Method for constructing an in vitro model of immune-related interstitial pneumonia

Abstract

The present invention provides a method for constructing an in vitro model of immune-related interstitial pneumonia (CIP), and the CIP in vitro model is divided into an in vitro model at the cell (sphere) level and an in vitro model at the organoid level. The method for constructing the CIP model at the cell level includes treating a co-culture system of lung cancer cells and activated PBMC cells with PD-1 monoclonal antibodies, and obtaining a culture supernatant containing PMBCs in the co-culture system. The culture supernatant is used as a "conditioned medium" for the culture of lung epithelial cells or lung epithelial cell spheres. The method for constructing the CIP model at the organoid level includes treating normal lung tissue organoids with PD-1 monoclonal antibodies and activated PBMC cells. The in vitro CIP model established by the present invention can better simulate the in vivo pathogenesis and is suitable for in vitro experiments for studying the mechanism of immune-related interstitial pneumonia.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing an in vitro model of immune-related interstitial pneumonia. Background Art

[0002] Immune checkpoint inhibitors are currently common anti-tumor immunotherapies that can achieve lasting anti-tumor effects in a considerable number of patients. However, about 20% of patients experience grade 3-5 severe immune-related adverse events while benefiting from clinical anti-tumor effects. For patients with severe adverse events, regardless of the anti-tumor efficacy of immunotherapy, they are required to permanently stop immunotherapy. Therefore, immune-related adverse reactions limit the further development of immunotherapy and put these patients with well-controlled tumors at risk of poor control of the primary disease again. Faced with the increasing number of immune-related adverse events and the poor clinical prognosis caused by them, there is an urgent need for scientific research to explore their pathogenesis and develop preventive and mitigating drug regimens. However, since there is no recognized in vitro model to verify these pathogenesis and the discovered therapeutic targets cannot be verified, the first-line treatment for adverse reactions is currently limited to the use of steroids. It is worth noting that the mortality rate of patients who do not respond well to steroid treatment is as high as 75%, which makes it urgent to develop in vitro models that can simulate the pathophysiological process of irAEs. Summary of the invention

[0003] In order to solve the above technical problems, the present invention uses lung cancer cells, cell spheroids and normal lung tissue organoids to construct an in vitro model of immune-related interstitial pneumonia. Specifically,

[0004] The first aspect of the present invention provides a method for constructing an in vitro model of immune-related interstitial pneumonia at the cellular level, the method comprising using conditioned medium for culturing lung epithelial cells or lung epithelial cell spheroids to obtain an in vitro model of immune-related interstitial pneumonia; the conditioned medium is the culture supernatant obtained after a co-culture system of lung cancer cells and activated PBMC cells treated with PD-1 monoclonal antibody.

[0005] In certain embodiments, the culture supernatant contains PBMCs.

[0006] In certain embodiments, the conditioned medium is used for culturing lung epithelial cells or lung epithelial cell spheroids for 12-36 hours, preferably about 24 hours.

[0007] In certain embodiments, the lung cancer is selected from A549, H520 and H1299.

[0008] In certain embodiments, the lung epithelial cells are selected from BEAS-2B and HPAE Pic.

[0009] In certain embodiments, the PD-1 monoclonal antibody is selected from pembrolizumab (Keytruda), nivolumab, sintilimab, toripalimab and / or carrelizumab.

[0010] In certain embodiments, the PBMC cells are activated by stimulation with IL-2, anti-CD28 and Y-27632.

[0011] The second aspect of the present invention provides a method for constructing an in vitro model of immune-related interstitial pneumonia at the organoid level, comprising co-culturing lung cancer paracancerous tissue organoids with activated PBMC cells, adding PD-1 monoclonal antibody, and culturing to obtain an in vitro model of immune-related interstitial pneumonia.

[0012] In certain embodiments, the PD-1 monoclonal antibody is selected from pembrolizumab (Keytruda), nivolumab, sintilimab, toripalimab and / or carrelizumab.

[0013] In certain embodiments, the PBMC cells are activated by stimulation with IL-2, anti-CD28 and Y-27632.

[0014] The third aspect of the present invention provides an in vitro model of immune-related interstitial pneumonia at the cellular level constructed according to the method described in the first aspect of the present invention, or an in vitro model of immune-related interstitial pneumonia at the organoid level constructed according to the method described in the second aspect of the present invention.

[0015] The fourth aspect of the present invention provides the use of the in vitro model of immune-related interstitial pneumonia according to the third aspect of the present invention in the study of the mechanism of immune-related interstitial pneumonia.

