Tuen Mun

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Lung cancer shows substantial genetic and phenotypic heterogeneity across individuals, driving a need for personalised medicine. Here, we report lung cancer organoids & normal bronchial organoids established from patient tissues comprising five histological subtypes of lung cancer and non-neoplastic bronchial mucosa as in vitro models representing individual patient. The lung cancer organoids recapitulate the tissue architecture of the primary lung tumours & maintain the genomic alterations of the original tumours during long-term expansion in vitro. The normal bronchial organoids maintain cellular components of normal bronchial mucosa. Lung cancer organoids respond to drugs based on their genomic alterations: a BRCA2-mutant organoid to lớn olaparib, an EGFR-mutant organoid to erlotinib, & an EGFR-mutant/MET-amplified organoid khổng lồ crizotinib. Considering the short length of time from organoid establishment to drug testing, our newly developed model may prove useful for predicting patient-specific drug responses through in vitro patient-specific drug trials.

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Lung cancer is the leading cause of cancer mortality worldwide1. Lung cancer is histologically diverse and includes three major types (adenocarcinoma, squamous cell carcinoma, and small cell carcinoma) and several less frequent types (including adenosquamous carcinoma & large cell neuroendocrine carcinoma). Standard molecular-targeted therapies have been developed according lớn genetic alterations, such as EGFR mutations và ALK fusions2, & expression of biomarkers, such as programmed death-ligand 1 (PD-L1)3. Classical in vitro models, including cancer cell lines, have been essential lớn investigate molecular-targeted therapy based on genetic alterations, as they provided considerable advantages in terms of manipulation, time, & throughput4. However, large-scale genomic analyses of lung cancer demonstrate phenotypic and genomic diversity between individual patients represented by intertumoural và intratumoural heterogeneity5. Cancer cell lines vày not generally maintain their original heterogeneity and three-dimensional (3D) organ structure; therefore, they are fundamentally limited in representing the complexity of lung cancer. Patient-derived xenograft models (PDXs) recapitulate the original cancer in terms of tissue structure4,6,7 and maintain the genetic and histological characteristics of the original cancer for up khổng lồ 14 passages8. However, PDXs have several disadvantages: establishing a PDX model has a low success rate (average 30–40%)7 và requires a long time (from 2 lớn 10 months)6,7,9. In addition, a PDX mã sản phẩm is costly và resource-intensive, limiting statistical power, as well as the potential for high throughput studies10.

Recently, tissue-specific stem cells derived from several adult human organs, such as colon11, stomach12, liver13, pancreas13, & lung14, have been cultured in 3d conditions using hydrogel with collagen or other ECM components, such as Matrigel. In these conditions, cells proliferate và give rise to lớn differentiated progeny that undergo self-organisation15. This characteristic arises from specific organoid culture conditions, which promote stem cell proliferation and differentiation16. Therefore, organoids represent a functional unit that consists of a hierarchy of stem cells and differentiated cells16,17. Human cancer also proliferates and organises into specific tissue architecture in vivo, so cancer cells derived from several human primary tumour samples, including colon18, pancreas19, prostate20, liver21, breast22, and bladder23 cancers, have been used to lớn establish organoids that successfully recapitulate the cancer tissue architecture. Moreover, cancer organoids require less time to become established than PDXs và have been shown khổng lồ stably maintain morphological & genetic features even after long-term expansion11,22. Therefore, cancer organoids derived from human cancer tissues have been suggested as alternative in vitro models that maintain the characteristics of the original tumours and can potentially serve as avatars for selecting anticancer therapeutics24 & biobanking for individual patients.

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Organoid models for both tracheobronchial and alveolar tissue have been developed from adult stem cells or pluripotent stem cells25,26,27 và shown lớn recapitulate the epithelial organisation of these two distinct tissue types in vitro14,26,28. As personalised models for lung cancer, in vitro tissue culture29 or tumour spheroid culture10,30 using a 3d culture system has been studied khổng lồ predict response to lớn anticancer therapy, but have limited growth or recapitulation of original tumour architecture23. More recently, lung cancer organoids (LCOs) culture using the airway organoid culture system has been reported. But there were some limitation khổng lồ lung cancer specific growth và to represent variety of lung cancer31. Here, we report the derivation of organoids from primary lung cancer tissues (LC tissues) và paired non-neoplastic airway tissues, creating a biobank of 80 LCO lines from five subtypes of lung cancer và five normal bronchial organoids (NBOs). We demonstrate that our newly established biobank of LCOs and NBOs faithfully maintain the histological and genetic characteristics of their respective parental tissues and have potential for use in patient-specific drug trials và proof-of-concept studies on targeted therapy and resistance mechanisms.


