Methods for delaying differentiation of cancer stem cells

Abstract

The present disclosure relates to a serum-free conditioned medium for cancer stem cells (CSCs), which is characterized by comprising a combination of a basal medium and supplements primarily containing serum replacement, alanylglutamine, a ROCK inhibitor, and TGF-β. The present disclosure also provides a multi-step screening method for establishing stable and proliferative cancer stem cell lines using the serum-free conditioned medium, and the cancer stem cell lines obtained by such a method. The isolated cancer stem cell lines are highly pure, can proliferate significantly millions of times faster than cells obtained by conventional screening methods, and can stably maintain CSC stemness in vitro for a long period of time.

Description

[Technical Field]

[0001] The present disclosure relates to a method for screening and culturing multiple cancer stem cells (CSCs). More specifically, the present disclosure relates to a conditioned medium for selecting and culturing CSCs, and a quadruple sorting technology platform for isolating stable CSC lines from CSCs using the conditioned medium. [Background technology]

[0002] Most cancer cells (approximately 98% or more) lack tumorigenicity, drug resistance, and metastatic potential. However, a very small percentage (0.01-2%) of cancer cells possess the ability to self-multiply, proliferate, and differentiate into multiple lines of cells. This population of cancer cells, defined as cancer stem cells (CSCs), is responsible for tumor drug resistance, recurrence, and metastasis. If we can separate CSCs from normal cancer cells and establish reliable tumor models, we can elucidate the functional characteristics of CSCs, the mechanisms of tumorigenesis, and tumorigenesis / metastasis. This allows us to target and eliminate the CSC population. For researchers studying CSCs, this represents a reliable strategy for effectively treating malignant tumors.

[0003] There are three common methods for isolating CSCs: culture-based methods, magnetic cell sorting (MACS), and fluorescence-activated cell sorting (FACS). Culture-based methods are based on the proliferation properties of CSCs. Differentiated tumor cells die under serum-free and non-adherent culture conditions, whereas CSCs survive and proliferate to form floating tumor spheres.

[0004] CSCs express specific surface markers, such as CD44 and CD133, which can be identified using various techniques, including flow cytometry, immunocytochemistry, immunofluorescence, and Western blot (WB). Conventional surface antigen screening methods, which use fluorescent antibodies targeting stem cell antigens such as CD44, CD24, and CD133, are rapid methods for sorting CSCs. However, different CSCs have different surface antigens. Therefore, cancers with unknown surface antigens cannot be sorted, making sorting and identification of tumorigenic and metastatic CSCs difficult. Furthermore, the cost of screening antibodies is high, and repeated screening processes are required. Only a small number of CSCs (hundreds or thousands) can be sorted, and CSCs differentiate within a short period of time (approximately 5 days). As a result, CSC characteristics cannot be maintained, and CSC proliferation is unstable. As a result, CSCs isolated using conventional antibody-based screening methods are prone to differentiation and unstable, losing their characteristics after several days of culture.

[0005] To solve the above problems, the present disclosure develops a conditioned medium for the screening and culture of CSCs. Based on the behavioral screening of CSCs, a screening technology platform is established that enables rapid testing according to different needs and realizes unlimited culture of highly pure and stable CSCs. Summary of the Invention

[0006] In one aspect, the present disclosure relates to a serum-free conditioned medium for CSCs, comprising a basal medium and a combination of supplements added to the basal medium. The combination of supplements comprises 10-30% by volume of serum replacement, 1-10 mM pyruvate, 2-20 μM ROCK inhibitor, and 0.2-10 ng / ml TGF-β1. In one specific embodiment of the present disclosure, the combination of supplements further comprises 5-20 ng / ml of fibroblast growth factor (FGF), 0.1-5.0 mM MEM non-essential amino acid solution, 20-100 μg / ml of L-ascorbic acid, and 5-20 mM lactic acid. In one preferred embodiment of the present disclosure, the supplement combination further comprises 15-25% by volume of knockout serum replacement, 2-5 mM L-alanyl-L-glutamine, 0.5-2 mM MEM non-essential amino acid solution, 5-15 ng / ml of fibroblast growth factor, 20-60 μg / ml of L-ascorbic acid, 5-15 mM of lactic acid, 5-10 μM of Rho-associated protein kinase kinase (ROCK) inhibitor, and 1-5 ng / ml of TGF-β1.

[0007] In one specific embodiment of the present disclosure, the basal medium may be any known medium used in cell culture, including, but not limited to, DMEM, DMEM / F12, RPMI 1640, MEM, etc. In one specific embodiment of the present disclosure, the basal medium and supplement complex further comprises a 1000-fold diluted lipid concentrate solution having a defined chemical composition and a 100-fold diluted SPITE medium supplement solution.

[0008] In another aspect, the present disclosure provides a method for forming a CSC line, comprising a CSC screening step of selecting CSCs with stem cell properties from a parent cancer cell population according to behavioral screening, and then culturing the selected CSCs in the serum-free conditioned medium of the present disclosure to expand the CSCs, thereby forming a CSC line.

