Organoids can be used to model and study the initiation and progression of lung diseases. Combined with CRISPR/Cas9 gene editing technology, lung organoids are well suited to identify genes that play important roles in disease development. As a striking example, Na et al. (2022) generated an organoid-derived SCLC model and found that Kmt2c deficiency promoted SCLC tumorigenesis and metastasis. This study was the first to reveal the function of KMT2C gene in SCLC. Differently from previously reported in leukemia (Chen et al., 2014), KMT2C deficiency promotes SCLC tumorigenesis and metastasis through DNMT3A-mediated epigenetic reprogramming. Strikoudis et al. (2019) found that the deletion of HPS1, HPS2, or HPS4 in lung organoids generated from ESCs promoted fibrotic changes in lung organoids. However, the deletion of HPS8 did not. Zheng et al. (2022) identified DNAH9 mutation in primary cilia dyskinesia patient, and constructed Dnah9 knock-down organoids, which displayed typical Primary cilia dyskinesia phenotypes: reduced ciliary beating and increased secretion of inflammatory factors.

The mutations of the CFTR gene cause CF (Stutts et al., 1995). CF organoid models could contribute to CFTR gene functional studies. Organoids derived from patients with CF allow the assessment of CFTR function in an organoid swelling assay. Dekkers et al. (2013) found that the function of the CFTR F508del mutant protein was restored by CFTR-restoring compounds. Wong et al. (2022) revealed the conductance defect of the rare CFTR mutation R352Q. Organoids derived from CF patients are also used to generate biobank for the study of gene correction by adenine base editors and CFTR repair (Geurts et al., 2020). CFTR is an attractive target for gene editing approaches. Gene therapy has been used to correct the CFTR locus using CRISPR/Cas9 and homologous recombination in CF patient-derived organoids. In these studies, organoids are used as a tool to model lung cancer and fibrotic lung disease and identify disease-associated genes.

Because organoids represent most cellular components found in the organ of origin, they are widely applied to study the interactions between host and pathogen, particularly tropism and host immune response. Lung organoids derived from human PSCs have been used to study the cell tropism of SARS-CoV-2. Pei et al. (2021) demonstrated that SARS-CoV-2 infects ciliated, club, and AT2 cells. ASGR1 and KREMEN1 were identified as alternative receptors of SARS-CoV-2 and played essential roles in ACE2-independent virus entry (Gu et al., 2022). In SARS-CoV-2-infected airway organoids, HIF1ɑ signaling regulated glycolysis, and GW6471 blocked infection by inhibiting the HIF1ɑ-glycolysis axis (Duan et al., 2021). Using human ASC-derived lung organoids, Bui et al. (2019) reported that IBVs infected various cell types, including ciliated, club, goblet, and basal cells. Similarly, human airway organoids have been employed in studying the cell tropism of IAV (Hui et al., 2018). H1N1, H7N9, and H5N6 viruses infected ciliated cells and goblet cells, but not basal cells in organoids, as observed in bronchus explants.

On the other hand, these models that mimic the cellular response to pathogen infection are crucial for studying the pathogenesis of the pathogen and developing vaccines and therapeutics. Katsura et al. (2020) reported an alveolosphere culture system for the propagation and differentiation of human AT2 cells. This system also supports the immune response to SARS-CoV-2 infection, and transcriptome analysis of infected organoids mirrors inflammatory responses in COVID-19 lungs. Low-dose interferon pre-treatment has proved to block SARS-CoV-2 replication in organoids, suggesting an effective therapy for COVID-19. Similarly, Youk et al. (2020) developed a SARS-CoV-2 infection model in which the expression of interferon-associated genes (such as IFI27 and IFI6) and proinflammatory genes (such as DDX58 and IL6) significantly increased, indicating an innate immune response in organoids. In iPSC-derived lung organoids, interferons, cytokines, and chemokines are induced, and key genes of the inflammasome pathway are activated after infection (Tiwari et al., 2021). Salgueiro et al. (2022) found that cancer-derived lung organoids showed a more decreased innate immune response to influenza A virus than those derived from healthy tissue. Overall, these models offer a robust system for studying pathogen infection and developing effective therapies (Rodrigues Toste de Carvalho et al., 2021).

