Construction method and application of TRM cell and lung cancer organoid co-culture model

Abstract

The invention discloses a construction method and application of a TRM cell and lung cancer organoid co-culture model, and the method comprises the following steps: respectively digesting lung cancer para-carcinoma tissue and lung cancer tissue into single cells by using first digestive juice, then sorting by using a flow antibody to obtain TRM cells, and carrying out fluorescence labeling by using DIO dye; extracting tumor cells from lung cancer para-carcinoma tissues and lung cancer tissues and culturing the tumor cells into lung cancer organs; the method comprises the following steps: digesting lung cancer organs into uniform cell masses by using TryplE digestive enzyme; the method comprises the following steps: mixing a lung cancer organoid cell block mass and TRM cells according to a preset ratio, resuspending by using matrigel, blowing and beating on ice, inoculating into a pore plate, placing in a 37 DEG C incubator, adding a lung cancer organoid culture medium after the gel is solidified, and culturing for one day to obtain the TRM cell and lung cancer organoid co-culture model. According to the invention, interaction between tumor tissue-derived macrophages and tumor cells can be better reduced, so that research on targeted macrophage drugs can be better carried out.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing a TRM cell and lung cancer organoid co-culture model and its application. Background Art

[0002] Lung cancer is one of the most common cancers. With the popularization of chest CT screening, more early lung cancer patients have been discovered and early intervention has been carried out, which has reduced the mortality rate of lung cancer to a certain extent. However, the 5-year survival rate of most lung cancer patients after diagnosis is still low.

[0003] Therefore, there is an urgent need to identify new therapeutic targets and prognostic therapeutic factors for lung cancer. Tumor-associated macrophages are very important non-cancerous cells associated with tumor progression, playing an important role in carcinogenesis, neoangiogenesis, neurogenesis, immunosuppressive TME (tumor immune microenvironment) remodeling, recurrence, chemotherapy resistance and metastasis.

[0004] In addition to circulating monocyte-derived macrophages, there are also unique tissue-resident macrophages (TRM) in tissues. Resident macrophages in different tissues show completely different chromatin landscapes, transcriptional profiles and functional characteristics. In lung tissue, they are called alveolar macrophages and interstitial macrophages. Studies have shown that this type of tissue-specific macrophage plays a different role in lung cancer than monocyte-derived macrophages. How to develop a model that can restore the interaction between tumor tissue-derived macrophages and tumor cells, so as to better conduct research on macrophage-targeted drugs, is a technical problem that technicians in this field need to solve. Summary of the invention

[0005] The present invention provides a method for constructing a co-culture model of TRM cells and lung cancer organoids and its application, so as to develop a model that can restore the interaction between tumor tissue-derived macrophages and tumor cells, thereby better conducting research on macrophage-targeted drugs.

[0006] One aspect of the present invention provides a method for constructing a co-culture model of TRM cells and lung cancer organoids, comprising: Step S1, digesting the paracancerous tissue and lung cancer tissue into single cells with the first digestion solution, and then sorting them with flow cytometry antibodies to obtain TRM cells, and fluorescently labeling them with DIO dye to make them have green fluorescence; Step S2, extracting tumor cells from lung cancer tissue and culturing them into lung cancer organoids; Step S3, using TryplE digestive enzyme to digest the lung cancer organoids into uniform cell masses; Step S4, the lung cancer organoid cell clumps are mixed with TRM cells in a preset ratio, resuspended with matrix gel, inoculated into a well plate after pipetting on ice, and placed in a 37°C incubator. After the gel solidifies, lung cancer organoid culture medium is added. After one day of culture, a co-culture model of TRM cells and lung cancer organoids is obtained.

[0007] In the above construction method, step S1 specifically includes: Step S101, washing the paracancerous lung cancer tissue and the lung cancer tissue with ice-cold washing solution, placing them on ice, and cutting the tissues into small pieces; Step S102, resuspending the tissue pieces with the first digestion solution, shaking and digesting them at 220 rpm in a shaker at 37°C for 40 minutes, filtering them with a 70 μm filter, and grinding the unbroken tissue with a syringe handle; Step S103, centrifuge at 500 g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10-15 minutes, then centrifuge at 500 g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S104, add FCR blocker, incubate at room temperature for 15 minutes, and add phosphate buffer to wash once; Step S105, adding flow cytometry antibodies to label CD45, CD3, CD14, and CD11b, and incubating at 4°C for 30 minutes; Step S106, after washing once with phosphate buffer, resuspending with phosphate buffer, performing flow cytometry to separate CD45 + Cells, CD3 - Cells, CD14 + Cells, CD11b + Cells, namely, TRM cells are obtained; Step S107, fluorescently labeling the TRM cells with DIO dye to make them have green fluorescence.