[0009] In some specific embodiments of the present disclosure, the CSC line is a stable tumorigenic CSC line for long-term culture. The CSC screening step includes four stages: a first stage, tumorigenicity screening; a second stage, self-proliferation screening; a third stage, therapy resistance screening; and a fourth stage, single-cell recurrence screening. In a preferred embodiment of the present disclosure, in the tumorigenicity screening stage, a suspension of parent cancer cells is cultured in a 1-1.5% agar medium by volume to simulate an in vitro tumorigenic environment and select highly tumorigenic cancer cells. In one preferred embodiment of the present disclosure, in the self-proliferation screening stage, 3D growth technology is used to select CSCs that can survive in suspension and form tumorspheres. In a further preferred embodiment of the present disclosure, in the therapy resistance screening stage, a channel blocker is used to block the drug exclusion channel possessed by drug-resistant CSCs. A simulated fluorescent dye is used as a drug to select CSCs that can emit fluorescence. In a further preferred embodiment of the present disclosure, in the single-cell recurrence screening stage, the selected cancer cells are made into a single-cell suspension and cultured in CSC serum-free conditioned medium. A CSC line that can form the largest tumor from individual cells in the shortest time is selected.

[0010] In some specific embodiments of the present disclosure, the CSC line is a stable metastatic CSC line for long-term culture. The CSC screening step includes four stages: a first stage, metastatic CSC screening; a second stage, self-renewal screening; a third stage, therapy resistance screening; and a fourth stage, single-cell recurrence screening. In a preferred embodiment of the present disclosure, the metastatic CSC screening stage simulates transwell invasion as an in vitro tumor metastasis environment to select cancer cells with epithelial-mesenchymal transition (EMT) and high invasive potential. In another preferred embodiment of the present disclosure, the self-renewal screening stage uses 3D growth technology to select CSCs that can survive in suspension and form tumor spheres. In a further preferred embodiment of the present disclosure, the therapy resistance screening stage uses a channel blocker to block the drug exclusion channel possessed by drug-resistant CSCs. A simulated fluorescent dye is used as a drug to select CSCs that can emit fluorescence. In a further preferred embodiment of the present disclosure, the single-cell recurrence screening stage involves forming a single-cell suspension of selected cancer cells and culturing them in a serum-free conditioned medium. A CSC line that can form the largest tumor from individual cells in the shortest time is selected.

[0011] In some specific embodiments of the present disclosure, the CSC line is a stable, radiation-resistant CSC line for long-term culture. The CSC screening step includes four stages: a first stage, radiation resistance screening; a second stage, self-proliferation screening; a third stage, therapy resistance screening; and a fourth stage, single-cell recurrence screening. In a preferred embodiment of the present disclosure, the radiation resistance screening stage involves irradiating cancer cells with radiation (50-60 Gy) 25-30 times to select radiation-resistant cancer cell lines. In another preferred embodiment, the self-proliferation screening stage involves using 3D growth technology to select CSCs that can survive in suspension and form tumorspheres. In a further preferred embodiment of the present disclosure, the therapy resistance screening stage involves using a channel blocker to block the drug exclusion channel possessed by drug-resistant CSCs. A simulated fluorescent dye is used as a drug to select CSCs that can emit fluorescence. In a further preferred embodiment of the present disclosure, the single-cell recurrence screening stage involves forming a single-cell suspension of selected cancer cells and culturing them in serum-free conditioned medium. A CSC line capable of forming the largest tumor from individual cells in the shortest time is selected.

[0012] In a specific embodiment of the present disclosure, the CSC line is a stable drug-resistant CSC line for long-term culture. The CSC screening step includes four stages: a first stage, drug resistance screening; a second stage, self-renewal screening; a third stage, therapy resistance screening; and a fourth stage, single-cell recurrence screening. In one embodiment of the present disclosure, the drug resistance screening stage involves applying a personalized cancer chemotherapy drug to cancer cells 15 to 30 times, preferably 20 times, to select drug-resistant cancer cells. In a preferred embodiment, the drug dose in each round is gradually increased from 100 to 300 nM to 1 to 30 μM. In another preferred embodiment of the present disclosure, the self-renewal screening stage uses a 3D growth technique to select CSCs that can survive in suspension and form tumor spheres. In a further preferred embodiment of the present disclosure, the therapy resistance screening stage uses a channel blocker to block the drug exclusion channel possessed by drug-resistant CSCs. A simulated fluorescent dye is used as a drug to select CSCs that can emit fluorescence. In a further preferred embodiment of the present disclosure, in the single-cell recurrence potential screening step, the selected cancer cells are made into a single-cell suspension and cultured in a serum-free conditioned medium, and a CSC line capable of forming the largest tumor from individual cells in the shortest time is selected.