It is becoming well established that interactions between the stroma and tumor cells play a critical role in tumor growth and progression (Bussard et al., 2016). As described by Lambrechts, 52 stromal subtypes have been identified in human lung tumors, including tumor-associated fibroblasts, endothelial cells, and tumor-infiltrating immune cells (Lambrechts et al., 2018). However, little is known about the mechanisms of interactions between the stroma and tumor cells. The organoid in vitro co-culture systems provide a novel method for studying these cell-cell interactions. Chan et al. developed an in vitro 3D NSCLC model comprising human lung adenocarcinoma cells, fibroblasts, endothelial cells, and NSCLC cancer stem cells. This model recapitulates the tumor microenvironment characteristics and tumor cell heterogeneity (Chan et al., 2016). Using the alginate microencapsulation strategy, Rebelo et al. (2018) established a 3D cell model enclosing three cellular components: NSCLC cells, cancer-associated fibroblasts, and monocytes. The “3D-3-culture” model recreated the activation of monocytes into tumor-associated macrophages in vitro and demonstrated the macrophage plasticity in response to chemotherapy and immunotherapy. Similarly, by co-culturing AT2 cells with macrophages in an organoid system, Lechner et al. (2017) revealed that type 2 innate lymphoid cells and lung macrophages supported alveolar epithelial stem cell proliferation and differentiation. Ferreira et al. (2018) engineered hybrid 3D multicellular tumor spheroids that combined A549, fibroblasts, and human mesenchymal stem cells from bone marrow (hBM-MSCs) with bioinstructive hyaluronan microparticles. In this in vitro model, hBM-MSCs exhibit dynamic interactions with cancer cells and fibroblasts, as observed in vivo. To study tumor metastasis, Ramamoorthy et al. (2019) developed a multicellular lung organoid model that resembles the architecture of metastatic tumors in vivo. This study provides an in vitro model for studying tumor metastasis and has significant utility in developing ideal therapeutic agents. These findings demonstrate the ability of organoids to recapitulate the interactions of tumor cell-stroma cell, suggesting the potential use of organoid models to develop novel therapies targeting the tumor microenvironment.

During the past decades, scientists have used the traditional 2D culture cell lines to screen antitumor drugs, most of which have proved ineffective in clinical studies (Caponigro and Sellers, 2011). The current research leads us to believe that the organoid technology can fill the gap between classical 2D cell lines and clinical trials. Shi et al. (2020) established a protocol for developing NSCLC organoids to lay the foundation for drug screening and biomarker identification. Jabs et al. (2017) developed an automated microscopy-based assay to resolve drug-induced cell death and proliferation inhibition. This has been used to screen clinically relevant drugs for lung cancer treatment. Kim et al. (2019) created a biobank of 80 lung cancer organoid lines from five subtypes of lung cancer that can be used for anticancer drug screening. Based on genomic alterations, organoids derived from different patients respond differently to drugs: A BRCA2-mutant organoid responds to olaparib, an EGFR-mutant organoid responds to erlotinib, and an EGFR-mutant/MET-amplified organoid responds to crizotinib. Because p.M965I is non-pathogenic to BRCA2 function, organoids with the BRCA2 p.M965I mutation had a higher olaparib IC50 than organoids with the BRCA2 p.W2619C mutation. Using SCLC patient-derived organoids, Choi et al. (2021b). developed a novel CDK7 inhibitor, YPN-005, which showed potent anticancer effects compared to the CDK7 inhibitor THZ1. Human PSC-derived lung organoids infected with SARS-CoV-2 can serve as valuable in vitro platforms for drug screening to identify candidate COVID-19 therapeutics. Through high-throughput screening, some FDA-approved drugs, including imatinib, mycophenolic acid, and quinacrine dihydrochloride, have proven to be entry inhibitors of SARS-CoV-2 (Han et al., 2021). Human airway epithelium organoids also were used to test the treatment effect of molnupiravir against different SARS-CoV-2 variants (Lieber et al., 2022). Saul et al. (2021) revealed that Pan-ErbB inhibitors rescued cells from SARS-CoV-2 replication, inflammation and lung injury. Enzalutamide, a second-generation antiandrogen agent, effectively inhibited SARS-CoV-2 infection in human prostate cells. However, enzalutamide showed no antiviral activity in human lung organoids, suggesting its inappropriateness in treating COVID-19 through targeting lung cells (Li et al., 2021b).

In addition to lung cancer and infectious diseases, 3D lung organoids also have been used to develop novel therapeutic strategies for IPF and CF. Using an in vitro IPF model, Surolia et al. (2019) observed that vimentin intermediate filament assembly regulated fibroblast invasion in fibrogenic lung injury. Following treatment with withaferin A, an inhibitor of vimentin intermediate filament assembly, 14 of 15 patient pulmospheres demonstrated inhibition of invasion. Chemical screening performed in IPF organoids showed that ALK5 inhibitor and integrin ɑVβ6 antagonist ameliorated the fibrogenic changes (Suezawa et al., 2021). A multimodal iPSC-derived organoid platform was generated for CF drug testing. This platform can be used to accelerate therapeutic development for CF caused by rare variants (Berical et al., 2022). Sodium/glucose cotransporters were revealed as potential therapeutic targets for CF (Hirai et al., 2022).

