Transforming tumoroids derived from ALK-positive pulmonary adenocarcinoma to squamous cell carcinoma in vivo

Approximately 3–5% of non-small cell lung cancers (NSCLC) harbor ALK fusion genes and may be responsive to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors. There are only a few reports on cell lines with EML4-ALK variant 3 (v3) and tumoroids that can be subject to long-term culture (> 3 months). In this study, we established tumoroids (PDT-LUAD#119) from a patient with lung cancer harboring EML4-ALK that could be cultured for 12 months. Whole-exome sequencing and RNA sequencing analyses revealed TP53 mutations and an EML4-ALK v3 mutation. PDT-LUAD#119 lung tumoroids were sensitive to the ALK tyrosine kinase inhibitors (ALK TKIs) crizotinib, alectinib, entrectinib, and lorlatinib, similar to NCI-H3122 cells harboring EML4-ALK variant 1 (v1). Unexpectedly, clear squamous cell carcinoma and solid adenocarcinoma were observed in xenografts from PDT-LUAD#119 lung tumoroids, indicating adenosquamous carcinoma. Immunostaining revealed that the squamous cell carcinoma was ALK positive, suggesting a squamous transformation of the adenocarcinoma. Besides providing a novel cancer model to support basic research on ALK-positive lung cancer, PDT-LUAD#119 lung tumoroids will help elucidate the pathogenesis of adenosquamous carcinoma. Supplementary Information The online version contains supplementary material available at 10.1007/s13577-024-01085-8.


Introduction
In recent years, the identification of lung cancers harboring rare driver mutations has increased, leading to a considerable increase in the number of approved targeted therapies.This approach has substantially improved the prognosis of patients with lung cancer [1].Anaplastic lymphoma kinase (ALK) rearrangements are detected in 3-5% of all non-small cell lung cancers (NSCLCs), and ALK tyrosine kinase inhibitors (TKIs) improve the prognosis of patients with ALKpositive NSCLC [2].EML4 is located on chromosome 2p21 and contains 26 exons.There are various fusion breakpoints in multiple exons of EML4 in EML4-ALK-positive lung cancer.Among these, EML4-ALK variant 1 (v1), where exons 20-29 of ALK fuse with exons 1-13 of EML4, and variant 3 (v3), where exons 20-29 of ALK fuse with exons 1-6 of EML4, are the most commonly observed variants, constituting approximately 75-80% of the total variants [3].Furthermore, patients with lung cancer harboring EML4-ALK v3 or TP53 mutations have a poor prognosis [4,5].
In recent years, organoid research has been conducted using pluripotent stem cells and biopsy and surgical specimens [6,7].In cancer research, organoids have emerged as novel preclinical models derived from various malignant tumors, serving as alternatives to traditional twodimensional cell cultures and genetically engineered mouse models [8].In our previous studies, we have highlighted the clinical application of lung tumoroids derived from surgically removed lung cancer tissues and malignant pleural effusions, demonstrating their utility in personalized medicine [9].Although several cell lines have been used in the study of EML4-ALK-positive lung cancer, the number of cancer models remains limited.There is a need to develop preclinical cancer models that further reflect the characteristics of patient tumors to analyze their pathogenesis and develop therapies for these types of tumors.
In the present study, we established tumoroids, PDT-LUAD#119, from a patient with NSCLCs harboring EML4-ALK v3, characterized the tumoroids, and evaluated their utility as a preclinical model.We also discuss the adenocarcinoma-to-squamous cell carcinoma transformation observed in an in vivo xenograft tumor derived from PDT-LUAD#119 lung tumoroids.

Cell lines and their culture conditions
NCI-A549 pulmonary adenocarcinoma cells harboring KRAS G12S and NCI-H2228 pulmonary adenocarcinoma cells harboring variant 3a and 3b (v3a/b) EML4-ALK fusion were obtained from the American Type Culture Collection (Manassas, VA, USA).NCI-H3122 pulmonary adenocarcinoma cells harboring variant 1 (v1) EML4-ALK fusion were sourced from the National Institute of Health (NIH) (Rockville, MD, USA).These cells were cultured as monolayers in the Roswell Park Memorial Institute (RPMI) 1640 medium for NCI-H2228 and NCI-H3122, or in Dulbecco's modified Eagle medium (DMEM) for NCI-A549.The media were supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin.All cells were maintained at 37 °C in an atmosphere of 5% CO 2 , authenticated using short tandem repeat analysis, and regularly tested for Mycoplasma contamination using the TaKaRa PCR Mycoplasma Detection Set (Takara Bio, Inc., Otsu, Japan).