[0013] In another aspect, the present disclosure provides a two-step method for rapidly selecting clinical CSCs, which includes the following first and second steps: the first step is stem cell gene transcription function screening, in which the octamer-binding transcription factor 4 (OCT4) gene is introduced into a clinical tumor sample using a lentiviral transduction system, and OCT4 gene-positive cancer cells are selected; and the second step is CSC screening, in which cancer cells with CSC properties are selected from OCT4 gene expression-positive cancer cells according to behavioral screening, and then cultured in a CSC serum-free conditioned medium.

[0014] In some specific embodiments of the present disclosure, the OCT4 gene is linked to a reporter gene. In one embodiment, the reporter gene is a fluorescent protein kinase reporter gene, preferably a green fluorescent protein kinase reporter gene. In another embodiment, the reporter gene is a luciferase reporter gene. In a further embodiment, the reporter gene is an antibiotic reporter gene.

[0015] In some specific embodiments of the present disclosure, the CSCs are clinical tumorigenic CSCs, and the CSC screening step comprises a tumorigenicity screening step. In another embodiment, the CSCs are clinical metastatic CSCs, and the CSC screening step comprises a metastatic invasion ability screening step. In a further embodiment, the CSCs are clinical radiation-resistant CSCs, and the CSC screening step comprises a radiation resistance screening step. In a further embodiment, the CSCs are clinical drug-resistant CSCs, and the CSC screening step comprises a drug resistance screening step.

[0016] In another aspect, the present disclosure provides isolated CSC lines obtained by the above-described selection method. In some specific embodiments of the present disclosure, the isolated CSC lines can stably grow and maintain CSC characteristics for four months or more. In one specific embodiment, the isolated CSC lines can stably grow and maintain CSC characteristics for three months or more.

[0017] In another aspect, the present disclosure provides a method for selecting a cancer drug, comprising treating an isolated CSC line of the present disclosure with a candidate drug and testing the inhibitory effect of the candidate drug on the CSC line. In a specific embodiment of the present disclosure, the isolated CSC line is transduced by linking an OCT4 promoter with a reporter gene. In a specific embodiment of the present disclosure, the reporter gene is a green fluorescent protein kinase reporter gene. In a specific embodiment of the present disclosure, the reporter gene is a luciferase reporter gene. In a specific embodiment of the present disclosure, the reporter gene is an antibiotic reporter gene. [Brief explanation of the drawings]

[0018] [Figure 1] This is a schematic diagram showing the change in the percentage of CD44-positive cells during the culture period. CSCs isolated by a conventional CD44 antibody screening method and CSCs isolated by the method of the present disclosure are cultured in a conventional cell culture medium or a serum-free conditioned medium for at least 100 days. The cultured CSCs are sampled at regular intervals, and the percentage of the cell population with high CD44 antigen expression among the cultured CSCs is examined. [Figure 2A] 1 shows a tumorigenic CSC line (PANC-1 TCSC) for long-term culture obtained by applying the quadruple sorting method provided by the present disclosure to parental pancreatic cancer cells. [Figure 2B] Soft agar cell colonies grown during the tumorigenicity screening step are shown. [Figure 2C] 1 shows tumor spheres formed during the self-renewal screening step. [Figure 2D] FIG. 1 shows the results of sorting side population cells obtained in the treatment resistance screening step. [Figure 2E] The largest single-cell colony selected in the single-cell recurrence potential screening step is shown. [Figure 3]This is a schematic diagram comparing the in vitro tumorigenicity of parental pancreatic cancer cells with that of isolated stem cell lines, using a soft agar assay to simulate an in vitro tumorigenic environment. Cells were seeded in 3x soft agar and cultured for 14 days. Cell colony formation was observed. The left panel shows the parental (PT) cancer cells, and the right panel shows the tumorigenic cancer stem cell (TCSC) line. [Figure 4A] 1 shows a tumorigenic CSC line (DLD-1 TCSC) for long-term culture obtained by applying the quadruple selection method of the present disclosure to parent colon cancer cells. [Figure 4B] Soft agar cell colonies formed during the tumorigenicity screening step are shown. [Figure 4C] 1 shows tumor spheres formed during the self-renewal screening step. [Figure 4D] FIG. 1 shows the results of sorting side population cells obtained in the treatment resistance screening step. [Figure 4E] The largest single-cell colony selected in the single-cell recurrence potential screening step is shown. [Figure 5A] 1 shows a metastatic CSC line (PANC-1 MCSC) for long-term culture obtained by applying the quadruple sorting method of the present disclosure to parental pancreatic cancer cells. [Figure 5B] This shows cell colonies with high invasive potential selected using transwell invasion in the metastatic potential screening step. [Figure 5C] 1 shows tumor spheres formed during the self-renewal screening step. [Figure 5D] FIG. 1 shows the results of sorting side population cells obtained in the treatment resistance screening step. [Figure 5E] The largest single-cell colony selected in the single-cell recurrence potential screening step is shown. [Figure 6] Schematic diagram showing the metastatic potential of cell lines observed using a migration assay, comparing the metastatic potential of parental cancer cells with that of isolated stem cell lines within 24 hours. [Figure 7A]1 shows a metastatic CSC line (DLD-1 MCSC) for long-term culture obtained by applying the quadruple sorting method of the present disclosure to parent colon cancer cells. [Figure 7B] This shows cell colonies with high invasive potential selected using transwell invasion in the metastatic potential screening step. [Figure 7C] 1 shows tumor spheres formed during the self-renewal screening step. [Figure 7D] FIG. 1 shows the results of sorting side population cells obtained in the treatment resistance screening step. [Figure 7E] The largest single-cell colony selected in the single-cell recurrence potential screening step is shown. [Figure 8] Schematic diagram showing the percentage of a cell population with high CD44 levels among all cultured CSCs cultured for 35 days. CSCs isolated by a conventional CD44 antibody screening method and CSCs isolated by the method disclosed herein were cultured in conventional cell culture medium or serum-free CSC conditioned medium, respectively. The cultured CSCs were examined on day 35 to determine the percentage of CSCs with high CD44 levels among all cultured CSCs. [Figure 9A] FIG. 1 is a schematic diagram showing the levels of stem cell-related genes in CSCs after one month of culture. [Figure 9B] This is a schematic diagram showing the levels of epithelial-mesenchymal transition (EMT)-related genes in CSCs after one month of culture. CSCs isolated by a conventional CD44 antibody screening method and CSCs isolated by the disclosed method were cultured for one month in conventional cell culture medium or serum-free CSC conditioned medium, respectively. The relative RNA expression levels of stem cell-related genes, such as OCT4, Sox2, Klf4, c-Myc, Nanog, and Lin28, and EMT-related genes, such as Twist, Snail, Slug, Zeb1, E-cad, N-cad, Vim, and FN1, were then quantitatively analyzed. DETAILED DESCRIPTION OF THE INVENTION

