Patent Application
20210207082
This application is based on and claims priority under 35 U.S.C. § 119 to Korean Patent Application No. 10-2019-0172372, filed on Dec. 20, 2019, in the Korean Intellectual Property Office, the disclosure of which is incorporated by reference herein in its entirety.
The present disclosure relates to a spherical 3D tumor spheroid and a method for evaluating efficacy of a tumor therapeutic agent using the same.
Currently, most of pre-clinical tests in vitro for development of new anticancer drugs are conducted using cells cultured in typical tissue culture plates of a two dimensional (2D) environment. To obtain results prior to in vivo tests for drug development, presumably more than 70% of cancer researchers still depend on two dimensional (2D) culture systems. However, in vitro cells of 2D culture systems grow on a planar surface as a monolayer, and thus may not be suitable for drug screening, making it difficult to accurately reflect cells existing in an in vivo three dimensional (3D) environment. In this regard, the predictive efficacy of in vivo drug evaluation has proven to be insufficient, resulting in a costly failure in clinical tests due to a huge financial loss.
To overcome limitations of 2D culture systems in pre-clinical stages, attention has recently been focused on 3D culture technique as a tool for improving physiological relevance of in vitro models for evaluating drug efficacy. The in vitro models equipped with 3D platforms, including multicellular spheroids, scaffolds and biochips, have been developed in many researches. The 3D culture platform has led to development of new, more physiological human normal tissues and tumor models. Many researchers have established in vitro models of tumors and living organs associated with drug metabolism of a human body, including liver, heart, viscera and kidney.
An object of the present disclosure is to provide a spherical 3D tumor spheroid and a method for evaluating efficacy of a tumor therapeutic agent using the same.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments of the disclosure.
An aspect provides a spherical 3D tumor spheroid comprising a core part including a tumor cell group; and a peripheral part comprising an adipose-derived stromal cell group and an extracellular matrix component and surrounding the core part.
Another aspect provides a tumor cell model including the 3D tumor spheroid.
Another aspect provides a screening composition including the cell model.
Another aspect provides a composition for evaluating a tumor therapeutic agent comprising the 3D tumor spheroid.
The cells of the tumor cell group and the adipose-derived stromal cell group are not particularly limited so long as the cells are derived from individuals having tissues from which tumors may be generated, and examples thereof may include cells derived from humans, mice, rabbits, dogs, pigs or monkeys, specifically from humans.
The cells of the tumor cell group are not particularly limited so long as the cells are obtained from tumor cell lines that can be easily available in the art. For examples the tumor cell group comprising, but not limited to, a breast cancer cell, a lung cancer cell, a fibrosarcoma cell, a stomach cancer cell, an oral cavity cancer cell, a prostate cancer cell, a liver cancer cell, an ovarian cancer cell, a thyroid cancer cell, a uterine cancer cell, a glioblastoma cell, a melanoma cell, a gingiva cancer cell, a tongue cancer cell, a pancreatic cancer cell, a kidney cancer cell, a bone cancer cell, a testicular cancer cell, a mesothelioma cell, a lymphoma cell, a brain tumor cell, a colon cancer cell or a bladder cancer cell.
According to a specific embodiment, the tumor cell group may include MDA-MB-231, MDA-MB-468, BT-549, MCF-10A, MCF-7, A549, HT1080, MKN45 or SK-BR-3 cell.
The extracellular matrix component may be a product expressed by an interaction between the tumor cell group and the adipose-derived stromal cell group. The extracellular matrix component may include components existing in the entire peripheral part, including a surface of the spheroid, and may be configured to surround the entire core part. When an expression level of the extracellular matrix component is low, the extracellular matrix component may not be implemented so as to be similar to an actual tumor tissue known to have an over-expressed extracellular matrix component, and thus making it difficult to use the extracellular matrix component in screening a cancer or tumor therapeutic agent or evaluating efficacy thereof.
The extracellular matrix component may include collagen and fibronectin. The extracellular matrix component may be a product expressed by an interaction between the tumor cell group and the adipose-derived stromal cell group, as described above. The collagen may include collagen type 1, which plays a key role in preventing penetration of a cancer or tumor therapeutic agent. Thus, the collagen can serve as a main checkpoint in evaluating or screening efficacy of the cancer or tumor therapeutic agent using the spheroid.
The spheroid may have a diameter of 200 μm to 900 μm, 250 μm to 850 μm, 300 μm to 800 μm, 350 μm to 750 μm, 400 μm to 700 μm, 450 μm to 650 μm, or 500 μm to 600 μm, and the diameter of the spheroid is preferably 500 μm to 600 μm. When the diameter of the spheroid is within this range, the 3D tumor spheroid is able to similarly mimic an in vivo tumor while maintaining an appropriate size as an in vitro 3D tumor spheroid.
