescence. In a preferred embodiment of the present invention, the single-cell recurrence ability screening is to prepare a single-cell suspension of the selected cancer cells and culture them in the serum-free exclusive medium for cancer stem cells of the present invention, and screen out the cancer stem cell line that can form the largest tumor from a single cell in the shortest time.

[0011] In some specific embodiments of the present invention, the cancer stem cell line is a stable radioresistant cancer stem cell line for long-term culture. The cancer stem cell screening step includes a four-stage screening step: the first stage, radioresistance ability screening; the second stage, self-renewal ability screening; the third stage, treatment resistance screening; and the fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present invention, for the radioresistance ability screening, the cancer cells are irradiated with radiation 25 to 30 times (50 to 60 Gy), and a pure strain of cancer cells with radioresistance ability is screened out. In a preferred embodiment of the present invention, for the self-renewal ability screening, 3D amplification technology is used to screen out cancer stem cells that can survive in suspension and form tumor spheres. In a preferred embodiment of the present invention, for the treatment resistance screening, a channel blocker is used to block the drug efflux channels specific to drug-resistant cancer stem cells, and a fluorescent dye is used to simulate the drug, and the fluorescent cancer stem cells are sorted out. In a preferred embodiment of the present invention, for the single-cell recurrence ability screening, the selected cancer cells are made into a single-cell suspension and cultured in the serum-free exclusive medium for cancer stem cells of the present invention, and a cancer stem cell line that can form the largest tumor from a single cell in the shortest time is screened out.

[0012] In some specific embodiments of the present invention, the cancer stem cell line is a stable drug-resistant cancer stem cell line for long-term culture. The cancer stem cell screening step includes a four-stage screening step: the first stage, drug resistance ability screening; the second stage, self-renewal ability screening; the third stage, treatment resistance screening; and the fourth stage, single-cell recurrence ability screening. In an embodiment of the present invention, for the drug resistance ability screening, a standardized chemotherapy drug for individual cancers is used for 15 to 30 times, preferably 20 times, and the chemotherapy drug is selected to screen out drug-resistant cancer cells. In a preferred embodiment of the present invention, the drug dose for each drug selection is gradually increased from 100 to 300 nM to 1 to 30 μM. In a preferred embodiment of the present invention, for the self-renewal ability screening, 3D amplification technology is used to screen out cancer stem cells that can survive in suspension and form tumor spheres. In a preferred embodiment of the present invention, for the treatment resistance screening, a channel blocker is used to block the drug efflux channels specific to drug-resistant cancer stem cells, and a fluorescent dye is used to simulate the drug, and the fluorescent cancer stem cells are sorted out. In a preferred embodiment of the present invention, for the single-cell recurrence ability screening, the selected cancer cells are made into a single-cell suspension and cultured in the serum-free exclusive medium for cancer stem cells of the present invention, and a cancer stem cell line that can form the largest tumor from a single cell in the shortest time is screened out.

[0013] On the other hand, the present invention provides a method for two-stage rapid screening of clinical cancer stem cells, comprising: First, the stem cell gene transcription function screening stage, in which a clinical tumor sample is transfected with the octamer-binding transcription factor 4 (OCT4) gene through a lentiviral transfection system, and cancer cells showing positive expression of the OCT4 gene are sorted out; and second, the cancer stem cell screening stage, in which the cancer cells sorted out and shown to be positive for the expression of the OCT4 gene are selected for cancer stem cells with cancer stem cell characteristics based on behavioral screening, and cultured in the serum-free exclusive medium for cancer stem cells of the present invention.

[0014] In some specific embodiments of the present invention, the OCT4 gene is combined with a reporter gene. In a specific embodiment of the present invention, the reporter gene is a fluorescent protein reporter gene, preferably a green fluorescent protein reporter gene. In a specific embodiment of the present invention, the reporter gene is a luciferase reporter gene. In a specific embodiment of the present invention, the reporter gene is an antibiotic reporter gene.

[0015] In some specific embodiments of the present invention, the cancer stem cell is a clinical tumor-forming cancer stem cell, and the cancer stem cell screening stage includes a tumor-forming ability screening step. In some specific embodiments of the present invention, the cancer stem cell is a clinical metastatic cancer stem cell, and the cancer stem cell screening stage includes a metastatic invasion ability screening step. In some specific embodiments of the present invention, the cancer stem cell is a clinical radioresistant cancer stem cell, and the cancer stem cell screening stage includes a radioresistance ability screening step. In some specific embodiments of the present invention, the cancer stem cell is a clinical drug-resistant cancer stem cell, and the cancer stem cell screening stage includes a drug resistance ability screening step.

[0016] On the other hand, the present invention provides an isolated cancer stem cell line, characterized in that it is isolated by the screening method described above. In a specific embodiment of the present invention, the isolated cancer stem cell line can be stably amplified and maintain cancer stem cell characteristics for more than 4 months. In a specific embodiment of the present invention, the isolated cancer stem cell line can be stably amplified and maintain cancer stem cell characteristics for more than 3 months.

