Cancer stem cell serum-free conditioned medium, and use thereof in a quadruple selecting method for cancer stem cell line, and the isolated cancer stem cell line

Abstract

The present disclosure relates to a cancer stem cell (CSC) serum-free conditioned medium, which is characterized by comprising a basal medium, and a supplement composition, mainly comprising a serum substitute, alanyl-glutamine, a ROCK inhibitor and TGF-β. The present invention also provides a multi-stage screening method for the establishment of a stable and proliferating cancer stem cell line by using the serum-free conditioned medium, and cancer stem cell lines obtained by such method. The isolated cancer stem cell line has highly purity, can be significantly expanded to million cells more than that obtained by conventional screening methods, and can maintain CSC sternness stably over a long period of time in vitro.

Description

CANCER STEM CELL SERUM-FREE CONDITIONED MEDIUM, AND USETHEREOF IN A QUADRUPLE SELECTING METHOD FOR CANCER STEM CELL LINE, AND THE ISOLATED CANCER STEM CELL LINEFIELD

[0001] The present disclosure relates to methods of screening and culturing a plurality of cancer stem cells (CSCs). More specifically, the present disclosure pertains to a conditioned medium for selecting and culturing the CSCs, as well as a quadruple selecting technique platform using the conditioned medium to isolate a stable CSC line from the CSCsBACKGROUND

[0002] Most cancer cells (approximately 98% or more) lack tumorigenicity, drug resistance, and metastatic ability. However, there is a very small proportion (0.01-2%) of cancer cells that possess self-renewal, proliferation, and multi-lineage differentiation potential. This population of cancer cells is defined as cancer stem cells (CSCs) resulting in drug resistance, recurrence, and metastasis of tumors. If CSCs can be separated from regular cancer cells and can be cultured to establish a reliable tumor model, functional characteristics, tumor formation mechanisms, and tumor formation metastasis of the CSCs can be elucidated. Thus, CSC population can be targeted and eliminated. For researchers studying CSCs, this is one of reliable strategies for effectively treating malignant tumors.

[0003] There are three common methods for isolating CSCs, namely a culture-based method, magnetic cell sorting (MACS), and fluorescence-activated cell sorting (FACS). The culture-based method is based on growth characteristics of the CSCs. Differentiated tumor cells die under serum-free and non-adherent culture conditions, and the CSCs survive and proliferate to form floating tumor spheres.

[0004] The CSCs express specific surface markers such as CD44, CD133, etc., which can be identified by using various techniques such as flow cytometry, immunocytochemistry, immunofluorescence, and western blotting (WB). Conventional surface antigen screening methods use fluorescent antibodies targeting stem cell antigens, such as CD44, CD24, and CD133, to sort CSCs, which is quick. However, different CSCs have different surface antigens. Therefore, cancer with unknown specific surface antigens cannot be sorted, and it is difficult to sort and identify tumorigenic CSCs and metastatic CSCs. In addition, cost of antibodies for screening is high, and a screening process must be repeated. Only a small number of CSCs (hundreds or thousands) can be sorted, and the CSCs undergo differentiation in a short time (approximately five days). Consequently, characteristics of the CSCs cannot be sustained, and proliferation of the CSCs is not stable. As a result, the CSCs isolated by traditional methods are easy to differentiate and unstable because they are obtained by conventional antibody-based screening methods. The CSCs lose their characteristics after being cultivated for a few days.

[0005] To solve the above issue, the present disclosure develops a conditioned medium for screening and cultivating CSCs. A screening technique platform, which allows for rapid testing according to different requirements and realizes unlimited culture of CSCs having high purity and stability, is established based on behavior screening of the CSCs.SUMMARY

[0006] In one aspect, the present disclosure relates to a CSC serum-free conditioned medium, including a base culture medium and a supplement combination added to the base culture medium. The supplement combination includes 10-30 vol% of serum substitute, 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 supplement combination furtherincludes 5-20 ng / ml fibroblast growth factor (FGF), 0.1-5.0 mM MEM non-essential amino acid solution, 20-100 pg / ml L-ascorbic acid, and 5-20 mM lactate. In one preferred embodiment of the present disclosure, the supplement combination further includes 15-25 vol% of KnockOut serum replacement, 2-5 mM L-alanyl-L-glutamine, 0.5-2 mMMEM non-essential amino acid solution, 5-15 ng / ml fibroblast growth factor, 20-60 pg / ml L-ascorbic acid, 5-15 mM lactic acid, 5-10 μM rho-associated protein kinase (ROCK) inhibitor, and 1-5 ng / ml TGF-β1.

[0007] In one specific embodiment of the present disclosure, the base culture medium may be any known culture medium used for cell culture, including, but not limited to, DMEM, DMEM / F12, RPMI 1640, MEM, etc. In one specific embodiment of the present disclosure, the base culture medium and the supplement combination further include a 1000X dilution of lipid concentrate with explicit chemical components and a 100X dilution of SPITE culture medium supplement.