[0019] Other features and advantages of the present disclosure are further illustrated and explained in the following examples, which are used only as auxiliary explanations and are not intended to limit the scope of the present disclosure.

[0020] First embodiment: Formation and preparation of CSC serum-free conditioned medium This disclosure provides a serum-free CSC conditioned medium for screening and culturing highly pure and stable CSCs. The medium contains a basal medium and a combination of supplements added to the basal medium. The supplement combination primarily contains 10-30% by volume of serum replacement, 1-5 mM pyruvate, 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 lactate, 2-10 μM ROCK inhibitor, and 0.2-10 ng / ml TGF-β1. The serum-free conditioned medium was prepared by mixing all medium components in a sterile serum bottle on a sterile work bench and then filtering the mixture through a 0.45 μm sterile filter into a sterile serum bottle.

[0021] Table 1 below shows the final concentrations of reagents added to the basal medium of the CSC serum-free conditioned medium (selected from DMEM, DMEM / F12, RPMI 1640, MEM, etc.).

[0022] [Table 1]

[0023] KnockOut TM Serum replacement (KnockOut TM KnockOut SR is a serum-free supplement that supports the growth of pluripotent stem cells (PSCs) on fibroblast feeder layers. TM SR is suitable for serum-free nutrition and culture of multiple types of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and can directly replace fetal bovine serum (FBS) in conventional laboratory solutions.

[0024] Pyruvate (also known as L-alanyl-L-glutamine, Ala-Glu, or GlutaMAX) is an advanced cell culture additive that can directly replace the essential nutrient L-glutamine in cell culture. During cell culture, cells gradually release peptidases into the medium, which hydrolyze L-alanyl-L-glutamine to L-alanine and L-glutamine. The cells then absorb and utilize these hydrolysis products.

[0025] MEM non-essential amino acid (NEAA) solution is derived from the MEM medium formulation and contains seven non-essential amino acids: L-alanine, L-glutamine, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine. NEAA effectively improves the medium composition, reduces the side effects of non-essential amino acids produced by cells during cell culture, and promotes cell growth and metabolism. NEAA is a commonly used additive in cell culture.

[0026] The chemically defined lipid concentrate is a concentrated lipid emulsion with a chemically defined composition. This media supplement is designed to reduce or replace fetal bovine serum in cell culture media. The chemically defined lipid concentrate 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 the expression of protein kinases in insect cell viruses.

[0027] SPITE Liquid Media Supplement is a multifunctional cell additive used to create serum-free media. Purified substances in SPITE Liquid Media Supplement can replace components normally provided by serum, such as insulin, transferrin, selenium, pyruvate, and ethanolamine.

[0028] ROCK inhibitors are selective ATP-competitive inhibitors of Rho-associated protein kinase (ROCK). ROCK inhibitors also inhibit myosin phosphorylation and Ca 2+ROCK inhibitors may be effective inhibitors of sensitization-induced smooth muscle contraction. Examples of ROCK inhibitors include Y-27632, fasudil, and H-1152. Cultivating human embryonic stem cells (hES) in serum-free suspension medium (SFEB) with 10 μM Y-27632 significantly reduced dissociation-induced apoptosis and improved sorting efficiency (from approximately 1% to approximately 27%).