[0008] In the above construction method, the components and contents of the cleaning solution are: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X.

[0009] In the above construction method, the composition and content of the first digestion solution are: 1640 culture medium, 1X; collagenase IV, 100U; DNAse I, 50μg / mL.

[0010] In the above construction method, step S2 specifically includes: Step S201, washing the lung cancer tissue with ice-cold washing solution, placing it on ice, and cutting the tissue into small pieces; Step S202, resuspending the tissue pieces with the second digestion solution, shaking and digesting them at 220 rpm in a shaker at 37°C for 40 minutes, filtering them with a 70 μm filter, and grinding the unbroken tissue with a syringe handle; Step S203, centrifuge at 500g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10-15 minutes, then centrifuge at 500g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S204, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend the precipitate with matrix gel, inoculate into a well plate after pipetting on ice, place in a 37°C incubator, add lung cancer organoid culture medium after the gel solidifies, and culture to obtain lung cancer organoids.

[0011] The above construction method, wherein the composition and content of the second digestive solution are: collagenase 1, 2 mg / mL; TryplE digestive enzyme, 30 mM EDTA.

[0012] The above-mentioned construction method, wherein the components and contents of the lung cancer organoid culture medium are: Advanced DMEM / F12, 1X; N-Acetylcysteine, 1.25mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; FGF2, 20 ng / mL; B27, 1X.

[0013] In the above construction method, the preset ratio is: Lung cancer organoid cell clusters: TRM cells = 3:1.

[0014] Another method of the present invention provides the use of the TRM cell and lung cancer organoid co-culture model obtained by the above-mentioned construction method in the efficacy evaluation of targeted drugs.

[0015] According to the construction method and application of the TRM cell and lung cancer organoid co-culture model provided by the present invention, the present invention digests the paracancerous tissue and lung cancer tissue into single cells with the first digestive fluid, and then sorts them with flow cytometry antibodies to obtain TRM cells, and co-cultures the obtained TRM cells with lung cancer organoids to obtain a TRM cell and lung cancer organoid co-culture model, which is more in line with the original immune microenvironment of lung cancer patients, can better restore the model of interaction between tumor tissue-derived macrophages and tumor cells, better reflects the actual situation of patients, and is conducive to the development of tumor macrophage targeted drugs. When applied, paracancerous macrophages can be used as a control at the same time to better evaluate the efficacy of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the flow cytometry sorting diagram of TRM cells; Figure 2This is a co-culture model of TRM cells and lung cancer organoids, and a bright field image of lung cancer organoids; Figure 3 This is a diagram of the co-culture model of TRM cells and lung cancer organoids, and the statistical results of death cells in lung cancer organoids. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0018] An embodiment of the present invention provides a method for constructing a co-culture model of TRM cells and lung cancer organoids, the method comprising the following steps S1 to S4: Step S1, digesting the paracancerous lung cancer tissue and lung cancer tissue into single cells using the first digestion solution, then sorting them using flow cytometry antibodies to obtain TRM cells, and fluorescently labeling them with DIO dye to give them green fluorescence, the components and content of the first digestion solution are: 1640 culture medium, 1X; collagenase IV, 100U; DNAse I, 50μg / mL.

[0019] Wherein, step S1 specifically includes: Step S101, washing the paracancerous tissue and lung cancer tissue with ice-cold washing solution, placing them on ice, and cutting the tissue into small pieces, wherein the washing solution comprises: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X; Step S102, resuspending the tissue pieces with the first digestion solution, shaking and digesting them at 220 rpm in a shaker at 37°C for 40 minutes, filtering them with a 70 μm filter, and grinding the unbroken tissue with a syringe handle; Step S103, centrifuge at 500 g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10 minutes, then centrifuge at 500 g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S104, add FCR blocker, incubate at room temperature for 15 minutes, and add phosphate buffer to wash once; Step S105, adding flow cytometry antibodies to label CD45, CD3, CD14, and CD11b, and incubating at 4°C for 30 minutes; Step S106, after washing once with phosphate buffer, resuspending with phosphate buffer, performing flow cytometry to separate CD45 + Cells, CD3 - Cells, CD14 +Cells, CD11b + Cells, namely, TRM cells are obtained; Step S107, fluorescently labeling the TRM cells with DIO dye to make them have green fluorescence.