[0017] On the other hand, the present invention relates to a method for screening cancer drugs, which includes treating the isolated cancer stem cell line of the present invention with a candidate drug, and detecting the inhibitory effect of the candidate drug on the cancer stem cell line. In a specific embodiment of the present invention, the isolated cancer stem cell line is transfected with an OCT4 gene bound to a reporter gene. In a specific embodiment of the present invention, the reporter gene is a green fluorescent protein reporter gene (GFP). In a specific embodiment of the present invention, the reporter gene is a luciferase reporter gene. In a specific embodiment of the present invention, the reporter gene is an antibiotic reporter gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram showing the change of the percentage of CD44-positive cell population during the long-term tracking of cancer stem cells in culture. Cancer stem cells isolated by the traditional CD44 antibody sorting method or the cancer stem cells isolated by the present invention are respectively cultured in a general traditional cell culture medium or a serum-free exclusive culture medium for cancer stem cells for at least 100 days. The cancer stem cells cultured at a fixed culture day are sampled, and the percentage of cells showing a high CD44 expression level (CD44high) is tested.

[0019] Figure 2A It is for the parental pancreatic cancer cells to obtain the tumorigenic cancer stem cell line (PANC-1TCSC) for long-term culture through the four-stage screening method of the present invention; Figure 2B It is the soft agar cell colonies generated in the first tumorigenic ability screening stage; Figure 2C It is the tumor spheres formed in the second self-renewal ability screening stage; Figure 2D It is the sorting result diagram of side population cells obtained in the third treatment resistance screening stage; and Figure 2E It is the largest single-cell colony picked out in the fourth single-cell recurrence ability screening stage.

[0020] Figure 3 It is a schematic diagram comparing the in vitro tumorigenicity of parental cancer cells and isolated stem cell lines by simulating the environment of tumor formation in vitro using soft agar assay; the cells are inoculated in 3-fold diluted agar, and after 14 days of culture, the formation of cell colonies is observed. The left figure is parental cancer cells (PT); the right figure is the tumorigenic cancer stem cell line (TCSC).

[0021] Figure 4A It is for the parental colorectal cancer cells to obtain the tumorigenic cancer stem cell line (DLD-1TCSC) for long-term culture through the four-stage screening method of the present invention; Figure 4B It is the soft agar cell colonies generated in the first tumorigenic ability screening stage; Figure 4CThe tumor spheres formed in the second self-renewal ability screening stage; Figure 4D The sorting result diagram of side population cells obtained in the third treatment resistance screening stage; and Figure 4E The largest single-cell colony picked out in the fourth single-cell recurrence ability screening stage.

[0022] Figure 5A The metastatic cancer stem cell line (PANC-1MCSC) for long-term culture was obtained by subjecting parental pancreatic cancer cells to the four-stage screening method of the present invention; Figure 5B The cell colonies with high invasion ability were screened out by using an invasion chamber in the first metastasis ability screening stage; Figure 5C The tumor spheres formed in the second self-renewal ability screening stage; Figure 5D The sorting result diagram of side population cells obtained in the third treatment resistance screening stage; and Figure 5E The largest single-cell colony picked out in the fourth single-cell recurrence ability screening stage.

[0023] Figure 6 The metastasis ability diagram of the cell line was observed by using a migration assay to compare the metastasis abilities of parental cancer cells and isolated cancer stem cell lines during 24 hours.

[0024] Figure 7A The metastatic cancer stem cell line (DLD-1MCSC) for long-term culture was obtained by subjecting parental colorectal cancer cells to the four-stage screening method of the present invention; Figure 7B The cell colonies with high invasion ability were screened out by using an invasion chamber in the first metastasis ability screening stage; Figure 7C The tumor spheres formed in the second self-renewal ability screening stage; Figure 7D The sorting result diagram of side population cells obtained in the third treatment resistance screening stage; and Figure 7E The largest single-cell colony picked out in the fourth single-cell recurrence ability screening stage.

[0025] Figure 8 is a schematic diagram of the percentage of cell populations highly expressing the surface antigen CD44 (CD44 high cells) among the overall cultured cells after 35 days of culturing cancer stem cells. Cancer stem cells isolated by the traditional CD44 antibody sorting method or the present invention were respectively cultured in a general traditional cell culture medium or a serum-free exclusive medium for cancer stem cells, and tested on the 35th day. The percentage of cells showing a high CD44 expression level (CD44 high ) among the overall cultured cells.