The spheroid may have a roundness of 0.90 or greater. In addition, the spheroid may have a sphericity of 0.90 or greater. Preferably, the roundness of the spheroid is 0.99 or greater and the sphericity thereof is 0.99 or greater. To be used as a model for drug efficacy evaluation or drug screening, it is very important to stably form a spheroid having the same type and shape. Given that uniformity and consistency in forming a spheroid are very important particularly for establishing metabolic activity, the spheroid may have a perfect, uniform, and stable spherical shape.
The spheroid may be formed by co-culturing the tumor cell group and the adipose-derived stromal cell group at a cell density of 7:3 to 3:7, preferably 1:1, which may be based on the effect of the spheroid having increasing viability and decreasing drug permeability as the cell density is closer to 1:1. In such a case, by using a spheroid having the tumor cell group and the adipose-derived stromal cell group co-cultured at an appropriate cell density ratio within the range of 7:3 to 3:7, the efficacy of a cancer or tumor therapeutic agent may be screened or comparatively tested. As an example of the comparative test, when the co-cultured cell density ratio of the tumor cell group and the adipose-derived stromal cell group is 7:3 or 1:1, whether or not the spheroid penetrates the core of the candidate drug can be observed. For example, when the first drug succeeds in penetrating into the core portion only at 7:3, and the second drug succeeds in penetrating into the core portion in both 7:3 and 1:1 cases, it is determined that the first drug has lower efficacy than the second drug in treating a cancer or tumor.
In a specific embodiment, provided is a method for evaluating efficacy of a cancer or tumor therapeutic agent comprising: treating target drugs with the spheroid; and analyzing distributions of the target drugs in a core part of the spheroid or analyzing cell viability in the spheroid.
In treating the drugs, a general method used in the art for evaluating the efficacy of a cancer or tumor therapeutic agent in vitro, or a method of adding a cancer or tumor therapeutic agent to may be used.
For example, the treatment of the drug may be in the form of a culture medium having a spheroid seeded therein for a predetermined period of time and then culturing.
In treating the target drugs, a plurality of drugs may be treated on a plurality of spheroids in one-to-one (1:1) correspondence, or a plurality of drugs may be treated on a single spheroid, but not limited thereto. Any method may be employed as long as there is no problem in performance of the following step of analyzing distributions of the target drugs in a core part of the spheroid or analyzing cell viability in the spheroid.
In analyzing a distribution of the target drug in the core part of each spheroid, any method that is known in the art as a method for tracking a moving path of the target drug or an extent of distribution thereof, may be used without particular limitation, and, for example, the distribution may be analyzed by fluorescence detected from the drug.
In analyzing cell viability in the spheroid, the viability may be analyzed by counting the number of cells living in the spheroid before and after drug treatment, but any method that is known in the art as a method for analyzing cell viability may be employed without any particular limitation.
The cell viability in the spheroid reflects the overall phenomenon in which the size or number of tumor cell groups according to treatment of the target drug. That is, the cell viability may reflect diverse and comprehensive phenomena including apoptosis, necrosis, etc.
Considering selective therapy on tumor cells of the cancer or tumor therapeutic agent, the viability of cells in the spheroid is preferably a viability of the tumor cell group in the core part of the spheroid.
When a distribution of one target drug in the core part of the spheroid is higher than that of the other target drug, or when the cell viability in the spheroid treated with the one target drug is lower than that in the spheroid treated with the other target drug, the method may further include determining that the one target drug has higher efficacy in cancer or tumor treatment than the other target drug. The distribution of a drug in the core part may be inversely proportional to the cell viability in the spheroid. Specifically, the higher the distribution of a drug in the core part, the better the drug is delivered by penetrating into the peripheral part, which may be based on the fact that the drug distributed in the core part reduces the viability of the tumor cell group existing in the core part.
In the method, when a distribution of the one target drug in the core part of the spheroid is not observed at all or when the cell viability in the spheroid or the cell viability of the tumor cell group in the core part is not significantly reduced with treatment of the one target drug, compared to that without treatment of the one target drug, it is obvious to those skilled in the art that the one target drug may be evaluated or screened to be a drug that is not suitable to be used as a cancer or tumor therapeutic agent.
Another aspect provides a method for screening a tumor treating material, comprising incubating the 3D tumor spheroid and a candidate material.
The tumor spheroid is the same as described above.
The method comprises incubating the 3D tumor spheroid and the candidate material.
The candidate material may mean a drug expected to prevent or treat a tumor or cancer. The candidate material may be an organic material or an inorganic material, and may be a single compound or a composite compound. In addition, the candidate material may be protein, peptide, DNA, RNA, etc., and all materials existing in nature may be used as candidate materials.
Next, the method may comprise measuring an expression level of a tumor biomarker of the cultured spheroid or determining whether cultured spheroid has survived or not.