Patient-derived tumoroid culture
Patient-derived lung adenocarcinoma (LUAD) tumoroids (PDT-LUAD#119) were developed using tumoroid culture systems, as described previously [9].The research protocol was approved by the Ethics Committee of the Kawasaki Medical School (reference number 3171-5).All participating patients signed an informed consent form that was authorized by the relevant authority.

Fluorescence in situ hybridization (FISH)
Alpha-satellite DNA for all chromosomes was produced as previously described [9] and labeled using Nick Translation Mix (Sigma-Aldrich, St. Louis, MO, USA) with rhodamine (orange).EML4 and ALK probes were obtained from CytoTest (Rockville, MD, USA).A standard protocol was utilized to conduct FISH [10], and the samples were examined under a fluorescence microscope (ECLIPSE Ni, DS-Qi2; Nikon, Tokyo, Japan).

Xenograft inoculation of lung tumoroids
Cells from PDT-LUAD#119 lung tumoroids (4.0 × 10 6 cells) were dissociated with TrypLE™ Express Enzyme (Thermo Fisher Scientific), and then combined with 50 μL of basement membrane extract type 2 (BME type 2) and subsequently injected subcutaneously into 5-week-old NOD/Shiscid/IL-2Rγnull (NOG) mice (Charles River Laboratories Japan, Atsugi, Japan).The mice were euthanized when the subcutaneous tumor diameter reached 15 mm.The duration from xenograft initiation to euthanasia was approximately 120 days.All experimental procedures were approved by the Animal Research Committee of Kawasaki Medical School (Reference Number: 23-047), and animal care and handling were conducted in accordance with committee regulations.

Luminescence cell viability assay
Tumoroids were enumerated and suspended in BME type 2; 4-μL droplets were seeded in clear-bottom, white-walled flat-bottom 96-well culture plates (PerkinElmer, Waltham, MA, USA), and then medium was added.Twenty-four hours post-seeding, ALK TKI inhibitors were added to the medium.Viability assessment was conducted 72 h posttreatment using the Celltiter-Glo R 2.0 Cell Viability Assay (Promega, Madison, WI, USA), following the manufacturer's instructions.Luminescence readings were obtained using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Rockford, IL, USA).

Clinical and pathological presentation of the patient harboring the parental lung cancer of PDT-LUAD#119
An 87-year-old woman who fell into a ditch and had difficulty walking on her own was admitted to the emergency room (ER) of our hospital.In addition to a bruise on her left buttock, a nodule was detected in the upper lobe of the right lung on computed tomography (CT) (Supplementary Fig. 1a).Following a thorough examination, the patient was suspected to have lung cancer (Supplementary Fig. 1b, cT1cN0M0 stage IA3), and right lower lobectomy via videoassisted thoracic surgery (VATS) was performed.Pathological examination revealed that the size of the tumor was 22 mm × 18 mm (Supplementary Fig. 1c and 1d), and it was diagnosed as solid-predominant pulmonary adenocarcinoma mixed with micropapillary adenocarcinoma (Fig. 1a-c).No EGFR mutations were found; however, ALK expression was detected in the cytosol based on immunohistochemical staining using anti ALK antibody (clone D5F3; Supplementary Fig. 1e, Fig. 1d, e).Nuclear NKX2-1 expression was partially detected (Fig. 1f, g); however, no p40 expression was observed in the primary tumor (Fig. 1h, i).These findings, including the results of hematoxylin and eosin staining, suggested that the primary tumor was pulmonary adenocarcinoma with negligible amounts of squamous cell carcinoma components.The tumor cells had metastasized to the hilar and mediastinal lymph nodes, resulting in a final diagnosis of stage IIIA (pT1N2M0, Fig. 1j, k).

PDT-LUAD#119 lung tumoroids were sensitive to ALK TKIs
We analyzed the viability of PDT-LUAD#119 lung tumoroids in addition to the other types of tumoroids and lung cancer cells used in this study.NCI-A549 pulmonary adenocarcinoma cells harboring KRAS G12S and PDT-LUAD#5 lung tumoroids harboring BRAF G469A were resistant to all four ALK TKIs (Fig. 4a-d).Crizotinib and entrectinib at higher concentrations inhibited the growth of NCI-A549 cells and PDT-LUAD#5 lung tumoroids compared to the other two kinds of ALK TKIs (alectinib and lorlatinib).Among lung cancer cells, NCI-H3122 cells with EML4-ALK v1 tended to be more sensitive to ALK TKIs than NCI-H2228 cells with EML4-ALK v3a/b.This finding is consistent with previous reports showing that lung cancers with EML4-ALK v3a/b are less sensitive to ALK TKIs than those with EML4-ALK v1 mutations.In contrast, PDT-LUAD#119 lung tumoroids showed relatively high sensitivity to ALK TKIs despite having EML4-ALK v3a/b.