[0026] Figure 9A The schematic diagram of the expression levels of stem cell stemness genes after culturing cancer stem cells for one month; Figure 9BSchematic diagram of the expression levels of epithelial-mesenchymal transition genes in cancer stem cells after one month of culture. Cancer stem cells isolated by the conventional CD44 antibody sorting method or the cancer stem cells isolated by the present invention were cultured in a general conventional cell culture medium or a serum-free exclusive medium for cancer stem cells for one month, and then the relative RNA expression levels of stemness genes (OCT4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes) and epithelial-mesenchymal transition (EMT) genes (Twist, Snail, Slug, Zeb1, E-cad, N-cad, Vim and FN1 genes) were quantitatively analyzed. Detailed implementation manners

[0027] Other features and advantages of the present invention will be further exemplified and described in the following implementation examples, and these implementation examples are only for auxiliary illustration and are not used to limit the scope of the present invention.

[0028] Example 1. Composition and preparation of serum-free exclusive medium for cancer stem cells

[0029] In the present invention, a serum-free exclusive medium for screening and culturing highly pure and highly stable cancer stem cells is provided, which comprises: a basal medium and a supplement composition added to the basal medium, mainly comprising: 10-30 vol% serum substitute, 1-5 mM alanyl-glutamine dipeptide, 0.1-5 mM MEM non-essential amino acid solution, 5-20 ng / ml fibroblast growth factor, 20-100 μg / ml L-ascorbic acid, 5-20 mM lactic acid, 2-10 μM ROCK inhibitor and 0.2-10 ng / ml TGF-β1. Among them, the serum-free exclusive medium is prepared by dispensing all the medium formulations into a sterilized serum bottle in a sterile operation bench and then performing sterile filtration through a 0.45 μm filter into a sterile serum bottle.

[0030] In Table 1 below, the composition reagents and final concentrations added to the basal medium (which can be selected from DMEM, DMEM / F12, RPMI 1640, MEM, etc.) in the serum-free exclusive medium for cancer stem cells of the present invention are exemplified.

[0031] Table 1

[0032]

[0033]

[0034] Knockout TM Serum substitute (Knockout TMSR is a serum-free additive that supports the growth of pluripotent stem cells (PSCs) cultured on fibroblast feeder layers; it is suitable for serum-free feeder cell-dependent culture of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) from multiple species and can directly replace FBS in existing technology experimental protocols.

[0035] Alanyl-glutamine (Ala-Glu, also known as GlutaMAX) is an advanced cell culture additive that can directly replace the essential nutrient element L-glutamine in cell culture. During cell culture, cells gradually release a peptidase into the culture medium, which hydrolyzes L-alanyl-L-glutamine into L-alanine and L-glutamine, and then the cells absorb and utilize these two hydrolysis products.

[0036] MEM Non-Essential Amino Acids (NEAA) is derived from the MEM medium formula and contains 7 non-essential amino acids, namely L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine. It can effectively improve the cell culture medium formulation, reduce the side effects of cells producing non-essential amino acids during cell culture, and promote cell proliferation and metabolism. It is one of the commonly used additives in cell culture.

[0037] Chemically defined lipid concentrate is a concentrated fat emulsion. The chemical components of this culture medium supplement are clearly defined and are designed to reduce or replace fetal bovine serum in cell culture media. It can be used for the growth and maintenance of animal cells such as CHO cells, hybridomas, and insect cells; for culturing hybridomas to produce monoclonal antibodies; and for protein expression of insect cell viruses, etc.

[0038] SPITE liquid media supplement is a general cell additive used for formulating serum-free media. The purified substances in the SPITE liquid media supplement can replace the components normally provided by serum, including insulin, transferrin, selenium, propionate, and ethanolamine.

[0039] ROCK inhibitor is a selective ATP-competitive inhibitor of Rho-associated protein kinase (ROCK) and can also act as an agonist-induced Ca for myosin phosphorylation and smooth muscle contraction 2+An effective inhibitor of sensitization. Optional ROCK inhibitors include Y-27632, Fasudil, and H-1152. It is known that treating human embryonic stem cells (hES) cultured in serum-free suspension medium (SFEB) with 10 μM Y-27632 can significantly reduce apoptosis induced by dissociation and improve the cloning efficiency (from ~1% to ~27%).

[0040] The cancer stem cells isolated by the four-stage cancer stem cell line sorting method of the present invention or the traditional CD44 antibody sorting method are cultured in a general traditional cell culture medium (such as DMEM + 10% FBS) or the serum-free exclusive culture medium for cancer stem cells of the present invention, and the cell growth and differentiation are monitored for at least 100 days (the culture conditions are at 37 °C, 5% CO2, and 21% O2). The cancer stem cells cultured at a fixed number of days of culture are sampled, and the percentage of cells showing a high CD44 expression level among the overall cultured cells is tested.

[0041] The results are as Figure 1 shown. Whether the cancer stem cells isolated by the four-stage cancer stem cell line sorting method of the present invention or the traditional CD44 antibody sorting method, compared with the use of a general traditional cell culture medium, the exclusive culture medium for cancer stem cells of the present invention can effectively slow down the differentiation of cancer stem cells. The cancer stem cell line obtained by the four-stage cancer stem cell line sorting method of the present invention, compared with those isolated by the traditional CD44 antibody sorting method, still maintains the characteristic of expressing the surface antigen CD44 of cancer stem cells after long-term culture for at least 100 days; while the cancer stem cell line isolated by the traditional CD44 antibody sorting method begins to differentiate around the 5th day of culture, gradually loses the characteristics of cancer stem cells and cannot continue to expand.