The biomarker is a material capable of differentially diagnosing individuals in a normal group and individuals having a tumor or cancer group, and may include all of organic biomolecules increasing or decreasing in the individuals having a cancer, such as polypeptide, protein, nucleic acid, gene lipid, glycolipid, glycoprotein or sugar. A change in the expression of a tumor or cancer biomarker may be an increased or a decreased expression.
When the expression of a tumor biomarker of the cultured spheroid is increased or decreased, or when the cultured spheroid is killed, the method provides screening a candidate material.
Another aspect provides a method for providing information on cell permeability of an anticancer drug using the 3D tumor spheroid.
The spheroid and the caner are the same as described above.
In the present specification, the term “anticancer drug” collectively refers to a drug that kills a cancer cell while preventing division of the cancer cell. The anticancer drug has a feature of penetrating into a surface of a cancer cell. Therefore, the information on the cell permeability of the anticancer drug may be a clue in predicting the effect of the anticancer drug. If the anticancer drug is assessed to have a high cell permeability, it may be determined that the anticancer drug is effective in treating a cancer.
The anticancer drug may be vatalanib, lenvatinib, dovitinib, ammonium-glycyrrhizinate, epirubicin, temsirolimus, lintitript (SR-27897), cabozantinib, tesmilifene (DPPE), KX-01 (KX2-391, tirbanibulin), rubitecan, bardoxolone-methyl, mifepristone, mitomycin-c, genistein, paclitaxel, carboxyamidotriazole, pazopanib, halofuginone, vinblastine, SGX523, lonafarnib, marimastat, patupilone (epothilone-b), derenofylline, lorlatinib, OSI-930, everolimus, capecitabine, tamoxifen, rucaparib, alisertib, canertinib, altretamine, etoposide (7-ethyl-10-hydroxycamptothecin), roquinimex, aminoglutethimide, talazoparib, imatinib, quinestrol, alectinib, verubulin, or tipifarnib.
The method may further comprise: inducing a regression analysis model considering various chemical features of various anticancer drugs or interactions thereof.
The method may further comprise performing regression analysis using the regression analysis model.
In the present specification, the term “regression analysis” refers to a statistical technique for estimating effects of one or more independent parameters on dependent parameters. For example, in a case of regression analysis with one independent parameter, a regression line may be obtained by constructing a distribution of points at which the independent parameter and dependent parameters meet. The regression line may be represented by: Y=a+bX1+cX2. Designing a regression analysis model may include a stepwise regression based assay, and utilization of a forward process as a method for selecting parameters.
The regression analysis may be performed using, as input parameters, one or more chemical features selected from the group consisting of molecular weight (M.W) of drug, distribution coefficient (Log P), water solubility (Log S), acid dissociation equilibrium constant (pKa), physiological charge, hydrogen acceptor count, hydrogen donor count, polar surface area, rotatable bond count, polarizability, refractivity, and number of rings.
The regression analysis may use, as an output parameter, a difference in viability (or permeability) between a 3D multicellular tumor spheroid and a 3D single cell spheroid.
A model of the regression analysis may include a regression analysis equation represented by Equation 1, and
the regression analysis equation may be an equation for deriving a predicted cell viability difference (or permeability):
PO=a+bK+cL+dM+eN+fO+gP+hQ+iR+jS [Equation 1]
wherein PO is a predicted cell viability difference (or permeability), K is M·W(g/mol), L is log P, M is log S, N is a hydrogen acceptor count (units), O is a hydrogen donor count (units), P is a polar surface area (Å2), Q is a rotatable bond count (units), R is refractivity (m3/mol), S is polarizability (Å3), and a to j are constant values: a=0.1235313827, b=0.0035738171, c=0.0340283393, d=0.0340283393, e=−0.000519426, f=0.0108241714, g=−0.002123276, h=0.0007481956, i=−0.0050597, and j=−0.018713752.
The regression equation may be an equation in which a difference value in the cell viability (or permeability) between the 3D multicellular tumor spheroid and the 3D single cell spheroid are normalized between 0 and 1. The difference value being closer to 1 may mean that the permeability predicted by the chemical features is more similar to actual permeability. The multiple regression analysis may be a linear fitting method, and may be performed by deriving a regression line by identifying a distribution of points where permeability values predicted by chemical features and actual permeability values meet each other. A determinant coefficient (R2) of the regression line may be 0.60 to 0.75 or 0.62 to 0.70.
The method may further comprise: comparing the derived cell viability difference (or permeability) with an actual output parameter; and assessing permeability of the anticancer drug on the basis of the comparison result.
In another aspect, provided is a use of the 3D tumor spheroid or a composition comprising the same in manufacturing a cell model.
In another aspect, provided is a use of the tumor spheroid or a composition comprising the same in manufacturing a screening composition of a tumor therapeutic material.
The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
Hereinafter, the present disclosure will be described in further detail with reference to embodiments. However, the present disclosure is not limited to the disclosed embodiments.