[0008] In another aspect, the present disclosure provides a method of forming a CSC line, including: a CSC screening step, where CSCs with stem cell characteristics are selected from a parent cancer cell population according to behavior screening. Then, selected CSCs are cultured in the serum-free conditioned medium of the present disclosure to proliferate 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: first stage, tumorigenicity screening; second stage, self-renewal ability screening; third stage, treatment resistance screening; and fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present disclosure, in the tumorigenicity screening stage, suspension of the parent cancer cell is cultivated in a 1- 1.5 vol% agar culture medium to simulate an in vitro tumor-forming environment, thereby selecting highly tumorigenic cancer cells. In one preferred embodiment of thepresent disclosure, in the self-renewal ability screening stage, a 3D proliferation technique is used to select CSCs able to survive and form a tumor sphere in a suspension state. In yet another preferred embodiment of the present disclosure, in the treatment resistance screening stage, a channel blocker is used to block a drug exclusion channel which CSCs having drug resistance have. CSCs able to generate fluorescence are selected by using a fluorescent dye which is simulated as a drug. In yet another preferred embodiment of the present disclosure, in the single-cell recurrence ability screening stage, the selected cancer cells are made into single-cell suspension which is cultivated in the CSC serum-free conditioned medium. A CSC line able to form a largest tumor from an individual cell in a 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: first stage, metastatic CSC screening; second stage, self-renewal ability screening; third stage, treatment resistance screening; and fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present disclosure, in the metastatic CSC screening stage, a transwell invasion is simulated as an in vitro tumor metastasis environment, thereby selecting cancer cells having epithelial-mesenchymal transition (EMT) ability and high invasion ability. In another preferred embodiment of the present disclosure, in the self-renewal ability screening stage, a 3D proliferation technique is used to select CSCs able to survive and form a tumor sphere in a suspension state. In yet another preferred embodiment of the present disclosure, in the treatment resistance screening stage, a channel blocker is used to block a drug exclusion channel that CSCs with drug resistance have. CSCs able to generate fluorescence are selected by using a fluorescent dye which is simulated as a drug. In yet another preferred embodiment of the present disclosure, in the single-cell recurrence ability screening stage, the selected cancer cells are made into single-cell suspension which is cultivatedin the serum-free conditioned medium. A CSC line able to form a largest tumor from an individual cell in a 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: first stage, radiation resistance screening; second stage, self- renewal ability screening; third stage, treatment resistance screening; and fourth stage, single-cell recurrence ability screening. In a preferred embodiment of the present disclosure, in the radiation resistance screening stage, cancer cells are irradiated with radiation (50-60 Gy) 25-30 rounds to select cancer cell lines with radiation resistance. In another preferred embodiment of the present disclosure, in the self-renewal ability screening stage, a 3D proliferation technique is used to select CSCs able to survive and form a tumor sphere in a suspension state. In yet another preferred embodiment of the present disclosure, in the treatment resistance screening stage, a channel blocker is used to block a drug exclusion channel which CSCs having drug resistance have. CSCs able to generate fluorescence are selected by using a fluorescent dye which is simulated as a drug. In yet another preferred embodiment of the present disclosure, in the single-cell recurrence ability screening stage, the selected cancer cells are made into single-cell suspension which is cultivated in the serum-free conditioned medium. A CSC line able to form a largest tumor from an individual cell in a 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: first stage, drug resistance screening; second stage, self-renewal ability screening; third stage, treatment resistance screening; and fourth stage, single-cell recurrence ability screening. In one embodiment of the present disclosure, in the drug resistance screening stage, individualized chemotherapy drugs for cancers are applied to cancer cells 15-30 rounds, preferably 20 rounds, to select drug-resistant cancer cells.In a preferred embodiment, drug doses for each round are gradually increased from 100-300 nM to 1-30 μM. In another preferred embodiment of the present disclosure, in the self-renewal ability screening stage, a 3D proliferation technique is used to select CSCs able to survive and form a tumor sphere in a suspension state. In yet another preferred embodiment of the present disclosure, in the treatment resistance screening stage, a channel blocker is used to block a drug exclusion channel which CSCs having drug resistance have. CSCs able to generate fluorescence are selected by using a fluorescent dye which is simulated as a drug. In yet another preferred embodiment of the present disclosure, in the single-cell recurrence ability screening stage, the selected cancer cells are made into single-cell suspension which is cultivated in the serum-free conditioned medium. A CSC line able to form a largest tumor from an individual cell in a shortest time is selected.

[0013] In another aspect, the present disclosure provides a two-stage method for rapidly selecting a clinical CSC, including: first stage, stem cell gene transcription functional screening, where a clinical tumor sample is transduced with an octamer- binding transcription factor 4 (OCT4) gene by using a lentiviral transduction system to select cancer cells positive for the OCT4 gene; and second stage, CSC screening, where cancer cells having CSC characteristics are selected from the cancer cells positive for the OCT4 gene expression according to behavior screening, and are cultivated in the CSC serum-free conditioned medium.