[0042] Example 2: Screening and establishing tumorigenic cancer stem cell lines (TCSC)

[0043] This example presents a four-stage method for screening stable tumorigenic cancer stem cell lines for long-term culture, including: the first stage, screening for tumorigenic ability; the second stage, screening for self-renewal ability; the third stage, screening for treatment resistance; and the fourth stage, screening for single-cell recurrence ability. In the first stage, the parental pancreatic cancer cells are separated from the culture dish with trypsin and suspended in fresh medium to form a single-cell suspension ( Figure 2AAs shown). After centrifugation, resuspend in 500-1000 μl of 5% FBS fresh culture medium. Take 1 ml of 1-1.5% agar to each well of the 6-well plate culture medium and let it stand for 3-5 minutes to allow the agar to solidify. Add 666 μl of cell suspension and 333 μl of 1-1.5% agar (to make a final 3-fold dilution of agar) to each well, then slowly shake the plate to mix it, and let it stand for 3-5 minutes to allow the agar to solidify (add the cell suspension to the well first to prevent the agar from solidifying). Then add 1 ml of 5% FBS culture medium to each well. Place the plate in a 37°C low-oxygen (5% O2) incubator for culture. After 1-2 weeks of culture, use a 1 ml tip to remove the cell colony from the soft agar. Transfer the colony to a 6-well plate for temporary expansion to provide enough cells for the next round of selection. The cells were inoculated and subjected to another round of soft agar selection. The soft agar selection was repeated five times. To select cells with high tumorigenicity, the number of cells inoculated was gradually reduced to 10 6 , 10 5 , 10 4 , 10 3 and 10 2 cells and performed 5 rounds of selection. Figure 2B It showed that in the first stage, soft agar was used to simulate the environment of tumor formation in vitro, and cancer cells with high tumor-forming ability were screened out.

[0044] Then, in the second stage, 1×10 4 The cells are dispersed into single cells and inoculated into a 6-well plate covered with 1-1.5% soft agar and containing serum-free culture medium. The 6-well plate is placed in a low-oxygen (5% O2) incubator at 37°C for culture. Cells that cannot attach to the soft surface will form tumor spheres after a few days in the suspension culture medium. Every four days, the spheres are separated into single cells using a quantitative pipette to form one. Cells that can form new spheres have anoikis resistance and long-term self-renewal capabilities. After 1-2 weeks of culture, the number of tumor spheres is collected, and the cell colonies are transferred to a 6-well plate for temporary expansion to provide enough cells for the next round of selection. Repeat the above tumor sphere screening steps three times. In order to select cells with high tumor sphere generation, the number of inoculated cells is gradually reduced to 10 4 , 10 3 and 10 2 cells and performed 3 rounds of selection. Figure 2CIt is shown that in the second stage, using 3D amplification technology, under suspension conditions, cancer stem cells with stem cell characteristics will form spheres (tumor spheres) like blastocysts. This environment is also similar to the situation where tumors circulate in blood vessels. Only stem cells can survive, so they are screened out.

[0045] Next, in the third stage, at least 5×10 6 cells (at most 10 7 cells) are suspended in 5 ml of culture medium for standby. The 5 ml cell suspension is divided into 4 ml and 1 ml, and then placed into two blue-capped sorting tubes. 100 μM verapamil (Sigma-Aldrich, St. Louis, MO) and 10 μM fumitremorgin C (FTC, Sigma-Aldrich) are added to the sorting tube containing 1 ml of cell suspension, and cultured at 37 °C for 5 minutes (protected from light). 4 μl and 1 μl of Hoechst 33342 (5 μM, Sigma-Aldrich, St. Louis, MO) are added to the sorting tubes containing 4 ml and 1 ml of cell suspension respectively, and then the sorting tubes are placed in a 37 °C incubator for 90 minutes, gently tapping the tubes every 15 minutes.

