A method for establishing a three-dimensional model of quiescent cells in colon cancer tumors based on matrix hardness

Abstract

The present invention is applicable to the field of bioengineering technology, and provides a method for establishing a three-dimensional model of colon cancer tumor quiescent cells (CQCs) based on matrix hardness. Alginate is sterilized by high-pressure steam and a solution is prepared. After mixing with Matrigel and a cell suspension, a CaCO3 suspension is added to adjust the hardness, and finally a tumor spheroid is cultured under specific conditions. The present invention constructs a three-dimensional cell culture system that is uniform and transparent, has a controllable gel time, a mild environment, and is suitable for cell growth, providing new possibilities for the establishment of an in vitro three-dimensional CQCs model; the present invention achieves a wide coverage of the hardness from healthy colon tissue to colon cancer tissue, can more realistically simulate the in vivo environment, and improves the stability of the model; the present invention discovers and proves for the first time that a hard matrix can promote the generation of CQCs-like characteristics in colon cancer cell spheroids, providing a new perspective and basis for the mechanism of tumor metastasis and recurrence.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology, and in particular relates to a method for establishing an in vitro three-dimensional tumor model. Background Art

[0002] Cancer quiescent cells (CQCs) refer to a population of cells that are dormant or non-proliferating in tumors. Their presence not only prolongs the latency of tumors, but also increases the drug resistance of tumors, affecting the effectiveness of treatment. Therefore, the study of CQCs is of great significance for understanding tumor biology, developing more effective treatment strategies, and preventing tumor recurrence and metastasis.

[0003] At present, the models used by researchers to explore CQCs mainly involve nutrient deprivation or chemotherapy treatment of cells in culture dishes. However, this two-dimensional model is very different from the real situation in vivo, making it difficult to verify scientific research results in vivo. In order to solve this problem, it is urgent to develop an in vitro three-dimensional CQCs model that can simulate the real tumor morphology.

[0004] The development of solid tumors is often accompanied by extracellular matrix hardening. The hardness of a healthy colon is usually less than 0.5 kPa, while the hardness of colon cancer tissue is above 1 kPa. There is currently no evidence that an increase in matrix hardness can induce the production of CQCs. The mechanism of action of CQCs in tumor recurrence and chemotherapy resistance is complex and diverse, involving multiple levels such as cell biology, molecular biology, and immunology. In order to deeply understand the biological characteristics and mechanism of action of CQCs, the present invention proposes a method for establishing a three-dimensional model of colon cancer CQCs based on matrix hardness. Summary of the invention

[0005] The purpose of the present invention is to provide a method for establishing a three-dimensional model of colon cancer CQCs based on matrix hardness, aiming to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Alginate is a natural polysaccharide extracted from brown algae. It has excellent biocompatibility and structural stability and can easily react with certain divalent cations (such as Ca 2+ ) bonded to form a gel. And by controlling the degree of ionic cross-linking, the hardness of the gel can be independently adjusted under the premise of constant concentration and pore size ( Figure 2 ). Chelating agents such as EDTA competitively bind to Ca 2 +, the gel can be decrosslinked and restored to a fluid state, releasing the cells growing therein without loss. However, Alginate does not have cell recognition sites and cannot provide adhesion and growth information for cells. The most widely used commercially available Matrigel® Matrix (Corning, LA, US) is the gold standard for three-dimensional cell culture in vitro. Its initial hardness is about 175 Pa, which is similar to normal colon tissue, but its mechanical properties are poor. The present invention uses Alginate as a cell growth scaffold with controllable hardness, and uses Matrigel (matrigel) to provide cell adhesion and growth information, to prepare a three-dimensional cell culture system with independently adjustable hardness. In addition, the present invention adjusts the hardness of the gel system by instantly mixing the CaCO3 suspension, thereby solving the problem of too fast and uneven gelation of the crosslinked system with CaCl2 or CaSO4.

