Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation

Characterization of a human lung cancer cell line is reported. This cell line was established from a patient referred to Nemazi Hospital of Shiraz University of Medical Sciences with a diagnosis of poorly differentiated carcinoma. Sterile sample from peritoneal effusion was taken and immediately cultured in RPMI-1640 medium containing 20% FBS, at 37 degrees C with 5% CO2. This cell line has been in continuous culture for more than one year and has been named as Mehr-80. Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immunohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for large cell carcinoma with neuroendocrine differentiation (LCCND). This cell line will be a valuable in vitro tool for further studies on lung cancers.     

肝癌高转移细胞株的肿瘤干细胞生物学特性研究

目的:建立高转移肝癌细胞株,研究其肿瘤干细胞生物学特征,为靶向人肝癌干细胞治疗提供有价值的细胞模型。方法:肝癌细胞系Bel7402接种裸鼠皮下,成瘤后,取小鼠肺部转移灶,通过机械分离法,获取肺部肝转移细胞,体外扩大培养后,再次接种裸鼠。如此反复接种裸鼠,获得稳定肺转移肝癌细胞株Bel7402-V13。采用无血清悬浮培养及PKH26染色确定Bel7402-V13中肿瘤干细胞的存在。流式细胞术分析亲本Bel7402和Bel7402-V13中肿瘤干细胞标志物ESA的表达情况,分选Bel7402和Bel7402-V13中ESA+细胞并进行体外生物学特征研究及裸鼠致瘤实验。结果:建立高转移Bel7402-V13细胞株,Bel7402-V13细胞无血清悬浮培养7d后形成的细胞球体中存在单个PKH26阳性细胞。与Bel7402中ESA+细胞相比,流式分析显示干细胞标志物ESA比例高转移Bel7402-V13显著提高5.67倍（17.6±0.4 vs 3.1±1.5）。Bel7402-V13中ESA+细胞具有更强的自我更新能力［成球率:（94.8±7.5）% vs （52.3±6.9）%,P<0.01］,侵袭能力提高1.51倍（397.7±79.4 vs 262.0±40.1,P<0.01）,耐药能力也显著增强（IC50:0.286 vs 0.196,P<0.01）,ESA+细胞Bel7402-V13在裸鼠皮下接种2×102个细胞3月即可致瘤（3/6）,而接种2×102个ESA+细胞Bel7402细胞3月才能致瘤（1/6）。结论:获得高转移肝癌细胞株Bel7402-V13,伴随ESA+细胞增加,其体内外功能显著增强,为肝癌干细胞的靶向治疗提供有价值的细胞模型。     

Establishment and characterization of a new cell line from primary human breast carcinoma

Summary A new cell line (BRC-230) was established from surgical material of primary ductal infiltrating breast carcinoma. The epithelial nature of this cell line was confirmed by ultrastructural analysis and demonstrated the retention of structural properties characteristic of the original tumor. The BRC-230 cell line induced tumor in athymic Cr1:nu/nu(CD-1)BR nude mice, it possessed an abnormal karyotype with a modal chromosome number between 60–61 with eight recurrent marker chromosomes, and it presented a doubling time of 30.5 hr. Scatchard analysis demonstrated that both primary tumor and BRC-230 cells were estrogen and progesterone receptor negative.Immunoenzymatic and radioimmunoassays showed a production of marker antigens (CEA, TPA, CA125, CA15-3, CA19-9) which was similar in the patient's serum and BRC-230 cells. Thein vitro drug sensitivity assay of the cell line and of the parental tumor tissue showed overlapping results to all tested antiblastic drugs. BRC-230 cells were resistant to 4-Idroperoxy-cyclophosphamide, Idarubicinol, Mitoxantrone, Etoposide, 4Epidoxorubicin, and Doxorubicin, showing a multiple drug resistance phenotype. Amplification or rearrangement of Her-2neu, Ha-ras, and C-myc genes was observed neither in the original tumor nor in BRC-230 cells; the mdr-1 gene was also present in a single copy.We conclude from these studies that the BRC-230 cell line maintains the same characteristics as the original tumor and may provide us with a good model to studyin vitro the biology of drug resistance of breast cancer.     

Delta‐like 3 localizes to neuroendocrine cells and plays a pivotal role in gastrointestinal neuroendocrine malignancy

Delta‐like 3 (DLL3) is a member of the Delta/Serrate/Lag2 (DSL) group of Notch receptor ligands. Five DSL ligands are known in mammals, among which DLL3 has a unique structure. In the last few years, DLL3 has attracted attention as a novel molecular targeting gene in neuroendocrine carcinoma of the lung due to its high expression. However, the expression pattern and functions of DLL3 in the gastrointestinal tract and gastrointestinal neuroendocrine carcinoma remain unclear. In this study, we examined the expression and role of DLL3 in the gastrointestinal tract, as well as in gastrointestinal neuroendocrine carcinoma. Immunohistochemical staining of the human normal gastrointestinal tract revealed that DLL3 localized in neuroendocrine cells. DLL3 showed intense staining in chromogranin A‐positive gastric cancer specimens. Real‐time quantitative RT‐PCR and western blotting analyses showed considerable upregulation of DLL3 in gastrointestinal neuroendocrine carcinoma cell lines. Immuno‐electron microscopy demonstrated abundant expression of DLL3 in neurosecretory granules in these cells. Furthermore, gene silencing of DLL3 caused significant growth inhibition through the induction of intrinsic apoptosis. Our findings suggest that DLL3 is expressed in neuroendocrine cells of the gastrointestinal tract and that it has a pivotal role in gastrointestinal neuroendocrine carcinoma cells. Based on these findings, further investigations are required to achieve a breakthrough in developing therapeutic strategies for gastrointestinal neuroendocrine carcinoma.     