[0046] After the cells are washed twice with PBS, 2 ml and 1 ml of culture medium containing propidium iodide (PI) (2 μg / ml, Sigma-Aldrich, used to exclude dead cells) are added to resuspend the untreated (without verapamil and FTC) and treated (pretreated with verapamil and FTC) cells respectively, and the cell suspension is passed through the screening procedure. Prepare a white-capped tube containing 1 ml of culture medium with 0.2% PSA and cover it with paraffin film. Cell sorting is performed using a BD FACSAria flow cytometer (BD Biosciences). After excitation with a UV488 laser, the main cell population containing Hoechst 33342 will show blue and red fluorescence. A part of the side population cells will emit Hoechst dye through their ABC transporters and show low fluorescence. When treated with verapamil and FTC to block the efflux of the fluorescent dye, these side population cells show high fluorescence. By comparing the parameters obtained when treated with verapamil and FTC or without verapamil and FTC, the side population cells are sorted out. The isolated cells are cultured in a 6-well plate and temporarily amplified in a 37 °C hypoxic (5% O2) incubator to provide enough cells for the next selection. Figure 2DIt is shown that in the third stage, a fluorescent dye (Hoechst dye) was used to simulate the drug. Cancer stem cells have a drug efflux pump that can expel the drug. The use of a pump blocker (verapamil) can cause cancer stem cells with drug resistance to fluoresce and be sorted out.

[0047] Finally, in the fourth stage, a single cell was seeded in a round-bottom, ultra-low attachment 96-well plate with serum-free medium. The plate was placed in a 37 °C hypoxic (5% O2) incubator. The 3D cell colonies with the largest size formed in the shortest time, with a diameter of approximately 250 - 300 μm, were selected and cultured in the CSC serum-free exclusive medium of the present invention (as described in Example 1). After long-term culture, a stable pancreatic cancer tumorigenic cancer stem cell line was obtained. Figure 2E It is shown that in the fourth stage, after simulating treatment, the recurrence ability of a few tumor cells to form new tumors again was examined, and cancer stem cells that could form the largest tumors in the shortest time from single cells were screened out. Then, the screened cancer stem cell line was cultured in the CSC serum-free exclusive medium of the present invention (as described in Example 1). After long-term culture, a stable pancreatic cancer tumorigenic cancer stem cell line (PANC-1TCSC) was obtained. Figure 3 It is shown that the tumorigenic cancer stem cell line obtained in the present invention has a high tumorigenic ability.

[0048] Figures 4A to 4E It is shown that the cell colony morphology at each stage of obtaining a colorectal cancer tumorigenic cancer stem cell line (DLD-1TCSC) from colorectal cancer parental cancer cells by the four-stage screening method as described above.

[0049] Example 3. Screening and establishing a metastatic cancer stem cell line (MCSC)

[0050] This example presents a four-stage method for screening a stable metastatic cancer stem cell line for long-term culture, including: the first stage, screening for metastatic invasion ability; the second stage, screening for self-renewal ability; the third stage, screening for treatment resistance; and the fourth stage, screening for single-cell recurrence ability. In the first stage, parental pancreatic cancer cells were detached from the culture dish with trypsin and suspended in fresh medium to form a single-cell suspension. Figure 5A). After centrifugation, resuspend in 500-1000 μl of 5% FBS fresh culture medium. Add 80 μl of Matrigel (Corning, NY) to the upper space (the dilution ratio of Matrigel to serum-free culture medium is 1:2). After the Matrigel solidifies, add 600 μl of 10% FBS fresh culture medium to the lower compartment. Add 200 μl of cell suspension to the upper Matrigel. Place the transwell plate in a 37°C hypoxic (5% O2) incubator, and the cells begin to invade from the upper space to the lower space. After 1-2 weeks, transfer the lower cells to a 6-well plate for temporary expansion to provide enough cells for the next round of screening. Inoculate cells for another round of metastasis and invasion selection. Repeat the above metastasis and invasion screening five times. In order to select cells with high metastasis and invasion ability, the number of inoculated cells is gradually reduced to 10 6 , 10 5 , 10 4 , 10 3 and 10 2 cells and performed 5 rounds of selection.

[0051] The second to fourth screening steps described in Example 2 were then performed, and the selected cancer stem cells were cultured in the serum-free culture medium for CSCs of the present invention (described in Example 1). A stable pancreatic cancer metastatic stem cell line (PANC-1 MCSC) was obtained through long-term culture. Figures 5B to 5E The morphology of cell colonies obtained at each screening stage is shown. Figure 6 As shown, the metastatic cancer stem cell line obtained by the present invention has a high metastatic ability.

[0052] Figures 7A to 7E The figures show the morphology of cell colonies obtained at each stage of obtaining a colorectal cancer metastatic stem cell line (DLD-1TCSC) from colorectal cancer parental cancer cells using the four-stage screening method described above.

[0053] Example 4: Screening and Establishment of Resistant Cancer Stem Cell Lines

[0054] This example presents a method for screening stable and resistant cancer stem cell lines for long-term culture, including: the first stage, screening for resistance (radiation resistance or drug resistance) ability; the second stage, screening for self-renewal ability; the third stage, screening for treatment resistance; and the fourth stage, screening for single-cell recurrence ability. For the screening of cancer stem cells with radiation resistance, in the first stage, an Elekta Synergy radiotherapy machine (Elekta, Sweden) is used to irradiate at a rate of 562 MU / min at 2 Gy / day. After 30 irradiations, a pure strain of cancer cells with radiation resistance is screened out. The cells are cultured in a 37°C hypoxic (5% O2) incubator. The cells are inoculated into a 96-well plate using serum-free medium at a rate of one cell per well. The Elekta Synergy radiotherapy machine (Elekta, Sweden) irradiates single cells at a rate of 10 Gy / day. The pure strain of cells that forms the largest size in the shortest time is selected. The plate is placed in a 37°C hypoxic (5% O2) incubator for temporary amplification to provide sufficient cells for the next screening.