[0008] The method for establishing a three-dimensional model of colon cancer CQCs based on matrix hardness comprises the following steps:

[0009] Step 1: Select Alginate sodium salt powder and sterilize it by high-pressure steam at a steam pressure of 103.4 kPa and a temperature of 121.3°C for 30 min; dry it after sterilization, add sterile ultrapure water to prepare a solution with a mass fraction of 2%; stir it at 90°C for 4 h until the solution is clear, then cool it to room temperature and store it at 4°C for future use; store Matrigel at 4°C overnight until it becomes liquid;

[0010] Step 2: Resuscitate the desired colon cancer cell line (such as HCT-116 or SW-620), perform normal adherent culture and cell subculture, and after two subcultures, digest the colon cancer cells when the cells reach 80% confluence density and are in good condition, centrifuge at 1000 rpm for 5 min to collect the cells; adjust the cell concentration to 1×10 with DMEM medium. 4 / mL to obtain a cell suspension; place a 50ml sterile centrifuge tube in an ice-water mixture to maintain an external low-temperature environment, and mix the cell suspension, Matrigel and Alginate in the centrifuge tube;

[0011] Step 3: Keep the low temperature environment, absorb the CaCO3 suspension and add it to the mixed solution obtained in step 2, and adjust the amount of CaCO3 suspension added to make the CaCO3 2+ The final concentration reached 7.5 mM (soft matrix) or 15 mM (hard matrix);

[0012] Step 4. Incubate the mixed solution at 37°C for 30 min to wait for complete cross-linking. After cross-linking is complete, add complete culture medium (90% DMEM medium + 10% fetal bovine serum) and culture at 37°C, 5% CO2 for 7 days until tumor spheres with a diameter of approximately 50 μm are formed for subsequent experiments.

[0013] Furthermore, in step 2, the volume ratio of the cell suspension, Matrigel and Alginate is 1:1:2.

[0014] Furthermore, in step 3, the preparation process of the CaCO3 suspension is: prepare a CaCl2 solution and a Na2CO3 solution with a concentration of 1 M, filter and sterilize both, and immediately mix the CaCl2 solution and the Na2CO3 solution to generate a CaCO3 suspension with a concentration of 0.5 M.

[0015] The three-dimensional model of colon cancer CQCs was established according to the method described above.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention provides a method for establishing a three-dimensional model of colon cancer CQCs based on matrix hardness. By cross-linking the Alginate (alginate) in the Matrigel-Alginate gel system with an instantly prepared CaCO3 suspension, a three-dimensional cell culture system with uniformity, transparency, controllable gelation time, mild environment and suitable for cell growth is constructed, which provides new possibilities for the establishment of an in vitro three-dimensional CQCs model.

[0018] 2. The Matrigel-Alginate gel system constructed by the present invention is not only uniform and transparent, but also allows the hardness of the gel to be independently regulated by the CaCO3 suspension, without changing the pores of the gel and the concentration of the polymer precursor, thereby achieving a wide range of hardness coverage from healthy colon tissue to colon cancer tissue. This feature enables the model to more realistically simulate the in vivo environment and improve the accuracy and reliability of the experiment. In addition, CaCO3 is slightly soluble in water, and by slowly releasing CaCO3 in the system, the hardness of the gel can be adjusted. 2+ Ions can slowly cross-link the gel into a uniform and stable state in about 30 minutes, greatly improving the stability of the model.

[0019] 3. This invention first discovered and proved that hard matrix can promote the generation of CQCs-like properties in colon cancer cell spheroids. This discovery provides a new perspective and basis for understanding how the tumor mechanical microenvironment affects the biological behavior of tumor cells, especially the mechanism of tumor metastasis and recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1It is a preparation flow chart of the present invention.

[0021] Figure 2 It is an Alginate gel cross-linked with different volumes of CaCO3 suspension and is in a uniform and transparent state.