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

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

[0016] In another aspect, the present disclosure provides a separated CSC line obtained by the selecting methods described above. In some specific embodiments of the present disclosure, the separated CSC line can be stably proliferated and sustain CSC characteristics for more than 4 months. In one specific embodiment, the separated CSC line can be stably proliferated and sustain CSC characteristics for more than 3 months.

[0017] In another aspect, the present disclosure provides a method of selecting a cancer drug, including treating the separated CSC line of the present disclosure with a candidate drug, and testing an inhibitory effect of the candidate drug on the CSC line. In a specific embodiment of the present disclosure, the separated CSC line is transduced with an OCT4 promoter combined with a reporter gene. In one specific embodiment of the present disclosure, the reporter gene is a green fluorescent protein reporter gene. In one specific embodiment of the present disclosure, the reporter gene is a luciferase reporter gene. In one specific embodiment of the present disclosure, the reporter gene is an antibiotic reporter gene.DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is a schematic diagram showing a percentage change in cells positive for CD44 during culture over an extended period. CSCs separated by a conventional CD44 antibody screening method and CSCs separated by a method of the presentdisclosure are respectively cultivated in conventional cell culture mediums or serum- free conditioned mediums for at least 100 days. The cultivated CSCs are sampled every certain of days, and a percentage of a cell population having a high expression of CD44 antigen in the cultivated CSCs is tested.

[0019] FIG. 2A shows a tumorigenic CSC line (PANC-1 TCSC) for long-term culture obtained by applying a quadruple selecting method provided by the present disclosure to parental pancreatic cancer cells. FIG. 2B shows a soft agar cell colony grown during a tumorigenicity screening stage. FIG. 2C shows a tumor sphere formed during a self-renewal ability screening stage. FIG. 2D shows a sorting result diagram of side population cells obtained during a treatment resistance screening stage. FIG. 2E shows a largest single-cell colony selected during a single-cell recurrence ability screening stage.

[0020] FIG. 3 are schematic diagrams showing a comparison between in vitro tumorigenicity of the parental pancreatic cancer cells and in vitro tumorigenicity of a separated steam cell line, where a soft agar assay is used to simulate an in vitro tumor- forming environment. Cells are seeded in 3-fold diluted soft agar and cultured for 14 days. Formation of cell colonies is observed. A left diagram shows the parental (PT) cancer cell, and a right diagram shows the tumorigenic cancer stem cell (TCSC) line.

[0021] FIG. 4A shows a tumorigenic CSC line for long-term culture (DLD-1 TCSC) obtained by applying the quadruple selecting method of the present disclosure to parental colorectal cancer cells. FIG. 4B shows a soft agar cell colony formed during the tumorigenicity screening stage. FIG. 4C shows a tumor sphere formed during the self-renewal ability screening stage. FIG. 4D shows the sorting result diagram of side population cells obtained during a treatment resistance screening stage. FIG. 4E shows a largest single-cell colony selected during a single-cell recurrence ability screening stage.

[0022] FIG. 5A shows a metastatic CSC line (PANC-1 MCSC) for long-term culture obtained by applying the quadruple selecting method of the present disclosure to parental pancreatic cancer cells. FIG. 5B shows a cell colony having high invasion ability selected by using a transwell invasion during a metastatic ability screening stage. FIG. 5C shows a tumor sphere formed during the self-renewal ability screening stage. FIG. 5D shows a sorting result diagram of side population cells obtained during the treatment resistance screening stage. FIG. 5E shows a largest single-cell colony selected during the single-cell recurrence ability screening stage.

[0023] FIG. 6 shows a metastatic ability diagram of a cell line observed by using a migration assay, which compares metastatic ability of parental cancer cells and metastatic ability of a separated stem cell line within a 24-hour period.

[0024] FIG. 7A shows a metastatic CSC line for long-term culture (DLD-1 MCSC) obtained by applying a quadruple selecting method of the present disclosure to parental colorectal cancer cells. FIG. 7B shows a cell colony having high invasion ability selected by a transwell invasion during the metastatic ability screening stage. FIG. 7C shows a tumor sphere formed during the self-renewal ability screening stage. FIG. 7D shows a sorting result diagram of side population cells obtained during the treatment resistance screening stage. FIG. 7E shows a largest single-cell colony selected during the single-cell recurrence ability screening stage.

[0025] FIG. 8 is a schematic diagram showing a percentage of a cell population having high level of CD44 in entire cultivated CSCs which have been cultivated for 35 days. CSCs separated by a conventional CD44 antibody screening method and CSCs separated by a method of the present disclosure are respectively cultivated in conventional cell culture mediums or the CSC serum-free conditioned mediums. The cultivated CSCs are tested on day 35 to obtain the percentage of CDs having high level of CD44 in the entire cultivated CSCs.