[0016] The fifth aspect of the present invention provides use of the in vitro model of immune-related interstitial pneumonia according to the third aspect of the present invention in screening drugs for the prevention and / or treatment of CIP.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention successfully constructed an in vitro model of immune checkpoint inhibitor-associated interstitial pneumonia for the first time. It pioneered the use of the "conditional culture" co-culture model of lung epithelial cells or cell spheres and PBMCs, as well as the co-culture model of immune checkpoint inhibitor antibodies, PBMCs, and normal lung organoids. The in vitro CIP model at the cell (sphere) and organoid levels obtained by the present invention is suitable for in vitro experiments for the study of the mechanism of immune-related interstitial pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0020] Figure 1 Shows the process of constructing an in vitro model of immune-related interstitial pneumonia at the organoid level.

[0021] Figure 2 Showing proliferation of patient-derived normal lung tissue organoids.

[0022] Figure 3 Validation of the successful construction of a CIP (immune checkpoint inhibitor-associated pneumonia / immune-associated interstitial pneumonia) model in vitro by co-culturing organoids with PBMCs. A. Damage to organoids under bright field microscopy (20×), and the survival rate of CIPO was detected by ATP luminescence. B. HE and Masson staining of organoids (40×). C. α-SMA immunofluorescence staining and mean fluorescence intensity analysis of CIPO (20×). PBMC, peripheral blood mononuclear cells.

[0023] Figure 4 Overview of the procedures showing the generation of a CIP (Immune Checkpoint Inhibitor Associated Pneumonia) in vitro model of lung epithelium conditional co-culture with PBMCs. A. 3D model: lung epithelial spheroid conditional co-culture. B. 2D model: conditional co-culture with lung epithelial cells. CIP, Checkpoint Inhibitor Associated Pneumonia.

[0024] Figure 5 The results show that the CIP model was successfully constructed in vitro by conditional co-culture of lung epithelial cell lines and PBMCs. A. Observation of 3D spheroids under bright field microscopy. B. Detection of the expression of α-SMA and vimentin in 3D spheroids by QPCR. C. Detection of the expression of IL6, IL1β and MPO in the culture supernatant after conditioned culture of 3D spheroids by ELISA. D. Detection of cell activity in the 2D cell model by cck8. E. Detection of the expression of Fibronectin in 2D cells by western blot. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0027] Example 1 Experimental method

[0028] 1.1 Extraction and stimulation of PBMCs:

[0029] 8 ml of fresh blood sample from healthy volunteers was collected and centrifuged by gradient centrifugation (TBD, Catalog No. LTS1077). The uppermost buffy coat layer was collected and washed with PBS to obtain PBMC. Then, the cells were cultured in RPMI 1640 medium containing 10% FBS, penicillin and streptomycin (1%), and 1 ng / ml IL-2 (STEMCELL TECHNOLOGIES, Catalog No. 78036.1), 5 μg / ml anti-CD28 (eBioscience, Catalog No. 16-0289-81) and 10 μM Y-27632 (APExBIO, Catalog No. A3008) were added to the medium for 24 hours to expand and activate PBMC.

[0030] 1.2 Construction and co-culture of spheroids:

[0031] On the first day, 1.5 × 10 6 BEAS-2B cells were inoculated in RPMI1640 medium in ultra-low adhesion 24-well plates (Corning) and cultured for 2 days. The cells were observed and photographed under an inverted microscope (IX73, N25014). When more than 80% of the spheroids had a diameter >100 μm, subsequent conditioned culture experiments (i.e., "spheroids well plates") were performed. At the same time, 3.5×10 5 A549 lung cancer cells / well were seeded in an adherent six-well plate, and the culture medium was RPMI1640. On the second day, the activated PBMCs in Example 1.1 and 20 μg / ml of pembrolizumab were added at a co-culture ratio of 1:10 (A549:PBMC) and co-cultured for 24 hours. 5 BEAS-2B cells / well were seeded in adherent six-well plates, and the culture medium was RPMI1640. On the third day, the PBMCs co-cultured with A549 were aspirated together with the culture medium and added to the spheroids well plate / BEAS-2B six-well plate at a co-culture ratio of 1:10 (BEAS-2B:PBMC) for conditional co-culture, and the co-culture lasted for 24 hours.