[0022] Figure 3 This is a study on the inhibition of the growth of three-dimensional tumor spheroids by hard matrix; a is the rheometer detection of different final concentrations of Ca 2+ Resulting in changes in gel hardness; b is the diameter of HCT-116 tumor spheroids grown in soft / hard matrices measured on days 0, 1, 3, and 7; c is the diameter of SW-620 tumor spheroids grown in soft / hard matrices measured on days 0, 1, 3, and 7.

[0023] Figure 4 The study on the increased invasion of tumor spheroids grown in hard matrices; a shows the morphology of colon cancer tumor spheroids grown in soft / hard matrices through bright field observation, cytoskeleton staining and HE staining, respectively; b shows the number of invasive cell clusters of HCT-116 tumor spheroids grown in soft / hard matrices; c shows the number of invasive cell clusters of SW-620 tumor spheroids grown in soft / hard matrices; scale bar = 50 um, * p < 0.05.

[0024] Figure 5 Expression detection of Ki67 and E-cadherin in tumor spheroids grown in soft / hard matrices; a is immunofluorescence staining of Ki67 and E-cadherin in tumor spheroids; b is Ki67 expression statistics of HCT-116 tumor spheroids grown in soft / hard matrices; c is E-cadherin expression statistics of HCT-116 tumor spheroids grown in soft / hard matrices; d is Ki67 expression statistics of SW-620 tumor spheroids grown in soft / hard matrices; e is E-cadherin expression statistics of SW-620 tumor spheroids grown in soft / hard matrices; scale bar = 20 um, * p < 0.05.

[0025] Figure 6 Figure 3 is a study on the cell cycle arrest of tumor spheroids caused by hard matrix; a is a schematic diagram of the principle of the FUCCI cell cycle indicator system; b is the confocal microscope z-axis three-dimensional imaging of tumor spheroids grown in soft / hard matrices; c is the statistical proportion of cells in the G0 / G1 phase, S phase and G2 / M phase in HCT-116 tumor spheroids grown in soft / hard matrices; d is the statistical proportion of cells in the G0 / G1 phase, S phase and G2 / M phase in SW-620 tumor spheroids grown in soft / hard matrices.

[0026] Figure 7This is a study on the chemotherapy resistance of tumor spheroids caused by hard matrix; a is the live / dead staining of tumor spheroids 24 hours after treatment with chemotherapy drugs; b is the statistical proportion of dead cells in HCT-116 tumor spheroids grown in soft / hard matrices; c is the statistical proportion of dead cells in SW-620 tumor spheroids grown in soft / hard matrices; scale bar = 50 um, * p < 0.05.

[0027] Figure 8 Heat map of CQCs-related differentially expressed genes in transcriptomics of HCT116 tumor spheroids grown on soft / hard matrices. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0030] At present, the understanding of CQCs is not completely clear, but CQCs are believed to have the following five characteristics: 1) arrested proliferation; 2) low metabolic rate; 3) cell cycle arrest; 4) chemoresistance; 5) cancer stem cells (CSCs)-like properties. Some characteristics of CQCs overlap with those of CSCs, such as the expression of stem cell markers and the occurrence of epithelial-mesenchymal transition (EMT). CQCs play an important role in tumor latency and drug resistance. Based on the following examples, we show that hard matrix increases CQCs-like properties in colon cancer cell spheres.

[0031] like Figure 1 Shown is a flow chart for the preparation of tumor spheroids with a diameter of approximately 50 μm. The prepared tumor spheroids are used for subsequent experiments.