[0029] CSCs isolated by the quadruple sorting method of the present disclosure or by the conventional CD44 antibody screening method were cultured in conventional cell culture media (e.g., DMEM + 10% FBS) or the serum-free CSC conditioned medium of the present disclosure under standard culture conditions (37°C, 5% CO2, 21% O2) for at least 100 days. The cultured CSCs were sampled at regular intervals, and the proportion of cells with high CD44 levels among the cultured CSCs was examined.

[0030] The results are shown in Figure 1. Regardless of whether the CSCs were obtained by the disclosed quadruple sorting method or the conventional CD44 antibody differentiation method, when cultured in the disclosed serum-free CSC conditioned medium, the differentiation of the CSCs was effectively delayed compared to when cultured in conventional cell culture media. The CSCs obtained by the disclosed quadruple sorting method maintained the CD44 surface antigen characteristic even after long-term culture for at least 100 days. However, the CSCs obtained by the conventional CD44 antibody isolation method began to differentiate around day 5 of culture. The CSCs gradually lost their CSC characteristics and were unable to proliferate.

[0031] Second embodiment: Selection and generation of tumorigenic cancer stem cell (TCSC) lines This embodiment provides a quadruple selection method for CSC lines for long-term culture, including a first stage: tumorigenicity screening, a second stage: self-renewal screening, a third stage: therapy resistance screening, and a fourth stage: single-cell recurrence screening. In the first stage, parental pancreatic cancer cells are detached from the culture plate using trypsin and suspended in fresh medium to form a single-cell suspension (see Figure 2A). After centrifugation, the single-cell suspension is resuspended in 500–1000 μl of 5% FBS fresh medium. 1 ml of 1–1.5% agar is added to each well of a 6-well plate and allowed to stand for 3–5 minutes to solidify. 666 μl of the cell suspension and 333 μl of 1–1.5% agar (3-fold dilution) are added to each well, and the plate is gently shaken to mix the cell suspension and agar. The plate is then allowed to stand for 3–5 minutes to solidify the agar (the cell suspension is added to the well first to prevent the agar from solidifying). Then, 1 ml of 5% FBS medium was added to each well, and the plate was placed in a 37°C low-oxygen (5% O2) incubator for incubation. After 1-2 weeks of incubation, cell colonies were removed from the soft agar using a 1 ml tip. The cell colonies were transferred to a 6-well plate and temporarily grown to provide sufficient cells for the next round of selection. Cells were then seeded for further soft agar selection. The above soft agar selection was repeated five times. To select cells with high tumorigenicity, the number of inoculated cells was increased to 10. 6 , 10 5 , 10 4 , 10 3 From 10 2 The concentration was gradually reduced to 0.05, and five rounds of selection were performed. As shown in Figure 2B, in the first stage, soft agar was used to simulate an in vitro tumorigenic environment, and cancer cells with high tumorigenic potential were selected.

[0032] Then, in the second stage, 1 × 10 4The cells were dispersed into individual cells. Individual cells were seeded into 6-well plates covered with 11.5% agar and containing serum-free medium. The 6-well plates were cultured in a low-oxygen (5% O2) incubator at 37°C. Cells that could not adhere to the soft surface formed tumorspheres in suspension culture after several days. The tumorspheres were dissociated into individual cells using a volumetric pipette every four days to form new tumorspheres. Cells capable of forming new tumorspheres possess anoikis resistance and long-term self-proliferation capabilities. After 12 weeks of culture, the number of tumorspheres was recorded, and cell colonies were transferred to new 6-well plates for temporary expansion to provide sufficient cells for the next selection step. This tumorsphere selection process was repeated three times. To select cells with high tumorigenicity, the number of inoculated cells was increased to 10. 4 , 10 3 From 10 2 The concentration was gradually reduced to 0.05% and selection was performed three times. In the second stage, a 3D expansion technique was used, as shown in Figure 2C. Under suspension conditions, CSCs with stem cell properties assume a spherical shape (tumorsphere) resembling a blastocyst. This environment simulates the intravascular tumor situation, and only stem cells survive and are therefore selected.

[0033] Then, in the third stage, at least 5 × 10 6 cells (maximum 10 7 A 5 ml cell suspension was prepared and suspended in 5 ml of medium. The 5 ml cell suspension was divided into two portions, 4 ml and 1 ml, and each portion was placed in a blue sorting tube with a cap. 100 μM verapamil (Sigma-Aldrich, St. Louis, MO) and 10 μM fumitremordin C (FTC, Sigma-Aldrich) were added to the sorting tube containing 1 ml of cell suspension, and the sorting tube was incubated at 37°C for 5 minutes, protected from light. 4 μl and 1 μl of Hoechst 33342 dye (5 μM, Sigma-Aldrich, St. Louis, MO) were added to the sorting tube containing 4 ml and the sorting tube containing 1 ml of cell suspension, respectively. The tubes were then placed in a 37°C incubator for 90 minutes, during which time the tube was gently tapped every 15 minutes.