[0032] 1.3 Construction of organoids:

[0033] Normal lung tissue adjacent to the cancer of patients undergoing lung surgery was obtained from the operating room for organoid culture. All tissue samples were immediately stored under sterile conditions at 4°C in the laboratory on the day of collection for extraction and culture of PDO (patient-derived organoids). The tissue was first chopped and digested using a tissue digestion kit (Accuroid, Catalog No. K701), digested on a 37-°C shaker for 4 hours, and then the digestion system was filtered. After centrifugation of the filtrate, the precipitate was PDO. Subsequently, PDO was resuspended in matrix gel and cultured in a special organoid culture medium (Accuroid, Catalog No. M203). PDO was successfully amplified after 2-4 weeks.

[0034] 1.4 Detection of CIPO activity:

[0035] PDO was co-cultured with PBMC and PD1 antibody to generate CIPOs (checkpoint inhibitor pneumonitisorganoids, organoids damaged by inflammation). The activity detection of CIPOs was carried out by detecting ATP (Beyotime Biotechnology, Cat. No. C0061S). Simply put, 100ul of the CIPOs suspension to be tested was mixed with 100ul of the detection solution, incubated at room temperature for 25min, and chemiluminescence detection was performed and read in the Luminometer mode of a multifunctional microplate reader (TECAN, N34227). The amount of ATP was calculated based on the ATP standard curve to calculate the relative activity of the cells.

[0036] 1.5 HE staining and α-SMA immunofluorescence staining:

[0037] After co-culture, CIPOs were collected, dehydrated, fixed, and embedded in paraffin before sectioning. All tissue sections were 4 μm. HE staining was performed using hematoxylin (Biosharp, Catalog No. BL702B) and eosin (LEAGENE, Catalog No. DH0054), and collagen staining was performed using a Masson (LEAGENE, Catalog No. DC0033) kit and immunofluorescence (α-SMA, abcam, ab7817).

[0038] α-SMA and Masson markers show collagen deposition, and collagen is a marker for the occurrence of interstitial pneumonia. Therefore, high expression of α-SMA and Masson indicates the occurrence of interstitial pneumonia.

[0039] 1.6 Western blot:

[0040] After the conditioned co-cultured BEAS-2B cells were washed twice with ice PBS, the total protein of the cells was extracted using a protein extraction kit (Beyotime Biotechnology, Cat. No. P0033-100), and SDS-polyacrylamide gel electrophoresis (PAGE) analysis was performed. After incubation with the primary antibody Fibronectin (ab2413, abcam), the protein bands were detected using an infrared fluorescence scanning imaging system (LICOR Odyssey).

[0041] 1.7 Quantitative PCR:

[0042] The conditioned co-culture system of spheroids and PBMCs was filtered using a transwell chamber (NEST, catalog number 725301), and then the spheroids in the upper chamber were resuspended with PBS, transferred to an enzyme-free 1.5 ml EP tube, and centrifuged at 4°C to collect the conditioned co-cultured spheroids. The spheroid cells were lysed with Trizol reagent (Vazyme, catalog number R401) to extract total RNA, and then reverse transcribed with HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, catalog number R333-01), and q-PCR was performed on a real-time fluorescence quantitative PCR instrument (Roche, N19008) using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-02). The PCR primers are shown below, and the analysis data used the 2–ΔΔCT relative quantification method.

[0043]

[0044] 1.8 ELISA:

[0045] The supernatant was collected after spheroids were co-cultured. Human inflammatory factors IL6 (Biorbty, Cat#: orb775021), IL1β (Biorbty, Cat#: orb775135) and MPO (Biorbty, Cat#: orb774927) were detected using commercial ELISA kits according to the manufacturer's instructions, and data were collected on a multifunctional microplate reader (TECAN, N34227) at an OD of 450 nm.

[0046] Example 2 Experimental Results

[0047] 2.1 Organoid Conditioned Co-culture:

[0048] In order to simulate the scenario in which normal tissues in the body are attacked by activated immune cells, the co-culture system of organoids and immune cells was used for verification ( Figure 1 ). We collected extra paracancerous tissue from lung cancer patients who underwent surgery at Nanfang Hospital for organoid expansion and culture. The culture system still had good proliferation activity at the 4th generation and 36th day of the organoid ( Figure 2 ). When the organoids are successfully expanded and most of them have a diameter of 100 um, PBMCs that have been expanded and activated in advance (as described in Example 1.1) and 20 μg / ml of pembrolizumab are added for co-culture. The organoids damaged by inflammation in this system are checkpoint inhibitor pneumonitis organoids (CIPOs), which can reflect the effect of the immune microenvironment under the action of anti-PD1 on normal lung tissue.