[0026] FIG. 9A is a schematic diagram showing levels of stemness-related genes in CSCs after one month of culture. Figure 9B is a schematic diagram showing the levels of epithelial-mesenchymal transition (EMT)-related genes in CSCs after one month of culture. CSCs separated by a conventional CD44 antibody screening method and CSCs separated by a method of the present disclosure are respectively cultivated in conventional cell culture mediums or the CSC serum-free conditioned mediums for one month. Then, relative RNA expression levels of the yxymness-related genes, such as OCT4, Sox2, Klf4, c-Myc, Nanog, and Lin28, and relative RNA expression levels of the EMT-related genes, such as, Twist, Snail, Slug, Zebl, E-cad, N-cad, Vim, and FN1, are quantitatively analyzed.DETAILED DESCRIPTION

[0027] Other features and advantages of the present disclosure will be further exemplified and described in the following examples which are only used as auxiliary description and are not intended to limit the scope of the present disclosure.

[0028] First embodiment: formation and preparation of a CSC serum-free conditioned medium

[0029] The present disclosure provides a CSC serum-free conditioned medium for screening and culturing high-purity and high-stability CSCs, including a base culture medium and a supplement combination added to the base culture medium. The supplement combination mainly includes: 10-30 vol% of serum substitute, 1-5 mM pyruvate, 0.1-5 mM MEM non-essential amino acid solution, 5-20 ng / ml fibroblast growth factor, 20-100 pg / ml L-ascorbic acid, 5-20 mM of lactate, 2-10 μM ROCK inhibitor, and 0.2-10 ng / ml TGF-β1. The serum-free conditioned medium is prepared by mixing all medium components in a sterilized serum bottle on a sterile operating table, and then filtering the mixture through a 0.45 μm sterile filter into a sterile serum bottle.

[0030] In Table 1 below, final concentrations of reagents added to the base culture medium (selected from DMEM, DMEM / F12, RPMI 1640, MEM, etc.) of the CSC serum-free conditioned medium are as shown in table 1 below.

[0031] Table 1

[0032] The Knockout™ serum replacement (Knockout™ SR) is a serum-free additive that supports growth of pluripotent stem cells (PSC) on fibroblast feeder layers. The Knockout™ SR is suitable for serum-free nourishment and culture of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from multiple species, and can directly replace fetal bovine serum (FBS) in conventional experimental solutions.

[0033] The pyruvate (L-alanyl-L-glutamine, Ala-Glu, also known as GlutaMAX) is an advanced cell culture additive that can directly replace nutrient L-glutamine which is essential in cell culture. During cell culture, cells gradually release a peptidase into aculture medium, which hydrolyzes L-alanyl-L-glutamine into L-alanine and L- glutamine. The cells then absorb and utilize these hydrolysis products.

[0034] The MEM non-essential amino acid (NEAA) solution is derived from a MEM culture medium formulation and includes seven non-essential amino acids: L-alanine, L-glutamine, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine. NEAA can effectively improve composition of a culture medium, reduce side effects of non- essential amino acids generated by cells during cell culture, and enhance proliferation and metabolism of the cells. NEAA is a commonly used additive in cell culture.

[0035] The chemically defined lipid concentrate is a concentrated lipid emulsion with chemically defined composition. This culture medium supplement is designed to reduce or replace fetal bovine serum in cell culture medium. The chemically defined lipid concentrate can be used for 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.

[0036] The SPITE liquid media supplement is a versatile cell additive used to form serum-free culture media. Purified substances in the SPITE liquid media supplement can replace components, such as insulin, transferrin, selenium, pyruvate, and ethanolamine, usually provided by serum.

[0037] The ROCK inhibitors are selective ATP-competitive inhibitors of Rho- associated protein kinases (ROCK). Also, The ROCK inhibitors may be effective inhibitors for myosin phosphorylation and Ca2+-sensitization-induced smooth muscle contraction. The ROCK inhibitors may include Y-27632, fasudil, and H-1152. It is known that using 10 μM Y-27632 to culture treat human embryonic stem cells (hES) cultured in serum-free suspension culture medium (SFEB) significantly reduces apoptosis induced by dissociation and improves selection efficiency (increased from about 1% to about 27%).

[0038] CSCs separated by the quadruple selecting method of the present disclosure or separated by a conventional CD44 antibody screening method are respectively cultivated in conventional cell culture mediums (such as DMEM+10%FBS) or the CSC serum-free conditioned medium of the present disclosure for at least 100 days under standard culture conditions (37°C, 5% CO2, 21% O2). The cultivated CSCs are sampled every certain of days, and a percentage of cells having high level of CD44 in the cultivated CSCs is tested.