Experimental Materials
1. Cell Lines
Human adipose-derived stromal cells (ASC) were purchased from Cefobio (Seoul) and were maintained in ASC growth culture media (Cefobio, Seoul, Korea) supplemented with 10% FBS, 1% L-glutamine and penicillin/streptomycin. The culture media were replaced every other day.
Bone marrow stromal cells (BMSC) and Human dermal fibroblast (FIB) were purchased from the Catholic University of Korea (Seoul, Korea) and were maintained in DMEM 1× culture media (Gibco™, Cat #11965092) supplemented with 10% FBS, 1% L-glutamine and penicillin/streptomycin. The culture media were replaced every other day.
MDA-MB-231 human breast cancer cell line, A529 human lung cancer cell line, HT1080 human fibrosarcoma cell line, MKN45 human stomach cancer cell line, SK-BR-3 human breast cancer cell line, and MCF-7 human breast cancer cell line were purchased from The Korean Cell Line Bank (KCLB, Seoul), and were maintained in RPMI 1640 culture media (Gibco™, Cat #11875-093) supplemented with 10% FBS, 1% L-glutamine and penicillin/streptomycin. The cell lines were cultured in an incubator being under 37° C. and 5% CO2 conditions. The culture media were replaced every other day.
2. Poly-HEMA
120 mg/mL of a poly-hydroxyethyl methacrylate (poly-HEMA) stock solution diluted with 95% ethanol was prepared and was turned over to then apply a vortex thereto, and, in order to obtain a working solution of poly-HEMA, 1 mL poly-HEMA stock solution was added to 23 mL of 95% ethanol to then adjust a final concentration to 5 mg/mL. A new working solution was prepared whenever a new plate was prepared.
3. Additional Information
Matrigel (Matrigel® Basement Membrane Matrix, Corning, Cat #354234), anticancer drug (doxorubicin, SIGMA, #D1515), cell viability assay (Real Time-Glo™ MT Cell Viability Assay, Promega, Cat #G9711), and cell apoptosis/necrosis assay (Real Time-Glo™ Annexin V Apoptosis and Necrosis Assay, Promega, Cat #JA1011), were used. In addition, doxorubicin and 44 anticancer drugs, for drug efficacy evaluation, were supplied from Korea Chemical Bank of Korea Research Institute of Chemical Technology, and a list of the 44 kinds of anticancer drugs is shown in
Experimental Method
1. Preparation of Non-Adsorptive Poly-HEMA Plate
60 μL of a poly-HEMA stock solution was pipetted to each well of a 96-well U-bottom plate and then evaporated in a 30° C. incubator with a lid for one week.
2. Formation of 3D Multicellular Tumor Spheroids
In order to form 3D multicellular tumor spheroids, suspensions of various cell types were prepared in growth culture media. Stromal cells were seeded to each well of a 96-well plate pre-coated with poly-HEMA at a density of 0.5×104 cells/well, and then incubated in a 5% CO2 incubator at 37° C. for 48 hours. Then, each 25 μL of tumor cells MDA-MB-231, A549, HT1080, MKN45, SK-BR-3, and MCF-7 was plated on the stromal cells in each well of the plate at a density of 0.5×104 cells/well. After seeding the cells, the plate was centrifuged at 1000 rpm for 2 minutes to collect cells from the center of the well. 50 μL of a 10% matrigel solution diluted in the growth medium was gently added to a plate on ice to prevent matrigel from being gelled. Next, the plate containing cells and matrigel was centrifuged at 1000 rpm for 2 minutes. The plate was centrifuged at 1000 rpm for 2 minutes and then incubated in a 5% CO2 incubator at 37° C. for 48 hours.
3. Morphological Analysis
The cells were seeded using a camera with a 5× objective lens attached to a microscope, and then images of all rotating ellipsoids were photographed for two days. All images were analyzed using software ImageJ (National Institutes of Health, Bethesda, Md., USA).
4. Scanning Electron Microscope (SEM) Analysis
After a two-day cell culture, 3D multicellular tumor spheroids were washed with PBS three times. To fix 3D spheroids, the 3D spheroids were treated with 2.5% glutaraldehyde at 4° C. for one hour and then fixed with 1% osmium tetroxide in deionized water for two hours. The fixed 3D spheroids were dehydrated twice (each 5 minutes) with a series of graded ethanols (30%, 50%, 70%, 80%, 90%, and 100%), and then treated with hexamethyldisilazane (HMDS) for two minutes, followed by vacuum drying overnight. Prior to use of a scanning electron microscope (SEM), the 3D spheroids were transferred to an adhesive carbon tape, and then sputter-coated with gold at 10 mA for 60 seconds. SEM images were photographed at 15 kV (Inspect F50).
5. Cell Labelling
The cells were labelled with chloromethyl fluorescein diacetate (CMFDA, molecular probe) as a cell tracker dye at room temperature for 30 minutes. To observe a distribution of co-cultured cells in 3D spheroids, before seeding on a plate, stromal cells and tumor cells were stained with a cell tracker green CMFDA dye and a cell tracker Red CMTPX dye. After forming 3D spheroids, the cells were observed using a confocal microscope (LSM700, Zeiss).