[0055] For the screening of cancer stem cells with chemotherapeutic drug resistance, after 20 administrations of chemotherapeutic drugs in the first stage, cancer cells with drug resistance are screened out. The drug dose selected each time is gradually increased from 100 - 300 nM to 1 - 30 μM. The cells are stored in a 37°C hypoxic (5% O2) incubator. Blood therapy drugs are used to establish drug-resistant cells. As listed in Table 2 below, the standard blood therapy drugs used to establish different types of drug-resistant cancer cells are enumerated.

[0056] Table 2

[0057]

[0058]

[0059] After 20 selections of chemotherapeutic drugs, the cells are inoculated into a 96-well plate in the form of one cell per well. The single cells are cultured with the highest dose of chemotherapeutic drugs. The pure strain of cells that forms the largest size in the shortest time is selected. The plate is placed in a 37°C hypoxic (5% O2) incubator for temporary amplification to provide sufficient cells for the next selection.

[0060] Next, the screening steps of the second to fourth stages described in Example 2 are carried out. Subsequently, the screened cancer stem cells are cultured in the CSC serum-free exclusive medium of the present invention (as described in Example 1), and stable radiation-resistant cancer stem cell lines and drug-resistant cancer stem cell lines are obtained through long-term culture.

[0061] Example 5. Establishing a rapid screening platform for clinical cancer stem cells

[0062] This example presents a two-stage rapid screening method for clinical cancer stem cells, including: a stem cell gene transcriptional function screening stage; and a cancer stem cell screening stage. In the first stage, a clinical tumor sample is transfected with the Octamer-binding transcription factor 4 (OCT4) gene through a lentiviral transfection system, and cancer cells showing positive for the OCT4 gene are sorted out to identify their stemness status.

[0063] For the screening of clinical tumorigenic stem cells, in the second stage, 1 ml of 1-1.5% agarose is added to each well of a 6-well plate culture medium, and it is left to stand for 3-5 minutes to solidify the agarose. 666 μl of cell suspension (10 3 ~10 4 OCT4-GFP positive cancer cells) and 333 μl of 1-1.5% agarose (resulting in a final three-fold dilution of agarose) are added to each well, and then the plate is gently shaken to mix and left to stand for 3-5 minutes to solidify the agarose. The cell clone that forms the largest size in the shortest time is selected and cultured in the serum-free exclusive culture medium for cancer stem cells of the present invention.

[0064] For the screening of clinical metastatic cancer stem cells, in the second stage, 80 μl of Matrigel (Corning, NY) is added to the upper space (the dilution ratio of Matrigel to serum-free culture medium is 1:2). After the Matrigel solidifies, 600 μl of fresh 10% FBS culture medium is added to the lower compartment. 200 μl of cell suspension is added to the upper Matrigel. The transwell plate is placed in a 37°C hypoxic (5% O2) incubator, and the cells start to invade from the upper space to the lower space. The metastatic cancer stem cells are cultured in the serum-free exclusive culture medium for cancer stem cells of the present invention.

[0065] For the screening of clinical radioresistant cancer stem cells, in the second stage, OCT4-GFP positive cancer cells are seeded in a 96-well plate at a density of 10 3 ~10 4 cells per well using serum-free culture medium. An Elekta Synergy radiotherapy machine (Elekta, Sweden) is used to irradiate single cells at a rate of 562 MU / min at 10 Gy / day. The cell clone that forms the largest size in the shortest time is selected. The screened radioresistant cancer cells are cultured in the serum-free exclusive culture medium for cancer stem cells of the present invention.

[0066] For the screening of clinically drug-resistant cancer stem cells, standard therapeutic chemotherapy drugs were used in the second stage to establish chemotherapy-resistant cancer stem cells. The OCT4-GFP-positive cancer cells were seeded in a 96-well plate using serum-free medium at a density of 10 3 ~10 4 cells per well. The highest dose of chemotherapy drug was administered to single cells. The cell clone with the largest size formed in the shortest time was selected. The cancer cells with chemotherapy drug resistance screened out were cultured in the serum-free exclusive medium for cancer stem cells of the present invention.

[0067] With the two-stage screening method of the present invention, not only can cancer stem cells existing in clinical specimens be quickly screened out, but the obtained clinical cancer stem cells can be used as a screening platform for clinically effective drugs. Taking this example as an illustration, if a candidate drug can target cancer stem cells and reduce their stemness, the cancer stem cells expressing OCT4-GFP will lose their fluorescence, thereby evaluating the clinical therapeutic effects of candidate drugs on various cancer stem cells.