[0039] Results are as shown in FIG. 1. No matter CSCs are obtained by the quadruple selecting method of the present disclosure or a conventional CD44 antibody separating method, differentiation of the CSCs effectively slows down when the CSCs are cultured in the CSC cell serum-free conditioned medium of the present disclosure compared to being cultured in conventional cell culture mediums. The CSCs obtained by the quadruple selecting method of the present disclosure sustain characteristics of surface antigen CD44 after at least 100 days of long-term culture. However, the CSCs obtained by the conventional CD44 antibody separating method begin to differentiate around day 5 of culture. The CSCs gradually lose characteristics of CSC and cannot proliferate.

[0040] Second embodiment: selecting and creating a tumorigenic cancer stem cell (TCSC) line

[0041] The present embodiment provides a quadruple selecting method for CSC line for long-term culture, including: first stage: tumorigenicity screening; second stage: self-renewal ability screening; third stage: treatment resistance screening; and fourth stage: single-cell recurrence ability screening. In the first stage, parental pancreatic cancer cells are separated from a culture plate with trypsin, and suspended in fresh medium to form single-cell suspension as shown in FIG. 2A. After centrifugation, the single-cell suspension resuspends in 500-1000 pl of 5% FBS fresh medium. 1 ml of 1- 1.5% agar is added into each well of a 6-well plate culture medium. Standing for 3-5minutes to solidify the agar. 666 pl of the cell suspension and 333 pl of the 1-1.5% agar (3-fold dilution) are added into each well. Then the plate is shaken slowly to mix the cell suspension with the agar. Standing for 3-5 minutes to solidify the agar (the cell suspension is first added into the well to prevent the agar from solidifying). After that, 1 ml of 5% FBS medium is added into each well, and the plate is positioned in a hypoxic (5% O2) incubator at 37°C for culture. After 1-2 weeks of culture, a 1 ml tip is used to remove a cell colony from a soft agar. The cell colony is transferred to a 6-well plate for temporary proliferation to provide enough cells for a next round of selection. Cells are seeded for another soft agar selection. The above soft agar selection is repeated five times. To select cells with high tumorigenicity, a number of inoculated cells is gradually reduced from 106, 105, 104, 103to 102, and selection is performed 5 times. As shown in FIG. 2B, in the first stage, the soft agar is simulated as an in vitro tumor-forming environment, and cancer cells with high tumorigenic ability are selected.

[0042] Then, in the second stage, 1x104of cells are dispersed into individual cells. The individual cells are seeded on a 6-well plate which is covered by 11.5% agar and has a serum-free medium. The 6-well plate is placed in a low-oxygen (5% O2) incubator at 37°C for culture. Cells unable to adhere to a soft surface form tumor spheres in a suspension culture after a few days. The tumor spheres are dissociated into individual cells by using a quantitative pipette every four days to form new tumor spheres. Cells able to form new tumor spheres have anoikis resistance ability and long-term self- renewal ability. After 12 weeks of culture, a number of tumor spheres is recorded, and a cell colony is transferred to a new 6-well plate for temporary proliferation, thereby providing sufficient cells for a next selection step. This tumor sphere selecting process is repeated three times. To select cells with high tumorigenic ability, a number of inoculated cells is gradually reduced from 104, 103to 102, and selection is performed 3 times. As shown in FIG. 2C, a 3D proliferation technique is used in the second stage.In a suspension condition, CSCs having stem cell characteristics have a spherical shape (tumor spheres), resembling blastocysts. This environment is simulated as a situation of tumors in blood vessels, where only stem cells survive and are therefore selected.

[0043] Then, in the third stage, at least 5x 106cells (up to 107cells) are prepared to be suspended in a 5 ml culture medium. 5ml of cell suspension is divided into 4 ml and 1 ml portions which are later placed in two capped blue sorting tubes. 100 μM verapamil (Sigma- Aldrich, St. Louis, MO) and 10 μM fumitremorgin C (FTC, Sigma-Aldrich) are added to the sorting tube having 1 ml of the cell suspension, and the sorting tube is incubated at 37°C for 5 minutes to be protected from light. 4 μl and 1 μl of Hoechst 33342 dye (5 μM, Sigma-Aldrich, St. Louis, MO) are added to a sorting tube having 4 ml of cell suspension and a sorting tube having 1 ml of cell suspensions, respectively. The tubes are then placed in an incubator at 37°C for 90 minutes for culture. Meanwhile, the tubes are gently tapped every 15 minutes.