6. Immunofluorescence Staining
Section samples for immunofluorescence staining were washed with distilled water to remove OCT compounds, and were then allowed to pass through 0.25% triton X-100 in PBS at room temperature for 15 minutes. The samples were washed with PBS three times (5 minutes each time). After the samples were blocked with 3% bovine serum albumin (BSA) at room temperature for one hour, the samples were incubated with a mouse anti-collagen type I antibody (1:200), rabbit anti-fibronectin antibody (1:200) at 4° C. overnight. The samples were washed with PBS three times, and then incubated with Alexa Fluor® 488-donkey anti-mouse IgG (1:500) for one hour. The samples were washed with PBS and then observed using a confocal microscope.
7. Cell Viability Assay
For comparison of anticancer drug efficacy for 3D multicellular tumor spheroids, the spheroids were treated with 10 μg/mL drugs (doxorubicin, epirubicin, topotecan, or 44 types of anticancer drugs shown in
8. Cell Apoptosis and Necrosis Assays
Apoptotic and necrotic cells in spheroids were assayed using Real Time-Glo™ Annexin V apoptosis and necrosis assays. Real Time-Glo™ Annexin V apoptosis and necrosis assays were performed in combination with a cell viability assay. The spheroid in the culture medium (50 μL) formed by a two-day 3D cell culture, were transferred to a multi-walled plate having opaque walls, and then treated with 50 μL of 2× drug (doxorubicin) until a total concentration reached 10 μM. Thereafter, 2X apoptosis and necrosis formulations of the same volume (100 μL) were added to each well according to standard protocol proposed in manufacturer's instructions. Luminescence and fluorescence values were measured using a Glomax-Multi-Microplate reader (Promega, Glomax Discovery) for an integration time of 0.25-1.0 second per well.
9. Treatment of Anticancer Drug
Doxorubicin, epirubicin, topotecan, or 44 types of anticancer drugs shown in
10. Statistical Analysis
Statistical analysis of data was performed by an ANOVA one-way test using prism software (Graph Pad). Statistical significances were defined as *p<0.05, **p<0.01 and ***p<0.001. Multiple regression analysis was performed by stepwise regression using JMPpro statistical analysis software (JMP), and, for parameter selection, a forward process was utilized.
Experimental Results
1. Experimental Overview
The experimental overview is as shown in
2. Formation of 3D Multicellular Tumor Spheroids
(1) Co-Culture of Tumor Cells and Stromal Cells
To mimic a tumor microenvironment, tumor microenvironments for co-culturing three types of stromal cells and tumor cells were selected. Tumor cells on a 5% matrigel plate coated with poly-HEMA, and ASC, BMSC and FIB known as stromal cells in tumors, were co-cultured, yielding three types of multicellular tumor spheroids. During a 48-hour culture, 3D multicellular tumor spheroids were formed by self-organization of single cells on the plate.
(2) Diameters of 3D Multicellular Tumor Spheroids
During the next two-day culture period, irrespective of types of stromal cells co-cultured with tumor cells, three types of 3D multicellular tumor spheroids having a diameter of 500 μm to 600 μm were formed, (
(3) Roundness of 3D Multicellular Tumor Spheroids
The roundness represents a clear or smooth boundary (2D). The extent of roundness of a spheroid indicates a roundness of a projected region of the spheroid. The roundness is in a range of 0 to 1, and the closer to 1 the roundness is, the higher the roundness of a projected region of the spheroid is. The produced three types of spheroids have a roundness of 0.99 or greater, which is close to 1.0 (
(4) Sphericity of 3D Multicellular Tumor Spheroids
According to the sphericity, tumor spheroids may be classified as spherical tumor spheroids having a sphericity of 0.90 or greater (sphericity index; SI≥0.90) or non-spherical tumor spheroids having a sphericity of not greater than 0.90 (SI≤0.90). All of the produced three types of spheroids had a specificity index of greater than 0.99, that is, nearly close to 1.0. This suggests that all of the spheroids are formed in a spherical shape, that is, a three-dimensional well-defined shape (
3. 3D Multicellular Tumor Spheroid Assay
(1) Distribution of Tumor Cells in 3D Multicellular Tumor Spheroids
In order to mimic an interaction between a stromal cell and a cancer (tumor) cell, which is one of tumor microenvironment characteristics, 3D multicellular tumor spheroids were designed. Distributions of positions of the respective cell types (breast cancer cells and stromal cells) were identified from the 3D multicellular tumor spheroids using a cell tracker. Before seeding, stromal cells (ASC, BMSC and FIB) and human breast cancer cells (MDA-MB-231) were stained with a Cell Tracker™ Green CMFDA dye and a Cell Tracker™ Red CMTPX dye. After forming 3D multicellular tumor spheroids, distributions of cancer cells were observed in the spheroids using a confocal microscope.