[0049] After PDO and PBMC were co-cultured for 48 hours, the group with anti-PD1 showed that PDO was more damaged by PBMC and PDO activity was worse ( P =0.026)( Figure 3 A). Staining of organoid sections shows that the PDO in the group with anti-PD1 showed poor growth and a loose state ( Figure 3 B) and have more collagen deposition ( P =0.04)( Figure 3 C). The above results all showed that CIPOs were damaged by inflammation.

[0050] 2.2 Cell Conditions Co-culture:

[0051] Two conditional culture models were designed ( Figure 4 In brief, human primary PBMCs were extracted and co-cultured with lung cancer cells and PD1 antibody (pembrolizumab 20 μg / ml). After 24 hours, the supernatant of the culture system (containing PBMCs) was co-cultured with pre-cultured lung epithelial cell spheroids for 24 hours. After the culture, the inflammatory damage of the lung epithelial cell spheroids was detected. The results showed that the conditioned culture group containing PBMCs (i.e. Figure 5 The spheroids in the “PBMC+conditioned medium” were smaller and more loosely packed ( Figure 5 A), mRNA detection of interstitial fibrosis markers αSMA (in terms of numerical value) and Vimentin levels are higher ( P =0.0002)( Figure 5 B) ELISA detection of inflammatory factor IL6 in the co-culture supernatant ( P <0.0001), IL1β ( P <0.0001) and MPO ( P =0.0008) with a higher expression level ( Figure 5C). Similarly, after replacing the 3D sphere with a planar cell culture model, the CCK8 assay showed even poorer activity ( P =0.0006)( Figure 5 D), WB detected more Fibronectin protein expression ( Figure 5 E). Figure 5 The "PBMC" group represents the co-culture of activated PBMC with lung epithelial cells or lung epithelial cell spheres, and the "conditioned medium" group represents the co-culture of the supernatant after removing PBMC from the culture system supernatant with lung epithelial cells or lung epithelial cell spheres. The above results all show that the conditioned culture model has more severe inflammatory damage than the ordinary co-culture model.

[0052] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0053] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for constructing an in vitro model of immune-related interstitial pneumonia at the cellular level, characterized in that: The method comprises taking fresh blood samples from healthy volunteers, centrifuging them by gradient centrifugation, collecting the uppermost buffy coat layer, washing them with PBS to obtain PBMCs, and then culturing them in RPMI 1640 culture medium containing 10% FBS, penicillin and streptomycin (1%), and adding 1 ng / ml IL-2, 5 μg / ml anti-CD28 and 10 μM Y-27632 to the culture medium for 24 hours to amplify and activate PBMCs; On the first day, 1.5 × 10 6 BEAS-2B cells were seeded in ultra-low adhesion 24-well plates in RPMI1640 medium and cultured for 2 days. 5 A549 lung cancer cells / well were seeded in an adherent six-well plate, and the culture medium was RPMI1640; on the second day, the activated PBMCs and 20 μg / ml of pembrolizumab were added at a co-culture ratio of A549:PBMC=1:10, and co-cultured for 24 hours; at the same time, 3.5×10 5 BEAS-2B cells / well were seeded in adherent six-well plates, and the culture medium was RPMI1640; on the third day, the PBMCs co-cultured with A549 were aspirated together with the culture medium, and added to the 24-well plates or six-well plates seeded with BEAS-2B at a co-culture ratio of BEAS-2B:PBMC = 1:10 for conditional co-culture, and the co-culture lasted for 24 hours.

2. A method for constructing an in vitro model of immune-related interstitial pneumonia at the organoid level, characterized in that: The method comprises taking fresh blood samples from healthy volunteers, centrifuging them by gradient centrifugation, collecting the uppermost buffy coat layer, washing them with PBS to obtain PBMCs, and then culturing them in RPMI 1640 culture medium containing 10% FBS, penicillin and streptomycin (1%), and adding 1 ng / ml IL-2, 5 μg / ml anti-CD28 and 10 μM Y-27632 to the culture medium for 24 hours to amplify and activate PBMCs; Normal lung tissue adjacent to the cancer of patients undergoing lung surgery was obtained from the operating room. The tissue was cut into pieces and digested using a tissue digestion kit. The tissue was digested on a shaker at 37°C for 4 hours. The digestion system was then filtered and the filtrate was centrifuged. The precipitate was PDO. The PDO was then resuspended in matrix gel and cultured in a special organoid culture medium. After 2-4 weeks, PDO was successfully expanded. The expanded PDO, the activated PBMC and 20 μg / ml of pembrolizumab were co-cultured.

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