[0044] After cells are washed twice with PBS, 2 ml and 1 ml of culture medium containing propidium iodide (PI) (2 pg / ml, Sigma-Aldrich, used to exclude dead cells) are added into the tubes, thereby resuspending unmedicated cells (without verapamil and FTC) and medicated cells (verapamil and FTC). A selecting process is applied to cell suspension. A capped test tube containing 1 ml of medium having 0.2% PSA is prepared and covered with a paraffin film. Cell sorting is performed by using a BD FACSAria flow cytometer (BD Biosciences). After being excited with UV488 laser, a main cell population containing Hoechst 33342 emits blue and red fluorescence. Some of side population cells emit lower fluorescence due to the efflux of Hoechst dye through their ABC transporters. When verapamil and FTC are applied to prevent the efflux of fluorescence dyes, the side population cells emit high fluorescence. By comparing parameters obtained with or without verapamil and FTC, the side population cells can be sorted. Separated cells are cultured in a 6-well plate in a low-oxygen (5%O2) incubator at 37°C for temporary proliferation, thereby providing sufficient cells for a next selection step. As shown in FIG. 2D, in the third stage, A fluorescence dye (Hoechst dye) is used to simulate a drug. CSCs have a drug exclusion channel to exclude the dye. Thus, CSCs drug-resistant ability can be selected by using a channel blocker (verapamil).

[0045] Finally, in the fourth stage, a cell is seeded on a round-hole ultra-low attachment 96-well plate by using a serum-free medium. The plate is placed in a low- oxygen (5% O2) incubator at 37°C for culture. Cells forming a largest 3D cell colony having a diameter of around 250-300 μm in a shortest time are selected. Then, these cells are cultured in the CSC serum-free conditioned medium of the present disclosure as described in the first embodiment, and are long-term cultured to obtain a stable pancreatic CSC line. As shown in FIG. 2E, in the fourth stage, ability of a small number of tumor cells which form new tumors after treatment is simulated. A CSC line able to form a largest tumor in a shortest time from a cell is selected. The selected CSC line is subsequently cultured in the CSC serum-free conditioned medium of the present disclosure as described in the first embodiment. The selected CSC is long-term cultured to obtain a stable pancreatic tumorigenic CSC line (PANC-1 TCSC) as shown in FIG. 3. The pancreatic CSC line obtained by the present disclosure has high tumorigenic ability.

[0046] Colony morphologies of a colorectal tumorigenic CSC line (DLD-1 TCSC) obtained from parental colorectal cancer cells according to the above quadruple selecting method in each stage are as shown in FIGs 4A to 4E.

[0047] Third embodiment: selecting and forming a metastatic cancer stem cell (MCSC) line

[0048] The present embodiment provides a quadruple selecting method for long- term culture of a stable metastatic CSC line, including: first stage, metastasis invasionability screening; second stage, self-renewal ability screening; third stage, treatment resistance ability screening; and fourth stage, single-cell recurrence ability screening. In the first stage, parental pancreatic cancer cells are separated from a culture plate by using trypsin, and are suspended in a fresh culture medium to form single-cell suspension as shown in FIG. 5A. After centrifugation, the parental pancreatic cancer cells are suspended in a 500-1000 pl of 5% FBS fresh culture medium. 80 pl of matrigel (Corning, NY) is added to an upper chamber (a dilution ratio of the matrigel to the serum-free conditioned medium is 1 :2). After the Matrigel is solidified, 600 pl of 10% FBS fresh culture medium is added to a lower chamber. 200 pl of cell suspension is added to an upper Matrigel layer. A transwell plate is placed in a hypoxic (5% O2) incubator at 37°C. Cells begin to invade from the upper chamber to the lower chamber. After 1-2 weeks, the cells in the lower chamber are transferred to a 6-well plate for temporary proliferation, thereby providing sufficient cells for a next selection round. Another round of metastasis invasion ability screening is applied to the cells. The above steps are repeated five times to select cells having high metastasis invasion ability, and a number of seeded cells is gradually reduced from 106, 105, 104, 103, to 102.

[0049] Then, the screening steps of the second to fourth stages described in the second embodiment are performed. The selected CSCs are cultured in the CSC serum- free conditioned medium of the present invention described in the first embodiment. A stable pancreatic metastatic CSC line (PANC-1 MCSC) is obtained after long-term culture. FIGs. 5B to 5E show colony morphologies in each screening stage. As shown in FIG. 6, the metastatic cancer stem cell line obtained from the present invention has high metastatic ability.

[0050] Colony morphologies of a colorectal tumorigenic CSC line (DLD-1 TCSC) obtained from colorectal parent cancer cells according to the above quadruple selecting method in each stage are as shown in FIGs 7A to 7E.