Interestingly, the cancer cells and stromal cells were differently distributed in the 3D multicellular tumor spheroids according to the type of stromal cell (
(2) Surfaces of 3D Multicellular Tumor Spheroids
Surfaces of three types of 3D multicellular tumor spheroids were observed and compared, and, as a result, all of the three types of 3D multicellular tumor spheroids had surprisingly different surfaces according to the type of stromal cell (
The ASC+MDA-MB-231 spheroid looked as if it were surrounded by ECM components and had a smooth surface without any kind of cell found. Meanwhile, much more cancer cells were distributed on the surface of the FIB+MDA-MB-231 spheroid than on the surfaces of other spheroids, and cancer cells and stromal cells were uniformly distributed on the surface of the BMSC+MDA-MB-231 spheroid.
(3) Accumulation of ECMs on 3D Multicellular Tumor Spheroids
3D multicellular tumor spheroid sections were immunofluorescence stained on extracellular matrix (ECM) protein, collagen type 1 and fibronectin. Type 1 collagen and fibronectin were much more abundantly expressed in the ASC+MDA-MB-231 spheroid than the FIB+MDA-MB-231 spheroid and the BMSC+MDA-MB-231 spheroid (
Specifically, collagen type 1 was over-expressed on the surface of the ASC+MDA-MB-231 spheroid, compared to other regions of the ASC+MDA-MB-231 spheroid. This reflects a fact that collagen is an ECM protein present most abundantly on areas other than a primary breast cancer area. In addition, the smooth property of the ASC+MDA-MB-231 spheroid surface due to high ECM secretion may contributes to hiding shapes of single cancer cells on most parts of the spheroid surface.
As confirmed from the ASC+MDA-MB-231 spheroid, over-expression of ECMs is one of important factors in replication of a cancer tissue, and a cell-ECM interaction plays a key role in a tumor microenvironment. Specifically, expression levels of collagen type 1 and fibronectin were increased in breast cancer cells, which are associated with growth, metastasis and progression of a tumor. In addition, ECM components in the tumor microenvironment and over-expression of collagen type 1 and fibronectin play major roles in drug resistance as well as cancer progression. Particularly, the collagen type 1 is a drug resistance increasing factor. For these reasons, responses to the anticancer drugs may be predicted to be all different due to a noticeable difference in the expression level of ECM proteins in the three 3D multicellular tumor spheroids.
(4) Drug Penetration into 3D Multicellular Tumor Spheroids
To assess drug penetration into three types of 3D multicellular tumor spheroids, the spheroids were treated with doxorubicin as a chemotherapeutic formulation for two days. Doxorubicin is one of common chemotherapeutic agents used in treating a variety types of tumors. To confirm whether structural characteristics have impacts on anticancer drug penetration into spheroids, it was analyzed whether anticancer drug resistance, e.g., insufficient penetration of anticancer drug into the spheroids, was observed using natural red fluorescence of doxorubicin. Fluorescent images after the spheroids were treated with doxorubicin for 48 hours confirmed differential distributions of drugs according to the type of spheroid.
Confocal images of doxorubicin showed differences in the extent of doxorubicin penetrating into the spheroids (
(5) Drug responses to 3D Multicellular Tumor Spheroids
1) Cell viability of 3D Multicellular Tumor Spheroids
With regard to a low penetration level of the drug, the ASC+MDA-MB-231 spheroid treated with 10 μM doxorubicin for two days showed highest cell viability (56.67%), as compared with other types of spheroids. The cell viability of the BMSC+MDA-MB-231 spheroid was 48.33%, which is lower than that of the treated ASC+MDA-MB-231 spheroid, and the FIB+MDA-MB-231 spheroid observed to have a highest drug penetration level had a lowest cell viability (43%). There is a substantial difference in the cell viability between the ASC+MDA-MB-231 spheroid and the FIB+MDA-MB-231 spheroid (P=0.0024). These results suggest that the low level of a drug penetrating into a multicellular spheroid affects low drug efficacy or high drug resistance due to high viability (
In addition, when the same experiment was carried out on a 2D culture plate, the MDA-MB-231 co-cultured with fibroblast cells demonstrated a highest cell viability (
In 3D versus 2D comparison of cell viability, the three types of 3D spheroids were all assessed to have lower drug sensitivity than 2D monolayer spheroids. In addition, doxorubicin responses in three types of 3D breast cancer models showed a propensity opposite to that of 2D cell models, confirming that the structural characteristics of the 3D tumor models in combination with differences in the drug efficacy for the 3D breast cancer models caused drug responses completely different from that for the 2D models.
This result presents a significance in the structural characteristic of a 3D tumor model, suggesting that a structural effect exerted by a 3D cell model, not by a 2D cell model, for screening the efficacy of an anticancer drug, may change the efficacy and propensity of drug.