LCOs are established from five subtypes of cancer tissues

To establish LCOs from LC tissues, we developed a 3 chiều culture protocol in Matrigel (Corning, NY, USA) using minimum basal medium (MBM), which is a suboptimal truyền thông inhibiting growth of normal cell due to depletion of Wnt3a & Noggin. MBM was modified from the truyền thông for lung tumour initiating cells containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) & N2 supplement for insulin & transferrin32,33. Lớn establish LCO in 3 chiều culture, surgically resected LC tissues were dissociated as individual cells or cell clusters, embedded in Matrigel, và submerged in MBM. Even though our MBM contained fewer reagents and growth factors compared lớn other protocols, which contain several reagents and growth factors including FGF1026,27,28, FGF427, FGF728, Noggin26,27,28, and Wnt3a26,27,28, LCOs derived from LC tissues consistently generated round shapes (Fig. 1a). As shown in Fig. 1b, these LCOs were cultured for long-term expansion over 6 months without any change in spherical organoid morphology, and maintained proliferation capacity measured by a marker Ki67 immunolabelling (Supplementary Fig. 1a–c). In addition, we tried organoid culture with a small biopsy tissue lớn determine whether our culture protocol is applicable to samples obtained from a non-operable clinical setting. As shown Supplementary Fig. 1d, the cells from a biopsy successfully cultured to khung organoids.


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LCOs established for the lung cancer biobank. a Bright-field microscopy images of LCOs cultured for 2 weeks. Scale bar, 100 μm. The information of LCOs in these images; LCO-05, LCO-36, và LCO-55; adenocarcinoma, LCO-13; squamous cell carcinoma, LCO-21; small cell carcinoma, LCO-29; large cell carcinoma. b Representative images of long-term cultured LCOs. This LCO was derived from LC-49. Scale bar, 200 μm. c Representative images of successful & failed 2D và 3D cultures derived from lung cancers with different tissue composition. Scale bar, 200 μm. d The graph showing the successful or failed cases according lớn cancer tissue quality and the establishment rate of each cancer models according khổng lồ lung cancer subtypes. Cytologic QC, cytologic unique check. e Pie chart showing the subtypes of established 80 LCOs for the lung cancer biobank. The information of 80 LCOs is shown in Table 2. f Bright-field microscopy images & H&E staining images of LCOs before freezing và after thawing. Scale bar, 200 μm. The information of LCOs in these images: LCO-28 — squamous cell carcinoma; LCO-29 — large cell carcinoma; LCO-51 — adenocarcinoma; LCO-75 — small cell carcinoma; LCO-86 — adenosquamous carcinoma


To assess the efficiency of generating LCOs in comparison with other patient-derived cancer models (2D cultured lung cancer cells & PDXs), we established these three models in parallel from 36 patient tumour tissues of five different histological subtypes as a starting cohort (Table 1). Two-thirds of each LC tissue was transplanted into an immunodeficient mouse to develop a PDX, and the remaining one-third was divided equally khổng lồ culture cells in 2D & LCOs. Due to variation in tumour purity, some samples failed to form LCOs (Fig. 1c). Samples that failed to establish LCOs also failed to establish PDXs (Table 1). Thus, we developed a tissue chất lượng evaluation protocol before culturing LCOs. After dissociating the LC tissues using collagenase, an aliquot of cell suspension was prepared on cytologic slides và stained with H&E to evaluate the proportion of viable epithelial-like cells (Supplementary Fig. 2). As a result, we grouped 23 epithelial cell predominant samples, four fibroblast predominant samples & nine acellular samples (Fig. 1d và Table 1). Epithelial cell predominant samples were used as starting material for 2 chiều cultured primary lung cancer cells (2D-LC cells), organoid culture và PDX. As a result, we successfully generated 2d cells from 23 samples (100%), LCOs from đôi mươi samples (87%), và PDXs from 10 samples (43%) (Fig. 1d và Table 1). There was no difference in the success rate of organoid culture among different lung cancer subtypes (Fig. 1d). Establishment of LCOs and PDXs took ~4 weeks and 3–6 months, respectively. Notably, H&E staining showed that LCOs had similar morphological features khổng lồ patient tissues (Fig. 2). Using this protocol, we banked 80 LCO lines, including the 20 LCOs generated above, from five subtypes of lung cancer: adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma, và small cell carcinoma (Fig. 1e and Table 2). One of key factors of the organoid biobank will be efficient reconstitution of cryopreserved organoids. Therefore, we performed thawing chạy thử for cryopreserved organoids in our biobank. Overall, 39 (70%) of 56 LCOs successfully reconstituted organoid morphology và histologic features of original tissues in our 3 chiều culture protocol (Fig. 1f, Tables 2 và 3).


Table 1 The list of lung cancer samples used for initial cytologic chất lượng check, 2 chiều cell culture, organoid culture và PDX

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LCOs recapitulate the characteristics of the original tissues. ad H&E-stained & IHC-stained images of LCOs & their original LC tissues. The enlarged images in xanh boxes of b showed cytoplasmic keratinisation of individual squamous carcinoma cells. Scale bars, 100 μm. e H&E and IHC-stained images of an adenosquamous carcinoma organoid (upper panel). Individual organoids expressed either adenocarcinoma marker (CK7) or squamous cell carcinoma markers (CK5/6 or p63). Some single organoids showed mixed cell pattern composed of CK7+ và CK7− cells, CK5/6+ & CK5/6− cells, & p63+ and p63− cells. Scale bar, 100 μm. Bright field microscopy & immunofluorescence (IF) images of an adenosqumaous carcinoma organoid originated from a single cell (lower panel). The immunofluorescence staining was performed at Day 18 after seeding. The organoid from a single cell was composed of p63+/CK7− cells & p63−/CK7+ cells. The red arrow indicated a single cell seeded in a micro-well. Scale bar in bright-field microscopy images, 100 μm. Scale bar in IF images, 20 μm