[0051] Fourth embodiment: selecting and forming a CSC line having resistance

[0052] The present embodiment provides a quadruple screening method for the long- term culture of a CSC having stable resistance, including: first stage, resistance (radiation resistance or drug resistance) ability screening; second stage, self-renewal ability screening; third stage, treatment resistance screening; and fourth stage, single- cell recurrence ability screening. To screen CSCs having radiation resistance, in the first stage, an Elekta Synergy radiation machine (Elekta company, Sweden) is used to irradiate cancer cells at a rate of 562 MU / min with a dose of 2 Gy / day. After 30 round of irradiations, a cancer cell line having radiation resistance are selected. Cells are cultured in a 37°C hypoxic (5% O2) incubator. Cells are seeded in a 96-well plate in a one-cell-per-well way by using a serum-free culture medium. The Elekta Synergy radiation machine (Elekta company, Sweden) is used to irradiate single cells at a rate of 562 MU / min with a dose of 10 Gy / day. A largest cell light formed in a shortest time is selected. The plate was placed in a hypoxic (5% O2) incubator at 37°C for temporary proliferation, thereby providing enough cells for a next selection.

[0053] To screen CSCs having chemotherapy drug resistance, in the first stage, cancer cells are selected by being subjected to 20 rounds of chemotherapy drugs, and doses of drugs are gradually increased from 100-300 nM to 1-30 μM round by round. Cells are saved in a hypoxic (5% O2) incubator at 37°C. Blood therapy drugs are used to form cells having chemotherapy resistance. As shown in Table 2 below, standard chemotherapy drugs used to form different types of chemotherapy -resistant cancer cells are listed.

[0054] Table 2

[0055] After 20 rounds of chemotherapy drug selection, cells are seeded in a 96-well plate in a one-cell-per-well way. Individual cells are cultured with a highest dose of chemotherapy drug. A largest cell light formed in a shortest time is selected. The plate was placed in a hypoxic (5% O2) incubator at 37°C for temporary proliferation, thereby providing enough cells for a next selection.

[0056] Subsequently, selecting steps of the second to fourth stages as described in the second embodiment are performed. Then, the selected cancer stem cells are cultured in the CSC serum-free conditioned medium of the present invention as described in the first embodiment for long-term culture. Thus, stable CSCs having radiation resistance CSCs having drug resistance can be obtained.

[0057] Fifth embodiment: establishment of a clinical CSC platform for rapid screening

[0058] The present embodiment provides a two-stage method for rapidly screening clinical CSCs, including: stem cell gene transcription function screening stage; and CSC behavior screening. In the first stage, a clinical tumor sample is transfected with an octamer-binding transcription factor 4 (OCT4) gene using a lentiviral transfection system, and a plurality of cancer cells positive for the OCT4 gene expression are selected. Thus, the stemness status of the cancer cells can be identified.

[0059] To screen clinical tumor-initiating stem cells, in the second stage, 1 ml of 1- 1.5% agar is added to each well of a 6-well plate culture medium. Standing for 3-5 minutes to solidify the agar. Then, 666 pl of cell suspension (cancer cells positive for 103-104OCT4-GFP) and 333 μl of 1-1.5% agar (3-fold diluted) are added to each well. The plate is gently shaken to mix them. Stand for 3-5 minutes to solidify the agar. A largest cell formed in a shortest time is selected, and is cultured in the CSC serum-free conditioned medium of the present invention.

[0060] To screen clinical metastatic CSCs, in the second stage, 80 pl of Matrigel (Corning, NY) is added to an upper chamber (Matrigel is diluted with serum-free culture medium at a ratio of 1 :2). After Matrigel solidifies, 600 pl of 10% FBS fresh culture medium is added to a lower chamber. 200 pl of cell suspension is added to an upper Matrigel layer. Atranswell plate is placed in a hypoxic (5% O2) incubator at 37°C. Cells begin to invade from the upper chamber to the lower chamber. The metastatic CSCs are cultured in the CSC serum-free conditioned medium of the present disclosure.

[0061] To screen clinical radioresistant CSCs, in the second stage, cancer cells positive for OCT4-GFP are seeded in a 96-well plate at a density of 103-104cells per well by using a serum-free culture medium. The Elekta Synergy radiation machine (Elekta company, Sweden) is used to irradiate individual cells at a rate of 562 MU / min with a dose of 10 Gy / day. A largest cell light formed in a shortest time is selected. The selected cancer cells having radiation resistance are cultured in the CSC serum-free conditioned medium of the present disclosure.

[0062] To screen clinical drug-resistant CSCs, in the second stage, standard chemotherapy drugs are used to form chemotherapy-resistant CSCs. cancer cells positive for OCT4-GFP are seeded in a 96-well plate at a density of 103-104cells per well by using a serum-free culture medium. Individual cells are treated with a highest dose of chemotherapy drug. A largest cell light formed in a shortest time is selected.The selected cancer cells having chemotherapy drug resistance are cultured in the CSC serum-free conditioned medium of the present disclosure.

[0063] By using the two-stage selecting method of the present invention, CSCs of clinical specimens can be rapidly selected. Moreover, selected cancer stem cells can serve as a platform for screening effective drugs. In the present embodiment, if candidate drugs can target CSCs and reduce their stemness, CSCs positive for OCT4- GFP will lose fluorescence. Thus, a therapeutic effect of candidate drugs on different types of CSCs can be assessed.