2) Apoptosis or Necrosis of 3D Multicellular Tumor Spheroids
To investigate drug sensitivity depending on the viability against doxorubicin in the spheroids, Real Time-Glo™ Annexin V apoptosis and necrosis assays were performed. If apoptosis is induced by a drug response, a cell membrane is reversed to expose phosphatidyl-serine (PS), and an apoptotic cell can be analyzed by binding PS with Annexin V detected by a luminescence signal. A necrotic cell is detected by binding DNA with PI (green) that produces a fluorescence signal when the necrotic cell invades into a cell and a cell membrane loses integrity.
A fold change in the expression of Annexin V as an initial apoptosis marker correlates with viability. A highest increasing level of annexin V was observed from the FIB+MDA-MB-231 spheroid treated with doxorubicin. In addition, a lowest level of annexin V was observed from the ASC+MDA-MB-231 spheroid treated with doxorubicin. As confirmed from
4. Formation of 3D Multicellular Tumor Spheroids depending on Change in Conditions and Assays Thereof
(1) Change in Co-Culture Ratios
1) Accumulation of ECMs
Expression of extracellular matrix (ECM) protein in 3D multicellular tumor spheroids according to the co-culture ratio of ASC as a stromal cell and MDA-MB-231 as a breast cancer cell was investigated. 3D multicellular tumor spheroids were formed by varying co-culture ratios of ASC:MDA-MB-231 to 10:0, 3:7, 5:5, 7:3 and 0:10, sections of the 3D multicellular tumor spheroids were immunofluorescence stained to collagen type 1 and fibronectin. As a result, when the co-culture ratio of ASC:MDA-MB-231 was 5:5, expression levels of collagen type 1 and fibronectin were highest (
2) Drug Permeation and Cell Viability
To assess drug penetration into 3D multicellular tumor spheroids depending on the co-culture ratio, the spheroids were treated with doxorubicin as a chemotherapeutic agent for two days. Thereafter, actual images of the spheroids were observed, and the extent of a drug penetrating into the spheroids was assessed using natural red fluorescence of doxorubicin. As a result, fluorescent images of spheroids after the spheroids are co-cultured with doxorubicin for 48 hours showed differential drug distributions according to co-culture ratios. Specifically, the 3D multicellular tumor spheroid having a co-culture ratio of ASC:MDA-MB-231 being 5:5, was confirmed to have a drug less penetrating into the core thereof than other spheroids having different co-culture ratios (
Next, the viability of each of 3D multicellular tumor spheroids depending on the co-culture ratio was analyzed. As a result, the 3D multicellular tumor spheroid having a co-culture ratio of ASC:MDA-MB-231 being 5:5 was confirmed to show a relatively high viability compared to other spheroids having different co-culture ratios (
The results showed that the expression level of ECM was higher in the 3D multicellular tumor spheroid having stromal cells and breast cancer cells co-cultured in a 5:5 (1:1) ratio than in other spheroids having different co-culture ratios, suggesting that penetration of doxorubicin into the spheroid was inhibited by the high expression level of ECM. In addition, the viability was highest in the 3D multicellular tumor spheroid having the ASC:MDA-MB-231 co-culture ratio of 5:5, in which doxorubicin penetration was least, and thus drug penetration into the spheroid was highly inhibited, suggesting that the inhibited drug penetration may affect low efficacy or high resistance of drug.
(2) Change of Anticancer Drug
1) Cell Viability
To confirm drug penetration inhibiting effects of 3D multicellular tumor spheroids for various anticancer drugs, 44 anticancer drugs being in clinical use or clinical trial, except for doxorubicin, were treated for 48 hours, and viabilities of cells in the 3D multicellular tumor spheroid (ASC+MDA-MB-231) and the 3D single cellular tumor spheroid were comparatively analyzed (
The 3D single cell tumor spheroid was formed by a single culture of tumor cells on a 5% matrigel plate coated with poly-HEMA. 50 μL tumor cells were plated on each well of the plate at a density of 0.5×104 cells/well and then incubated in a 5% CO2 incubator at 37° C. for 48 hours. During a 48 hour culture, the 3D single cell tumor spheroid was formed by self-organization of the cells on the plate.