[0020] In this embodiment, the flow cytometry antibody is used for sorting. Figure 1 shown.

[0021] Step S2, extracting tumor cells from lung cancer tissue and culturing them into lung cancer organoids.

[0022] Wherein, step S2 specifically includes: Step S201, washing the lung cancer tissue with ice-cold washing solution, placing it on ice, and cutting the tissue into small pieces; Step S202, resuspend the tissue pieces with a second digestion solution, shake and digest at 220 rpm in a 37°C shaker for 40 minutes, filter with a 70 μm filter, and grind the unbroken tissue with a syringe handle, wherein the composition and content of the second digestion solution are: collagenase I, 2 mg / mL; TryplE digestive enzyme, 30 mM EDTA; Step S203, centrifuge at 500g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10-15 minutes, then centrifuge at 500g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S204, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the precipitate with matrix gel, blow on ice and inoculate into a well plate, place in a 37°C incubator, add lung cancer organoid culture medium after the gel solidifies, and culture to obtain lung cancer organoids, wherein the components and contents of the lung cancer organoid culture medium are: Advanced DMEM / F12, 1X; N-Acetylcysteine, 1.25mM; GlutaMax, 1X; HEPES, 10mM; penicillin / streptomycin, 100μg / mL; FGF2, 20 ng / mL; B27, 1X.

[0023] Step S3, using TryplE digestive enzyme to digest the lung cancer organoids into uniform cell clumps.

[0024] Step S4, the lung cancer organoid cell clumps and TRM cells are mixed according to a preset ratio. In this embodiment, the preset ratio is: lung cancer organoid cell clumps: TRM cells = 3:1; then resuspended with matrix gel, blown on ice and inoculated into a 48-well plate, 2 μL per well, placed in a 37°C incubator, and after the gel solidifies, lung cancer organoid culture medium is added. After one day of culture, a co-culture model of TRM cells and lung cancer organoids is obtained, and its bright field image can be referred to Figure 2 .

[0025] Based on the TRM cell and lung cancer organoid co-culture model obtained above, the present invention provides an application of the TRM cell and lung cancer organoid co-culture model in the efficacy evaluation of targeted drugs.

[0026] Specifically, in step S4, after one day of culture, after obtaining the co-culture model of TRM cells and lung cancer organoids, mololimab was added to the co-culture model of TRM cells and lung cancer organoids to block the inhibitory effect between TRM cells and tumor cell CD47-SIRPα; PI dye was added four days later to detect tumor cell death and calculate apoptosis data. For the results, please refer to Figure 2 and Figure 3 .

[0027] In addition, in this embodiment, a separately cultured lung cancer organoid was set up as a control group. Specifically, the digested lung cancer organoid cell mass was resuspended with matrix gel, inoculated into a 48-well plate, 2 μL per well, and placed in a 37°C incubator. After the gel solidified, lung cancer organoid culture medium was added. After one day of culture, lung cancer organoids were obtained. The bright field image can be found in Figure 2 After obtaining lung cancer organoids, Mololimab was also added. Four days later, PI dye was added to detect tumor cell death and calculate apoptosis data. For the results, please refer to Figure 2 and Figure 3 .

[0028] Result analysis: from Figure 1 It can be seen that the content of macrophages extracted from lung cancer tissue is about 58%, which is significantly higher than the content of macrophages extracted from adjacent cancer tissue (about 24%), indicating that the degree of macrophage infiltration in tumor tissue is higher than that in adjacent cancer tissue, and more macrophages can be obtained from tumor tissue.

[0029] Figure 2 In the middle, TMR cells show green fluorescence, and dead cells show red fluorescence. Figure 2 and Figure 3 It can be seen that compared with lung cancer organoids cultured alone, the co-culture model of TRM cells and lung cancer organoids with the addition of TRM cells showed an upward trend in the mortality rate of lung cancer organoids after four days. Although there was no obvious change, macrophages could be observed to gather next to the lung cancer organoids, indicating that TRM cells also have macrophage characteristics after extraction. Compared with PBMC-derived monocytes that have macrophage characteristics only after induction, TRM cells do not need to be induced, which is more convenient.

[0030] At the same time, when lung cancer organoids were co-cultured alone, the addition of mololimab had no obvious effect on their cells, but when mololimab was added after co-culture with TRM cells, TRM cells induced an increase in lung cancer organoid apoptosis, indicating that mololimab effectively blocked the inhibitory effect between CD47-SIRPα. Therefore, the present invention can achieve the efficacy evaluation of the targeted drug mololimab.