Xenografts derived from PDT-LUAD#119 lung tumoroids were diagnosed as adenosquamous carcinoma rather than pulmonary adenocarcinoma
To explore if the established tumoroids faithfully replicated the parental lung cancer pathology in vivo, patient-derived xenografts (PDXs) were established in NOD/Shi-scid/ IL-2Rγnull (NOG) mice through subcutaneous inoculation of PDT-LUAD#119 lung tumoroids.Unexpectedly, most xenografts from PDT-LUAD#119 lung tumoroids showed squamous cell carcinoma (Fig. 5a-h) mixed with adenocarcinoma in some parts (Fig. 5i-l) and finally diagnosed as adenosquamous carcinoma.Importantly, ALK expression was broadly positive, including the areas with squamous cell carcinoma (Fig. 5b, f and j).Although NKX2-1 expression was not detected (Fig. 5c, g), p40 expression was broadly observed in the xenografts (Fig. 5d, h).These results suggest that EML4-ALK v3-harboring pulmonary adenocarcinoma tumors might transform into squamous cell carcinoma during tumor formation in vivo.

Discussion
Yoshida et al. [17] reported the pathological features of ALK-positive lung cancer based on the examination of 54 patients who underwent surgical resection.They reported two distinctive findings in most ALK-positive tumors (78%): solid signet cell patterns and mucinous cribriform patterns, at least locally.In contrast, these features are rare in tumors with wild-type ALK (1%) [17].In our case, these characteristic initial findings of ALK-positive lung cancer were not observed in primary lung cancer.These features were also observed in lung cancers with other fusion genes [18].Therefore, future investigations may allow for the estimation of genetic subtypes based on histological findings.
Previous reports indicate that patients with EML4-ALK v1 show a better clinical response to ALK TKIs (such as crizotinib or alectinib) than patients with other types of ALK fusion variants [3].In contrast, patients with TP53 mutations and EML4-ALK v3 show a particularly poor prognosis [5].However, PDT-LUAD#119 lung tumoroids harboring EML4-ALK v3 and TP53 mutations were as sensitive to ALK TKI as NCI-H3122 cells with EML4-ALK v1 and TP53 mutations in the present study.Therefore, a response to an ALK-TKI may be expected if a patient from whom a tumor was established relapses with lung cancer.
In this study, a tumor from a patient with pulmonary adenocarcinoma carrying EML4-ALK v3 unexpectedly formed squamous cell carcinoma and adenocarcinoma after inoculation into NOG mice and was finally diagnosed as pulmonary adenosquamous carcinoma.According to WHO Classification 5th edition, pulmonary adenosquamous carcinoma is a cancerous tumor composed of squamous cell carcinoma and adenocarcinoma components, with each component accounting for over 10% of the entire tumor [19].One of the main mechanisms underlying the development of adenosquamous carcinoma is its transformation into squamous cell carcinoma.Adenosquamous carcinoma shares driver genes with adenocarcinoma but does not share them with squamous cell carcinoma.This finding suggests differentiation from adenocarcinoma to adenosquamous carcinoma, but not vice versa [20,21].Additionally, studies using genetically engineered mouse models indicate that the deletion of LKB1 transforms lung adenocarcinoma into squamous cell carcinoma [22].A limitation of the study is that only one type of tumoroid derived from a single patient with ALK-positive lung cancer was established.Therefore, further research is needed to determine the mechanism underlying the transformation of pulmonary adenocarcinoma carrying EML4-ALK into pulmonary adenocarcinoma and squamous cell carcinoma in mice.
There are only a few models for the carcinogenesis of adenosquamous carcinoma, and the PDT-LUAD#119 Yakuhin, Ltd., and Otsuka Pharmaceutical Co., Ltd., and personal fees from Yakult Honsha Co., Ltd.outside the submitted work.N. Takigawa reports grants and personal fees from Eli Lilly Japan, AstraZeneca, Daiichi-Sankyo Co., Ltd., Chugai Pharmaceutical, Taiho Pharmaceutical, Pfizer Inc., Japan, Boehringer-Ingelheim Japan, and Ono Pharmaceutical; grants from Kyowa Hakko Kirin, Nippon Kayaku Co., Ltd., and Takeda Pharmaceutical Co., Ltd.; and personal fees from MSD and Bristol-Myers Squibb Company, Japan, outside the submitted work.Fukazawa reports personal fees from Boehringer Ingelheim, Japan.No disclosures were reported by the other authors.

Fig. 1
Fig. 1 Pathological findings of the primary tumor.a Hematoxylin and eosin (HE)-stained tissue section of the parental lung cancer, revealing distinctive pathological features of solid adenocarcinoma.b, c Micropapillary adenocarcinoma was observed at the periphery of the tumor.Immunohistochemical staining showed widespread expres-

Table 1
Lung tumoroid media