When 30 out of a total of 44 types of anticancer drugs (about 68.18%) were treated, the 3D multicellular tumor spheroids showed a higher viability than the 3D single cell tumor spheroid, and when 14 anticancer drugs (about 31.82%) were treated, the 3D multicellular tumor spheroids showed a lower viability than the 3D single cell tumor spheroid (
2) Confirmation of Relevance of Anticancer Drug with Chemical Features and Cell Viability
To confirm which of major chemical features of an anticancer drug affects viability in a tumor spheroid and drug penetration, a multiple regression analysis method was performed. In detail, as shown in
The extracted chemical features may include molecular weight (M·W) of drug, distribution coefficient (Log P), water solubility (Log S), acid dissociation equilibrium constant (pKa), physiological charge, hydrogen acceptor count, hydrogen donor count, polar surface area, rotatable bond count, polarizability, refractivity, and number of rings, which were used as input parameters (
Thereafter, a difference in the viability (or permeability) between a 3D multicellular tumor spheroid and a 3D single cell spheroid was obtained from three independent experiments, and the obtained difference was used as an output parameter. Since a difference in individual chemical features of anticancer drugs could not perfectly account for cell viability, a multiple regression equation was derived in consideration of interactions among various factors as well as the respective chemical features.
PO=a+bK+cL+dM+eN+fO+gP+hQ+iR+jS
In the above equation, PO is a predicted cell viability difference (or permeability), K is M·W(g/mol), L is log P, M is log S, N is a hydrogen acceptor count (units), O is a hydrogen donor count (units), P is a polar surface area (Å2), Q is a rotatable bond count (units), R is refractivity (m3/mol), S is polarizability (Å3), and a to j are constant values: a=0.1235313827, b=0.0035738171, c=0.0340283393, d=0.0340283393, e=−0.000519426, f=0.0108241714, g=−0.002123276, h=0.0007481956, i=−0.0050597, and j=−0.018713752.
The present inventors compared the derived equation with the actual output parameter shown in
3) Penetration of Drug
Next, the extents of various types of anticancer drugs penetrating into 3D multicellular tumor spheroids were investigated. To this end, two chemo therapeutic agents, i.e., epirubicin and topotecan, used as therapeutic agents for various types of solid tumors, including a breast cancer, were used. Since epirubicin and topotecan naturally emit red fluorescence and green fluorescence, respectively, drug penetration can be detected by observing representation of fluorescence. Like in the previous case of doxorubicin, the FIB+MDA-MB-231 spheroid and the BMSC+MDA-MB-231 spheroid were used as control groups to be compared with the 3D multicellular tumor spheroid (ASC+MDA-MB231) in view of the extent of drug penetration.
These anticancer drugs were treated in the spheroids for 48 hours, and distributions of the anticancer drugs were analyzed using a fluorescence microscope. As a result, in both of the anticancer drugs, the 3D multicellular tumor spheroid (ASC+MDA-MB-231) showed statistically signifixcantly low drug permeability, compared to BMSC+MDA-MB-231 and FIB+MDA-MB-231 spheroid (
(3) Diversification of Tumor Cell
1) Distribution and Morphology of Tumor Cell
A solid tumor has a feature in that stromal cells are positioned on the surface of a tumor cell in a tumor microenvironment and ECMs are highly distributed on the surface of the tumor cell Therefore, an attempt was made to confirm whether such a feature is exhibited even in cases where 3D multicellular tumor spheroids are formed using solid tumor cells other than MDA-MB-231 breast cancer cells. To this end, A549 (lung cancer cell), HT1080 (fibrosarcoma cell), MKN45 (stomach cancer cell), SK-BR-3 (breast cancer cell) and MCF-7 (breast cancer cell) were used, and distributions of positions of the respective cell types ((solid tumor cells and stromal cells) were identified from the 3D multicellular tumor spheroids using a cell tracker. Before seeding, the stromal cells and solid tumor cells (A549, HT1080, MKN45, SK-BR-3, and MCF-7) were stained with a Cell Tracker™ Green CMFDA dye and a Cell Tracker™ Red CMTPX dye, respectively. After forming the 3D multicellular tumor spheroids, spheroid morphologies were observed using an optical microscope and distributions of cancer cells in the spheroids were observed in the spheroids using a confocal microscope.
When 3D tumor spheroids were formed using single tumor cells, no spheroids were formed in most of solid tumor cells, or spheroids, if any, were formed in an inconsistent shape. However, in cases of 3D multicellular tumor spheroids, spheroids having a consistently spherical shape were formed in all of five types of solid tumor cells (
2) Expression of ECM
Next, in the 3D multicellular tumor spheroids using solid tumors, expression levels of extracellular matrix (ECM) protein, collagen type 1 and fibronectin were identified by immunofluorescence staining. Three types of solid tumor cells (HT-1080, A549, and MKN45) were used. Whereas there were little expression levels of collagen type 1 and fibronectin in the 3D tumor spheroids using single tumor cells, collagen type 1 and fibronectin were highly expressed in all the 3D multicellular tumor spheroids using three types of solid tumor cells, compared to single cell spheroids (
The spherical 3D tumor spheroid according to an aspect has an appropriate diameter, roundness and specificity so as to be suitably used in vitro, and expresses an ECM structure similar to that of in vivo tumor, and thus may be used in evaluating the efficacy of drug for treating various types of tumors.
It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0172372 | Dec 2019 | KR | national |
| 10-2020-0176595 | Dec 2020 | KR | national |