[0031] In summary, according to the construction method and application of the TRM cell and lung cancer organoid co-culture model provided by the present invention, the present invention digests the paracancerous lung cancer tissue and lung cancer tissue into single cells with the first digestive fluid, and then sorts them with flow cytometry antibodies to obtain TRM cells, and co-cultures the obtained TRM cells with lung cancer organoids to obtain a TRM cell and lung cancer organoid co-culture model, which is more in line with the original immune microenvironment of lung cancer patients, can better restore the model of interaction between tumor tissue-derived macrophages and tumor cells, better reflects the real situation of patients, and is conducive to the development of tumor macrophage targeted drugs. When applied, paracancerous macrophages can be used as a control at the same time to better evaluate the efficacy of targeted drugs.

[0032] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a co-culture model of TRM cells and lung cancer organoids, characterized in that: include: Step S1, digesting the paracancerous tissue and lung cancer tissue into single cells with the first digestion solution, and then sorting them with flow cytometry antibodies to obtain TRM cells, and fluorescently labeling them with DIO dye to make them have green fluorescence; Step S2, extracting tumor cells from lung cancer tissue and culturing them into lung cancer organoids; Step S3, using TryplE digestive enzyme to digest the lung cancer organoids into uniform cell masses; Step S4, the lung cancer organoid cell clumps are mixed with TRM cells in a preset ratio, resuspended with matrix gel, inoculated into a well plate after pipetting on ice, and placed in a 37°C incubator. After the gel solidifies, lung cancer organoid culture medium is added. After one day of culture, a co-culture model of TRM cells and lung cancer organoids is obtained.

2. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 1, characterized in that: Step S1 specifically includes: Step S101, washing the paracancerous lung cancer tissue and the lung cancer tissue with ice-cold washing solution, placing them on ice, and cutting the tissues into small pieces; Step S102, resuspending the tissue pieces with the first digestion solution, shaking and digesting them at 220 rpm in a shaker at 37°C for 40 minutes, filtering them with a 70 μm filter, and grinding the unbroken tissue with a syringe handle; Step S103, centrifuge at 500 g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10-15 minutes, then centrifuge at 500 g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S104, add FCR blocker, incubate at room temperature for 15 minutes, and add phosphate buffer to wash once; Step S105, adding flow cytometry antibodies to label CD45, CD3, CD14, and CD11b, and incubating at 4°C for 30 minutes; Step S106, after washing once with phosphate buffer, resuspending with phosphate buffer, performing flow cytometry to separate CD45 + Cells, CD3 - Cells, CD14 + Cells, CD11b + Cells, namely, TRM cells are obtained; Step S107, fluorescently labeling the TRM cells with DIO dye to make them have green fluorescence.

3. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 2, characterized in that: The composition and content of the cleaning solution are: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X.

4. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 2, characterized in that: The composition and content of the first digestion solution are: 1640 culture medium, 1X; collagenase IV, 100U; DNAse I, 50μg / mL.

5. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 1, characterized in that: Step S2 specifically includes: Step S201, washing the lung cancer tissue with ice-cold washing solution, placing it on ice, and cutting the tissue into small pieces; Step S202, resuspending the tissue pieces with the second digestion solution, shaking and digesting them at 220 rpm in a shaker at 37°C for 40 minutes, filtering them with a 70 μm filter, and grinding the unbroken tissue with a syringe handle; Step S203, centrifuge at 500g for 5 minutes, discard the supernatant, wash twice with a washing solution, lyse with a red blood cell lysing solution for 10-15 minutes, then centrifuge at 500g for 5 minutes, discard the supernatant, and wash twice with a phosphate buffer; Step S204, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend the precipitate with matrix gel, inoculate into a well plate after pipetting on ice, place in a 37°C incubator, add lung cancer organoid culture medium after the gel solidifies, and culture to obtain lung cancer organoids.

6. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 5, characterized in that: The composition and content of the second digestion solution are: collagenase I, 2 mg / mL; TryplE digestive enzyme, 30 mM EDTA.

7. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 1, characterized in that: The components and contents of the lung cancer organoid culture medium are: Advanced DMEM / F12, 1X; N-Acetylcysteine, 1.25mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; FGF2, 20 ng / mL; B27, 1X.

8. The method for constructing a co-culture model of TRM cells and lung cancer organoids according to claim 1, characterized in that: The preset ratio is: Lung cancer organoid cell clusters: TRM cells = 3:

1.

9. Application of the TRM cell and lung cancer organoid co-culture model obtained according to the construction method according to any one of claims 1 to 8 in the efficacy evaluation of targeted drugs.

Images