[0034] After washing the cells twice with PBS, 2 ml of medium containing propidium iodide (PI) (2 μg / ml, Sigma-Aldrich, for dead cell exclusion) was added to the tubes for resuspension of drug-untreated cells (without verapamil and FTC) and drug-treated cells (with verapamil and FTC), respectively. The cell suspensions were then subjected to a selection process. Capped test tubes containing 1 ml of medium containing 0.2% PSA were prepared and covered with paraffin film. Cell sorting was performed using a BD FACSAria flow cytometer (BD Biosciences). Upon excitation with a UV488 laser, the primary cell population containing Hoechst 33342 emitted blue and red fluorescence. A portion of the side population cells emitted lower fluorescence due to efflux of Hoechst dye via ABC transporters. Adding verapamil and FTC to prevent efflux of the fluorescent dye resulted in high fluorescence in the side population cells. Side population cells can be sorted by comparing the parameters obtained with and without verapamil and FTC. The isolated cells were temporarily grown in a 6-well plate in a low-oxygen (5% O2) incubator at 37°C to provide sufficient cells for the next selection step. As shown in Figure 2D, in the third step, a fluorescent dye (Hoechst dye) is simulated as a drug. CSCs possess drug efflux channels to excrete the dye. Therefore, the drug resistance of CSCs can be selected using a channel blocker (verapamil).

[0035] Finally, in the fourth step, cells were seeded into round-hole ultra-low attachment 96-well plates using serum-free medium. The plates were cultured in a 37°C, low-oxygen (5% O2) incubator. Cells that formed the largest 3D cell colonies, approximately 250-300 μm in diameter, in the shortest time were selected. These cells were then cultured in the CSC serum-free conditioned medium of the present disclosure, as described in the first embodiment, and cultured long-term to obtain stable pancreatic CSC lines. As shown in Figure 2E, the fourth step simulates the ability of a small number of tumor cells to form new tumors after treatment. CSC lines that could form the largest tumors from single cells in the shortest time were selected. The selected CSC lines were then cultured in the CSC serum-free conditioned medium of the present disclosure, as described in the first embodiment. The selected CSCs were cultured long-term, resulting in a stable pancreatic tumorigenic CSC line (PANC-1 TCSC), as shown in Figure 3. The pancreatic CSC line obtained by the present disclosure has high tumorigenicity.

[0036] The colony morphology at each stage of the colon tumorigenic CSC line (DLD-1 TCSC) obtained from parent colon cancer cells according to the above-mentioned quadruple selection method is shown in Figures 4A to 4E.

[0037] Third embodiment: Selection and generation of metastatic cancer stem cell (MCSC) lines This embodiment provides a quadruple selection method for the long-term culture of stable metastatic CSC lines. It includes a first step for screening for metastatic invasion potential, a second step for screening for self-renewal potential, a third step for screening for therapeutic resistance, and a fourth step for screening for single-cell recurrence potential. In the first step, parental pancreatic cancer cells were detached from the culture plate using trypsin and suspended in fresh medium to form a single-cell suspension (shown in Figure 5A). After centrifugation, the parental pancreatic cancer cells were suspended in 500–1000 μl of 5% FBS fresh medium. 80 μl of Matrigel (Corning, NY) was added to the upper chamber (a 1:2 dilution ratio of Matrigel to serum-free conditioned medium). After the Matrigel solidified, 600 μl of 10% FBS fresh medium was added to the lower chamber. 200 μl of the cell suspension was added to the upper Matrigel layer. The transwell plate was placed in a 37°C low-oxygen (5% O2) incubator. The cells began to invade from the upper chamber to the lower chamber. After 1–2 weeks, the cells in the lower chamber were transferred to a 6-well plate for transient growth to provide sufficient cells for the next round of selection. The cells were then subjected to another screening for metastatic invasion potential. To select cells with high metastatic invasion potential, the above steps were repeated five times, with the number of seeded cells increased to 10. 6 , 10 5 , 10 4 , 10 3 From 10 2 was gradually reduced to

[0038] Next, the second to fourth screening steps described in the second embodiment are carried out. The selected CSCs are cultured in the serum-free CSC conditioned medium of the present disclosure described in the first embodiment. After long-term culture, a stable pancreatic metastatic CSC line (PANC-1 MCSC) is obtained. Figures 5B to 5E show the colony morphology at each screening stage. As shown in Figure 6, the metastatic cancer stem cell line obtained from the present disclosure has high metastatic potential.

[0039] The colony morphology at each stage of the colon tumorigenic CSC line (DLD-1 TCSC) obtained from parent colon cancer cells according to the above-described quadruple sorting method is shown in Figures 7A to 7E.