Owing to the heterogeneity of the disease, certain treatments work well for some patients but do not show promising results in others. Personalized medicine applies specific treatments to each patient. PDX model facilitates personalized medicine (Li et al., 2020; Xia et al., 2019; Aboulkheyr Es et al., 2018). However, they have several disadvantages, including a low success rate, high requirements for clinical samples, species differences, and most importantly, the inability to achieve high-throughput drug screening. Organoids derived from patients recapitulate the in vivo characteristics of genomic alterations, morphology, transcriptome, and drug response, even after long-term culture (Pasch et al., 2019). Thus, patient-derived organoids (PDOs) have been used as preclinical models to develop personalized medical strategies that provide support for clinical trials. Liello et al. reported a clinical case in which a patient with NSCLC responded significantly to the anti-PD-1 drug pembrolizumab. In concordance with the clinical response, in treating patient-derived 3D spheroids, researchers observed a high sensitivity to immunotherapy, whereas they did not see a significant response to standard cisplatin-based chemotherapy (Di Liello et al., 2019). Similarly, using organoids derived from a HER2-A775_G776YVMA-inserted advanced lung adenocarcinoma patient sample, Wang et al. (2019) demonstrated the antitumor activity of pyrotinib. In phase II clinical trial of pyrotinib 400 mg orally daily, 15 patients with HER2-mutant NSCLC showed an objective response rate of 53.3% and a median progression-free survival of 6.4 months. PDO-based drug testing revealed that the combination of FGFR and MEK inhibitors showed better treatment effects than a single FGFR inhibitor in FGFR1 amplified LUSC (Shi et al., 2020). PDO models from NSCLC patients with EGFR exon 20 insertions were used to characterize the antitumor activity of amivantamab. As observed in the organoid model, amivantamab has potent antitumor activity in NSCLC patients with EGFR exon20ins disease (Yun et al., 2020; Park et al., 2021). In LKB1-Deficient NSCLC organoids, the WEE1 kinase inhibitor AZD1775 alone and in combination with DNA agents has significant anticancer effects (Richer et al., 2017), thus providing a potential clinical application for AZD1775 in LKB1-Deficient NSCLC. Lung adenocarcinoma patient derived organoids were used to identify effective anti-cancer drugs poziotinib and pralsetinib for ERBB2 exon 20 insertions and RET fusions, respectively (Kim et al., 2021). Given the low success rates and the lengthy time in establishing PDOs, Hu et al. (2021) developed an integrated superhydrophobic microwell array chip, which could facilitate high-throughput 3D culture and drug test of lung cancer organoids within a week. For targeted therapy or chemotherapy, the drug test results are in good agreement with clinic outcomes. These findings highlight the important translational nature of organoid-based personalized medicine.

Over the past decade, immunotherapy has shown substantial clinical activity for a subset of cancer patients (Riley et al., 2019; O'Donnell et al., 2019). Immune checkpoint inhibitors (ICIs), particularly inhibitors of the PD-1 axis, have led to the increased overall survival of patients with NSCLC (Doroshow et al., 2019; Ko et al., 2018; Gandhi et al., 2018). However, the patient’s response to ICIs depends on the expression of target molecules and the tumor microenvironment. Platforms that allow the analysis and modeling of the complexity of the microenvironment would greatly contribute to the understanding of the critical mechanism that mediates a successful antitumor immune response (Dijkstra et al., 2018; Yuki et al., 2020; Wu et al., 2022). Several recent studies have successfully established in vitro culture systems to induce and analyze the tumor immune microenvironment (Bar-Ephraim et al., 2020). Lung organoids have also been used to study NSCLC immunotherapy in vitro. Dijkstra et al. (2018) developed a co-culture strategy of autologous tumor organoids and peripheral blood lymphocytes. This method allows tumor-reactive T cells to be enriched from the peripheral blood of patients with NSCLC or CRC. Furthermore, these T cells can kill the matched tumor organoids. This study provides a means to assess the sensitivity and resistance of tumor cells to immunotherapy (Cattaneo et al., 2020). In the same year, Neal et al. (2018) used a patient-derived organoid ALI system to model the tumor immune microenvironment, in which the original tumor T cell receptor spectrum was preserved and the immune checkpoint was blocked with anti-PD-1 and/or anti-PD-L1. Using this method, PDO tumor-infiltrating lymphocytes from NSCLC recapitulated the PD-1-dependent immune checkpoint. Thus, PDOs that incorporate immune and other stromal components may contribute to personalized immunotherapy for cancer. Hai et al. generated genetically engineered mouse lung organoid models of squamous cell lung cancer. Using this genetically defined mouse model and 3D tumor organoid culture system, they revealed that WEE1 inhibition can enhance the antitumor activity of anti-PD-1 monotherapy. Combined immune checkpoint blockade and WEE1 inhibition enhances NK and T cell recruitment in tumors and decreases immunosuppressive neutrophilic tumor infiltration (Hai et al., 2020), suggesting that organoid technology can also be used in the study of combinatorial immunotherapy.