[0032] Example 1: Hard matrix inhibits the growth of tumor spheroids;

[0033] like Figure 3As shown in a, the hardness of the matrix in which the cells grow is detected by rheometer (shown as Young's modulus). With the addition of CaCO3, the Young's modulus of the matrix gradually increases. The matrix with 7.5 mM CaCO3 added is a soft matrix, and its modulus is about 500 kPa, which is similar to that of healthy colon tissue. The matrix with 15 mM CaCO3 added is a hard matrix, and its modulus is about 1500 kPa, which is similar to that of colon cancer tissue. We tracked the growth of colon cancer HCT-116 and SW-620 cells grown in soft and hard matrices for one week (photographed the cell clusters in the gel on days 0, 1, 3, and 7 of cell inoculation), and measured and counted the diameters of the generated tumor spheroids, as shown in the figure. Figure 3 As shown in (b), the diameter of HCT-116 tumor spheroids in the hard matrix is ​​significantly smaller than that in the soft matrix. Figure 3 The results in (c) show that the diameter of SW-620 tumor spheroids in the hard matrix is ​​significantly smaller than that in the soft matrix, which indicates that the hard matrix inhibits the growth of tumor spheroids.

[0034] Example 2: Increased invasion of tumor spheroids grown in stiff matrices;

[0035] like Figure 4 As shown in a, through bright field observation and hematoxylin-eosin (HE) staining of colon cancer tumor spheroids grown in soft / hard matrices, it was found that colon cancer tumor spheroids grown in hard matrices were smaller, while colon cancer tumor spheroids grown in soft matrices were larger, and the internal self-organization was similar to a glandular cavity structure. The cytoskeleton was stained with green fluorescent phalloidin, and the cell nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI). It was found that the cytoskeleton of tumor spheroids in hard matrices was more diffuse, and there were more invasive cell clusters. The invasive cell clusters were counted. Figure 4 The results in (b) show that HCT-116 tumor spheroids in stiff matrix have more invasive cell clusters; consistent, Figure 4 The results in Figure c show that the SW-620 tumor spheroids in the stiff matrix have more invasive cell clusters. The above results indicate that the stiff matrix improves the invasiveness of tumor spheroids.

[0036] Example 3: Tumor spheroids grown in hard matrix have low proliferation and high invasion;

[0037] On the 7th day of cell inoculation, the cell clusters were released and fixed with paraformaldehyde. After frozen sections, the tumor spheroids were detected by immunofluorescence staining for the proliferation marker Ki67, and the cell nuclei were marked with DAPI. The results showed that the expression of Ki67, a proliferation-related indicator of tumor spheroids grown in hard matrix, was reduced (e.g. Figure 5 Middle a), in HCT-116 tumor spheroids ( Figure 5 b) and SW-620 tumor spheroids ( Figure 5 In (d), the hard matrix resulted in a significant decrease in the average optical density of Ki67, which is consistent with the conclusion in Example 1 that the hard matrix inhibits the growth of tumor spheroids. In addition, on the 7th day after cell inoculation, the cell clusters were released, fixed with paraformaldehyde, and stained for the epithelial cell marker E-cadherin after frozen sections. It was found that the hard matrix resulted in a significant decrease in the average optical density of HCT-116 ( Figure 5 c) and SW-620 ( Figure 5 e) The expression of epithelial cell marker E-cadherin in tumor spheroids is reduced. The loss of E-cadherin is the initial step of EMT, which means that the hard matrix may induce the occurrence of EMT, which is consistent with the conclusion in Example 2 that the hard matrix improves the invasiveness of tumor spheroids.

[0038] Example 4: Stiff matrix causes cell cycle arrest in tumor spheroids;

[0039] For the detection of cell cycle in tumor spheroids, we used Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) to transfect colon cancer HCT-116 and SW-620 cells. Figure 6 As shown in a, FUCCI fuses two cell cycle regulatory factors, Cdt1 and Geminin, with red fluorescent protein and green fluorescent protein respectively. The content of Cdt1 and Geminin will change with the operation of the cell cycle. The cell cycle stage of the cell can be determined by detecting the two fluorescent signals in the cell. Figure 6 As shown in (b), the tumor spheroids grown in soft matrix are composed of G0 / G1 phase cells inside, while the outside is surrounded by G2 / M phase cells that proliferate into the matrix. The number of G2 / M phase cells outside the tumor spheroids grown in hard matrix is ​​significantly reduced. The number of cells in each cycle was statistically analyzed and it was found that the hard matrix caused the HCT-116 tumor spheroids to be more stable. Figure 6 c) and SW-620 tumor spheroids ( Figure 6 D) The proportion of cells in the G0 / G1 phase increased significantly, indicating that the stiff matrix caused the arrest of cell cycle progression in the S phase.