[0040] Fourth embodiment: Selection and formation of resistant CSC lines This embodiment provides a quadruple screening method for the stable long-term culture of resistant CSCs. It includes a first stage: screening for resistance (radiation resistance or drug resistance), a second stage: screening for self-renewal, a third stage: screening for treatment resistance, and a fourth stage: screening for single-cell recurrence. To screen for radioresistant CSCs, in the first stage, cancer cells were irradiated with an Elekta Synergy radiotherapy system (Elekta, Sweden) at a dose rate of 562 MU / min at a dose of 2 Gy / day. After 30 irradiations, radioresistant cancer cell lines were selected. Cells were cultured in a hypoxic (5% O2) incubator at 37°C. Cells were seeded at one cell per well in a 96-well plate using serum-free medium. Individual cells were irradiated with an Elekta Synergy radiotherapy system (Elekta, Sweden) at a dose rate of 562 MU / min at a dose of 10 Gy / day. The largest cell colonies formed in the shortest time were selected. The plates were placed in a 37°C low oxygen (5% O2) incubator for temporary growth to provide enough cells for the next round of selection.

[0041] To screen for chemotherapy-resistant CSCs, in the first stage, cancer cells were exposed to 20 rounds of chemotherapy drugs, with the drug dose gradually increasing from 100–300 nM to 1–30 μM. The cells were stored in a 37°C hypoxic (5% O2) incubator. Hematological agents were used to generate chemotherapy-resistant cells. Table 2 below lists the standard chemotherapy agents used to generate different types of chemotherapy-resistant cancer cells.

[0042] [Table 2]

[0043] After 20 rounds of chemotherapy drug selection, cells were seeded into 96-well plates at one cell per well. Individual cells were cultured with the highest dose of chemotherapy drug. The largest cell colonies formed in the shortest time were selected. Plates were placed in a 37°C low-oxygen (5% O2) incubator for temporary growth to provide enough cells for the next round of selection.

[0044] Thereafter, the second to fourth selection steps described in Embodiment 2 are carried out. Next, the selected cancer stem cells are cultured for a long period of time in the CSC serum-free conditioned medium of the present disclosure described in Embodiment 1. This allows stable radioresistant CSCs and drug-resistant CSCs to be obtained. Fifth embodiment: Establishment of a clinical CSC platform for rapid screening This embodiment provides a two-step method for rapid screening of clinical CSCs, including a stem cell gene transcription function screening step and a CSC behavior screening step. In the first step, a clinical tumor sample is transfected with the octamer-binding transcription factor 4 (OCT4) gene using a lentiviral transduction system, and multiple cancer cells positive for OCT4 gene expression are selected. This allows the stemness of cancer cells to be identified.

[0045] To screen for clinical tumor-initiating stem cells, in the second step, 1 ml of 1–1.5% agar was added to each well of a 6-well plate and allowed to stand for 3–5 min to solidify the agar. Then, 666 μl of the cell suspension (10 3 ~10 4 OCT4-GFP-positive cancer cells and 333 μl of 1-1.5% agar (3-fold dilution) were added to each well, and the plate was gently shaken to mix. The plate was then left to stand for 3-5 minutes to allow the agar to solidify. The largest cells formed in the shortest time were selected and cultured in the CSC serum-free conditioned medium of the present invention.

[0046] To screen for clinical metastatic CSCs, in the second step, 80 μl of Matrigel (Corning, NY) was added to the upper chamber (Matrigel was diluted 1:2 with serum-free medium). After the Matrigel solidified, 600 μl of 10% FBS fresh medium was added to the lower chamber. 200 μl of cell suspension was added to the upper Matrigel layer. The transwell plate was placed in a 37°C low-oxygen (5% O2) incubator. Cells began to invade from the upper chamber to the lower chamber. Metastatic CSCs were cultured in the disclosed CSC serum-free conditioned medium.

[0047] In the second stage, to screen for clinical radiation-resistant CSCs, OCT4-GFP-positive cancer cells were cultured in serum-free medium at 10 s.p.m. in a 96-well plate. 3 ~10 4 The cells were seeded at a density of 1000 cells / well. Individual cells were irradiated with a dose of 10 Gy / day at a dose rate of 562 MU / min using an Elekta Synergy radiotherapy device (Elekta, Sweden). The largest cell colonies formed in the shortest time were selected. The selected radioresistant cancer cells were cultured in the CSC serum-free conditioned medium of the present disclosure.

[0048] To screen for clinically resistant CSCs, the second step involves generating chemotherapy-resistant CSCs using standard chemotherapy drugs. OCT4-GFP-positive cancer cells were cultured in serum-free medium at 10 ng / ml in a 96-well plate. 3 ~10 4 The cells were seeded at a density of 1000 cells / well. Individual cells were treated with the highest dose of chemotherapy. The largest cell colony formed in the shortest time was selected. The selected chemotherapy-resistant cancer cells were cultured in the CSC serum-free conditioned medium of the present disclosure.

[0049] The two-step selection method of the present disclosure allows for rapid selection of CSCs from clinical specimens. Furthermore, the selected cancer stem cells can serve as a platform for screening effective drugs. In this embodiment, if a candidate drug can target CSCs and reduce their stemness, OCT4-GFP-positive CSCs will lose fluorescence. Therefore, the therapeutic effects of candidate drugs on different types of CSCs can be evaluated.