[0068] Example 6. Purity and Characterization of Cancer Stem Cell Lines Isolated by the Four-Stage Screening Method of the Present Invention

[0069] The cancer stem cell lines established in Example 2 and Example 3, as well as the cancer stem cell lines sorted by commercially available CD44 antibody, were respectively seeded in a general cell culture medium and the serum-free exclusive medium as described in Example 1, and cultured in a 37°C hypoxic (5% O2) incubator for 35 days. Then the cells were taken out for CD44 surface antigen sorting, and the percentage of cells that could maintain high expression of CD44 surface antigen in the total cultured cells was detected.

[0070] The results are as Figure 8 shown. The cancer stem cell lines isolated by the four-stage screening method of the present invention can more stably and continuously express stem cell antigens compared with the cancer stem cell lines obtained by the traditional antibody sorting method (CD44-antibody sorting method). In addition, compared with culturing in a general cell culture medium, the serum-free exclusive culture medium for cancer stem cells of the present invention can effectively slow down the differentiation of cancer stem cells, and this effect is more significant in combination with the four-stage screening method of the present invention.

[0071] Furthermore, after culturing the cancer stem cell lines for 1 month, the expression of their stemness genes (OCT4, Sox2, Klf4, c-Myc, Nanog, and Lin28 genes) and epithelial-mesenchymal transition (EMT) genes (Twist, Snail, Slug, Zeb1, E-cad, N-cad, Vim, and FN1 genes) was detected as Figure 9A and Figure 9B shown. After culturing for one month, the cancer stem cell lines of the present invention still highly expressed stem cell stemness genes Figure 9A) and epidermal-mesenchymal transition genes ( Figure 9B ).

[0072] Example 7: Cancer stem cell lines as a drug screening platform

[0073] In this embodiment, some schemes using the cancer stem cell lines isolated by the present invention as a drug screening platform are listed. To facilitate in vitro or in vivo observation and identification of the stemness status of cancer stem cells, the isolated cancer stem cell lines are first transfected with OCT4-GFP or OCT4-Luc reporter genes using a lentivirus-based transfection system. If a candidate drug can effectively reduce the stemness of cancer stem cells (CSCs), CSCs expressing OCT4-GFP or OCT4-Luc will lose their fluorescence or luminescence (cold light) response. The reporter genes that can be used are not limited to GFP or Luc genes, and an antibiotic reporter gene can also be selected.

[0074] One approach involves forming tumor spheres from isolated cancer stem cell lines in 3D culture, then treating them with a nanodrug to observe whether the drug can effectively target and inhibit tumors. Cancer stem cell lines can also serve as a platform for evaluating the efficacy of drug combination therapies. Tumorigenic or metastatic cancer stem cells expressing OCT4-GFP or OCT4-Luc are treated with a combination of a known chemotherapeutic drug and a new drug (e.g., New Drug A, New Drug B, or New Drug C). The inhibitory effects of these drug combinations on parental cancer cells and cancer stem cells are then compared, thereby evaluating the efficacy of drug combination therapies or the therapeutic effects of new drugs on drug-resistant cancers.

[0075] For in vivo drug screening, tumorigenic cancer stem cell lines can be injected into zebrafish or mice to form tumors to study whether candidate drugs can effectively target and treat tumors. Alternatively, metastatic cancer stem cell lines can be injected into zebrafish or mice to study whether candidate drugs can effectively inhibit tumor metastasis.

[0076] The technical solution of this invention has successfully developed a cancer stem cell-specific culture medium, which is then used to screen and establish cancer stem cell lines with high purity (several dozen times higher stem cell gene expression than competing products), stability (effectively extending shelf life by a hundredfold), and high cell count (capable of expanding cell numbers by a million-fold). The isolated cancer stem cell lines can be supplied to pharmaceutical companies and academic research institutions, accelerating the development of new drugs that more specifically target cancer stem cells, thereby significantly reversing the current predicament in cancer treatment.

Claims

1. A method for screening cancer stem cell lines, characterized in that, Comprising the following steps: Behavior screening of cancer stem cells: screening cancer cells with cancer stem cell characteristics from a parental cancer cell population; Self-renewal ability screening: using 3D amplification technology to screen cancer stem cells that can survive in suspension and form tumor spheres from the cancer cells with cancer stem cell characteristics; Therapeutic resistance screening: blocking the drug efflux channels of the cancer stem cells with a channel blocker and applying a fluorescent dye to the drug efflux channels to screen cancer stem cells that can produce fluorescence; and Single-cell recurrence ability screening: preparing a single-cell suspension of the cancer stem cells that can produce fluorescence and culturing them in a culture medium to screen a cancer stem cell line that can form the largest tumor from a single cell in the shortest time, wherein The culture medium is a serum-free medium.