[0040] Example 5: Stiff matrix leads to chemotherapy resistance in tumor spheroids;

[0041] To evaluate the response of tumor spheroids grown on soft or hard matrices to the first-line drugs oxaliplatin and gemcitabine. Figure 7 The results in (a) suggest that tumor spheroids grown in stiff matrix showed fewer dead cells (shown in red) in response to both chemotherapy drugs. Figure 7 b) and SW-620 tumor spheroids ( Figure 7 c) Live / dead cell statistics revealed that the stiff matrix inhibited the mortality of tumor spheroids induced by chemotherapeutic drugs.

[0042] Example 6: Transcriptomics reveals CQCs-like features induced by stiff substrates;

[0043] Transcriptomic sequencing was performed on HCT-116 tumor spheroids grown in soft / hard matrices, and three independent samples were collected from each of the soft and hard matrix groups. A heat map of differentially expressed genes related to CQCs features in the samples was drawn, such as Figure 8 As shown, stiff matrix causes tumor spheroids to exhibit low metabolism, high invasiveness, stemness, and increased chemoresistance. These results suggest that it is feasible to construct a three-dimensional model of CQCs in vitro using extracellular matrix mechanics as a clue.

[0044] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for establishing a three-dimensional model of quiescent cells of a colon cancer tumor based on matrix hardness, characterized in that: The following steps are involved: Step 1: select alginate powder and sterilize it by high-pressure steam at a steam pressure of 103.4 kPa and a temperature of 121.3°C for 30 min; dry it after sterilization, add sterile ultrapure water to prepare a solution with a mass fraction of 2%; stir it at 90°C for 4 h until the solution is clear, then cool it to room temperature and store it at 4°C for future use; store the matrix gel at 4°C overnight until it becomes liquid; Step 2: Resuscitate the colon cancer cell line, perform adherent culture and cell subculture. After subculture twice, digest the cells when they reach 80% confluence and collect the cells by centrifugation at 1000 rpm for 5 min. Adjust the cell concentration to 1×10 4 / mL to obtain a cell suspension; place a 50ml sterile centrifuge tube in the ice-water mixture, and mix the cell suspension, matrix gel and alginate in the centrifuge tube; Step 3: Take the CaCO3 suspension and add it to the mixed solution obtained in step 2. Adjust the amount of CaCO3 suspension added to make the CaCO3 2+ The final concentration reached 15 mM to form a hard matrix; Step 4: Incubate the mixed solution at 37°C for 30 min to wait for complete cross-linking; After cross-linking, complete culture medium was added and cultured at 37°C in a 5% CO2 environment for 7 days until tumor spheres with a diameter of 50 μm were formed for subsequent experiments.

2. The method for establishing a three-dimensional model of colon cancer tumor quiescent cells based on matrix hardness according to claim 1, characterized in that: In step 2, the volume ratio of the cell suspension, matrix gel and alginate is 1:1:

2.

3. The method for establishing a three-dimensional model of colon cancer tumor quiescent cells based on matrix hardness according to claim 1, characterized in that: In step 3, the preparation process of the CaCO3 suspension is as follows: a CaCl2 solution and a Na2CO3 solution with a concentration of 1 M are prepared, both are filtered and sterilized, and the CaCl2 solution and the Na2CO3 solution are immediately mixed to generate a CaCO3 suspension with a concentration of 0.5 M.

4. A three-dimensional model of resting cells of a colon cancer tumor established according to the method for establishing a three-dimensional model of resting cells of a colon cancer tumor based on matrix hardness according to any one of claims 1 to 3.

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