[0050] Sixth embodiment: Evaluation of purity and characteristics of CSC lines isolated using the disclosed quadruple selection method The CSC lines formed in the second and third embodiments and the CSC line isolated using a commercially available CD44 antibody were seeded in a general cell culture medium and the serum-free conditioned medium of the present disclosure described in the first embodiment, respectively, and cultured for 35 days in a low-oxygen (5% O2) incubator at 37°C. The cells were then sorted by CD44 surface antigen, and the proportion of cells maintaining high CD44 surface antigen expression among all cultured cells was detected.

[0051] The results are shown in Figure 8. Compared with CSC lines isolated using conventional antibody selection methods (CD44 antibody selection methods), CSC lines isolated using the quadruple selection method of the present disclosure are able to express stem cell antigens more stably and for a longer period of time. Furthermore, when cultured in the serum-free CSC conditioned medium of the present disclosure, CSC differentiation is effectively delayed compared with when cultured in conventional cell culture media. This effect is more pronounced when the quadruple selection method of the present disclosure is applied.

[0052] As shown in Figures 9A and 9B, the CSC lines after one month of culture were further tested for stem cell genes (OCT4, Sox2, Klf4, c-Myc, Nanog, and Lin28 genes) and epithelial-mesenchymal transition (EMT) genes (Twist, Snail, Slug, Zeb1, E-cadherin, N-cadherin, vimentin, and FN1 genes). The CSC lines of the present disclosure maintained high expression of stem cell genes (Figure 9A) and EMT genes even after one month of culture.

[0053] Seventh embodiment: Use of CSC lines as a drug screening platform This embodiment demonstrates various scenarios in which CSC lines isolated by this disclosure can be used as a drug screening platform. To easily observe and identify the stemness state of CSCs in vitro or in vivo, isolated CSC lines are transduced with the OCT4-GFP or OCT4-Luc reporter gene using a lentivirus-based transduction system. If a candidate drug effectively reduces the stemness of CSCs, the OCT4-GFP- or OCT4-Luc-expressing CSCs lose fluorescence or luminescence (bioluminescence). The reporter gene is not limited to the GFP or Luc gene; other antibiotic resistance genes can also be used.

[0054] One example is forming tumor spheres from isolated CSC lines using a 3D culture method, treating them with nanodrugs, and observing whether the nanodrugs effectively target and inhibit tumors. CSC lines can also be used as a platform to evaluate the therapeutic efficacy of drug conjugates. For example, tumor-initiating or metastatic CSCs expressing OCT4-GFP or OCT4-Luc can be treated with a combination of known chemotherapeutic drugs and new drugs (e.g., Drug A, Drug B, or Drug C). By comparing the inhibitory effects of the drug conjugates on parental cancer cells with those on CSCs, the therapeutic efficacy of the drug conjugates and the therapeutic efficacy of new drugs against drug-resistant cancers can be evaluated.

[0055] To screen drugs in vivo, tumorigenic CSC lines can be injected into zebrafish or mice to generate tumors and determine whether candidate drugs can effectively target and treat tumors, and metastatic CSC lines can be injected into zebrafish or mice to determine whether candidate drugs can effectively inhibit tumor metastasis.

[0056] This disclosure successfully developed a CSC medium that can be used to screen and generate large numbers of CSC lines (capable of significant proliferation by millions of times) with high purity (stem cell gene expression several tens of times higher than that of competing products) and stability (effectively extending storage life by hundreds of times). The isolated CSC lines are available to pharmaceutical companies and research institutions, accelerating the development of CSC-targeted drugs to effectively solve the current challenges in cancer treatment.

Claims

1. 1. A method of delaying differentiation of a cancer stem cell (CSC) line, comprising culturing the CSC line in serum-free conditioned medium; The serum-free conditioned medium comprises: A basal medium; Includes a combination of supplements, The basal medium is selected from DMEM medium, DMEM / F12 medium, RPMI 1640 medium, or MEM medium; The combination of supplements comprises 10-30% by volume serum replacement, 1-10 mM pyruvate, 2-20 μM mpROCK inhibitor, and 0.2-10 ng / ml TGF-β1, 5-20 ng / ml fibroblast growth factor (FGF), 0.1-5.0 mM MEM non-essential amino acid solution, 20-100 μg / ml L-ascorbic acid, 5-20 mM lactic acid, 40-70 U / ml penicillin, 40-70 μg / ml streptomycin, 0.05-0.15 mM 2-mercaptoethanol, 1000-fold diluted lipid concentrate, and 100-fold diluted SPITE medium supplement.

2. The method of claim 1 , wherein the CSC line is a tumorigenic CSC line.

3. The method of claim 1 , wherein the CSC line is a metastatic CSC line.

4. The method of claim 1 , wherein the CSC line is a radioresistant CSC line.

5. The method of claim 1 , wherein the CSC line is a drug-resistant CSC line.

6. A method for selecting an anticancer drug, comprising: Culturing the drug candidate with a cancer stem cell (CSC) line; testing the inhibitory effect of the candidate drug on the CSC line; The CSC line is cultured according to the method of claim 1.

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