2. The method for screening a cancer stem cell line according to claim 1, wherein the cancer stem cell line is a tumor-forming cancer stem cell line, and the behavior screening of the cancer stem cells is tumor-forming ability screening, comprising: Culturing the suspension of the parental cancer cell population in an agarose medium of 1-1.5 vol% to simulate an in vitro tumor-forming environment, and screening the cancer cells with cancer stem cell characteristics having high tumor-forming ability.

3. The method for screening a cancer stem cell line according to claim 1, wherein the cancer stem cell line is a metastatic cancer stem cell line, and the behavior screening of the cancer stem cells is metastasis ability screening, comprising: Using a Transwell invasion chamber to simulate an in vitro tumor metastasis environment for the suspension of the parental cancer cell population, and screening the cancer cells with cancer stem cell characteristics having the ability of epithelial-mesenchymal transition (EMT) and high invasion ability.

4. The method for screening a cancer stem cell line according to claim 1, wherein the cancer stem cell line is a radioresistant cancer stem cell line, and the behavior screening of the cancer stem cells is radioresistance ability screening, comprising: Irradiating the parental cancer cell population with radiation and screening the cancer cells with cancer stem cell characteristics having radioresistance ability.

5. The method for screening a cancer stem cell line according to claim 1, wherein the cancer stem cell line is a drug-resistant cancer stem cell line, and the behavior screening of the cancer stem cells is drug resistance ability screening, comprising: Administering a chemotherapeutic drug to the parental cancer cell population and screening the cancer cells with cancer stem cell characteristics having drug resistance.

6. A serum-free medium, characterized in that, Comprising: A basal medium; and A supplement composition, wherein The basal medium is selected from DMEM, DMEM / F12, RPMI 1640 or MEM medium; and The supplement composition comprises: 10-30 vol% of serum replacement, 1-10 mM of alanyl-glutamine dipeptide, 2-20 μM of ROCK inhibitor, and 0.2-10 ng / ml of TGF-β1.

7. The serum-free medium according to claim 6, wherein the supplement composition further comprises: fibroblast growth factor at 5-20 ng / ml, MEM non-essential amino acid solution at 0.1-5.0 mM, L-ascorbic acid at 20-100 μg / ml, and lactic acid at 5-20 mM.

8. The serum-free medium according to claim 7, wherein the supplement composition further comprises: penicillin at 40-70 U / ml, streptomycin at 40-70 μg / ml, 2-mercaptoethanol at 0.05-0.15 mM, lipid concentrate diluted 1000-fold, and SPITE medium supplement diluted 100-fold.

9. A method for rapidly screening clinical cancer stem cells, characterized in that, comprising the following steps: Screening of stem cell gene transcription function: Transfecting a clinical tumor sample with an octamer-binding transcription factor 4 gene using a lentiviral transfection system to screen for cancer cells showing positive expression of the octamer-binding transcription factor 4 gene; and Screening of cancer stem cell behavior: Selecting cancer cells with cancer stem cell characteristics from the cancer cells showing positive expression of the octamer-binding transcription factor 4 gene based on behavior screening.

10. The method for rapidly screening clinical cancer stem cells according to claim 9, wherein the octamer-binding transcription factor 4 gene is bound to a reporter gene.

11. The method for rapidly screening clinical cancer stem cells according to claim 10, wherein the reporter gene is a fluorescent protein reporter gene.

12. The method for rapidly screening clinical cancer stem cells according to claim 11, wherein the fluorescent protein reporter gene is a green fluorescent protein reporter gene.

13. The method for rapidly screening clinical cancer stem cells according to claim 10, wherein the reporter gene is a luciferase reporter gene.

14. The method for rapidly screening clinical cancer stem cells according to claim 10, wherein the reporter gene is an antibiotic reporter gene.

15. The method for rapidly screening clinical cancer stem cells according to claim 9, wherein the cancer stem cell is a clinical tumor-forming cancer stem cell, and the behavior screening of the cancer stem cell is tumor-forming ability screening.

16. The method for rapidly screening clinical cancer stem cells according to claim 9, wherein the cancer stem cell is a clinical metastatic cancer stem cell, and the behavior screening of the cancer stem cell is metastatic invasion ability screening.

17. The method for rapidly screening clinical cancer stem cells according to claim 9, wherein the cancer stem cell is a clinical radioresistant cancer stem cell, and the behavior screening of the cancer stem cell is radioresistance ability screening.

18. The method for rapidly screening clinical cancer stem cells according to claim 9, wherein the cancer stem cell is a clinical drug-resistant cancer stem cell, and the behavior screening of the cancer stem cell is drug resistance ability screening.

19. A method for screening anti-cancer drugs, characterized in that, comprising: culturing a candidate drug with a cancer stem cell line; and detecting the inhibitory effect of the candidate drug on the cancer stem cell line, wherein the cancer stem cell line is the cancer stem cell line screened by the method for rapidly screening clinical cancer stem cells according to claim 9.

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