[0064] Sixth embodiment: assessment of purity and characteristic of a CSC line separated by using the quadruple selecting method of the present disclosure

[0065] The CSC lines formed in the second embodiment and the third embodiment and a CSC line separated by using commercially available CD44 antibody are respectively seeded in a general cell culture medium and the serum-free conditioned medium of the present disclosure as described in the first embodiment, and are cultured in a hypoxic (5% O2) incubator at 37°C for 35 days. Then, cells are sorted by a CD44 surface antigen, and a percentage of cells sustaining high CD44 surface antigen expression in entire cultured cells is detected.

[0066] Results are as shown in FIG. 8. The CSC line separated by the quadruple selecting method of the present disclosure can more stably and long-term express stem cell antigens compared to the CSC line separated by a conventional antibody selecting method (CD44-antibody selecting method). In addition, differentiation of the CSCs effectively slows down when the CSCs are cultured in the CSC cell serum-free conditioned medium of the present disclosure compared to being cultured in conventional cell culture mediums. This effect is more significant if the quadruple selecting method of the present disclosure is applied.

[0067] As shown in FIGs. 9A and 9B, stemness genes (OCT4, Sox2, Klf4, c-Myc, Nanog, and Lin28 genes) and epithelial-mesenchymal transition (EMT) genes (Twist, Snail, Slug, Zebl, E-cadherin, N-cadherin, Vimentin, and FN1 genes) of the CSC lines after 1 month of culture are further tested. The stemness genes (FIG. 9A) and EMT genes are still highly expressed by the CSC lines of the present disclosure after one month of culture.

[0068] Seventh embodiment: using CSC lines as drug screening platforms

[0069] In the present embodiment, various scenarios of using CSC lines separated by the present disclosure as drug screening platforms are presented. To easily observe and identify a stemness status of CSCs in vitro or in vivo, the separated CSC lines are transduced with OCT4-GFP or OCT4-Luc reporter genes by using a lentiviral-based transduction system. If a candidate drug effectively reduces stemness of the CSCs, the CSCs expressing OCT4-GFP or OCT4-Luc will lose fluorescence or luminescence (bioluminescence). The reporter genes are not limited to GFP genes or Luc genes, and other antibiotic resistance genes can also be used.

[0070] One example is using a 3D culture method to form tumor spheres from separated CSC lines, treating them with a nanodrug, and observing whether the nanodrug effectively targets and inhibits a tumor. The CSC lines can also be used as platforms to assess a therapeutic efficacy of drug combinations. For instance, OCT4- GFP or OCT4-Luc expressing tumor-initiating or metastatic CSCs can be treated with a known chemotherapy drug and a new drug (e.g., Drug A, Drug B, or Drug C) in combination. Inhibitory effects of the drug combinations on parent cancer cells and inhibitory effects of the drug combinations on the CSCs are compared, thereby evaluating therapeutic efficacy of the drug combinations or therapeutic effects of new drugs on drug-resistant cancers.

[0071] To screen drugs in vivo, tumorigenic CSC lines can be injected into zebrafish or mice to create tumors, thereby confirming whether candidate drugs can effectively target and treat the tumors. Also, metastatic CSC lines can be injected into zebrafish or mice to confirm whether candidate drugs can effectively inhibit tumor metastasis.

[0072] The present disclosure has successfully developed the CSC culture medium, which is used for screening and forming a great amount of CSC lines (can be significantly proliferated millions of times) having high purity (dozens of times higher than competing products in stem cell gene expression) and stability (effectively extending shelf life by hundreds of times). The separated CSC lines can be used by pharmaceutical companies and research institutions, thereby accelerating targeted drug development for CSCs to effectively solve difficulties in current cancer treatment.

Claims

AMENDED CLAIMS received by the International Bureau on 21 November 2024 (21.11.2024)Listing of Claims1. A method to slow down the differentiation of cancer stem cell (CSC) line, comprising: cultivating the CSC line in a serum-free conditioned medium, wherein the serum-free conditioned medium comprises: a base culture medium; and a supplement combination, wherein the base culture medium is selected from a DMEM culture medium, a DMEM / F12 culture medium, a RPMI 1640 culture medium, or a MEM culture medium; and wherein the supplement combination comprises 10-30 vol% of serum substitute, 1-10 mM pyruvate, 2-20 μM ROCK inhibitor, 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 lactate, 40-70 U / ml penicillin, 40-70 pg / ml streptomycin, 0.05-0.15 mM 2-mercaptoethanol, 1000-fold diluted lipid concentrate, and 100-fold diluted SPITE culture 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 of selecting an anti-cancer drug as recited in claim 1, comprising the following steps: culturing a candidate drug with a cancer stem cell (CSC) line; and testing an inhibitory effect of the candidate drug on the CSC line, wherein the CSC line is cultured according to the method of claim 1.

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