A pilot study of adjuvant chemotherapy with irinotecan and cisplatin for completely resected high-grade pulmonary neuroendocrine carcinoma (large cell neuroendocrine carcinoma and small cell lung cancer)

Background

Large cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC) are recognized as high-grade neuroendocrine carcinomas (HGNEC) of the lung. In patients with completely resected HGNEC, platinum-based adjuvant chemotherapy may be considered. However, the optimum chemotherapy regimen has not been determined. We conducted a multicenter single-arm phase II trial to evaluate irinotecan and cisplatin in postoperative adjuvant chemotherapy for HGNEC patients.

Patients and methods

Patients with completely resected stage I–IIIA HGNEC received four cycles of irinotecan (60 mg/m², day 1, 8, 15) plus cisplatin (60 mg/m², day 1). This regimen was repeated every 4 weeks. The primary endpoint was the rate of completion of chemotherapy (defined as having undergone three or four cycles), and secondary endpoints were the rate of 3-year relapse-free survival (RFS), rate of 3-year survival and toxicities.

Results

Forty patients were enrolled between September 2007 and April 2010. Patients’ characteristics were: median age (range) 65 [45–73] years; male 85%; ECOG-PS 1 60%; LCNEC 57% and SCLC 43%; stage IA/IB/IIB/IIIA 32/35/8/5%; 95% received lobectomy. The rate of completion of chemotherapy was 83% (90% C.I.; 71–90%). The rate of overall survival at 3 years was estimated at 81%, and that of RFS at 3 years was 74%. The rates of overall survival and RFS at 3 years were 86 and 74% among 23 LCNEC patients, and 74 and 76% among 17 SCLC patients, respectively. Nineteen patients (48%) experienced grade 3 or 4 neutropenia, but only five patients (13%) developed febrile neutropenia. Two patients (5%) developed grade 3 diarrhea, and four patients (10%) had grade 3 nausea. No treatment-related deaths were observed in this study. All 40 specimens were also diagnosed as HGNEC by central pathological review.

Conclusions

The combination of irinotecan and cisplatin as postoperative adjuvant chemotherapy was feasible and possibly efficacious for resected HGNEC.     

Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient

The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage- independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.     

Genomic profiling of high-grade large-cell neuroendocrine carcinoma of the colon

High-grade neuroendocrine carcinoma (HGNEC) of the colon is a rare and aggressive cancer that has a poor prognosis. Currently no standard treatment exists, and published case series report an overall survival of approximately one year with treatment. Typically patients receive treatment similar to that recommended for small-cell lung cancer, extrapolating from the similarity in cancer biology. Here we report a case of HGNEC of the colon with genomic profiling that identified a KRAS G12D mutation and a PI3K mutation that has not yet been reported in the literature for this tumor type.     

Establishment of a novel cell line derived from nasopharyngeal carcinoma

A novel epithelial cell line (designated as HNE-1), derived from nasopharyngeal carcinoma (NPC), was established and has passed more than 100 generations over one year. The NPC biopsy specimen was taken from a 27 year old man with poorly differentiated squamous cell carcinoma of the nasopharynx. The cultured cells showed polygonal shape and grew into multilayers under the inverted microscope. Electron microscopy showed that HNE-1 cells were characterized by bi-directional differentiation with some being poorly differentiated squamous carcinoma and the other poorly differentiated adenocarcinoma cells. A continuous positivity was showed by EBNA at subcultures 5-81. Tumorigenicity was demonstrated by heterotransplantation in BALB/c (nu/nu) mice, developing into well differentiated squamous carcinoma. Karyotype analysis showed aneuploidy with the modal chromosomal number 74-101 (5th-20th passages) and 15 marker chromosomes. The frequency of spontaneous sister chromatid exchange was very high in HNE-1 cells (87.6 +/- 0.4/cell).     

Establishment and characterization of patient‐derived organoids from a young patient with cervical clear cell carcinoma

Cervical clear cell carcinoma (cCCC) constitutes an extremely rare subtype of cervical cancer. Consequently, its pathogenesis remains largely unknown, with no cell lines established from primary tumors. Here, we report the first establishment of cCCC organoids, from biopsy samples of a 23‐year‐old patient diagnosed with cCCC. By applying a protocol that we recently optimized for gynecological tumors, we were able to propagate a patient‐derived cell line (PDC) for more than 6 months as organoids. This PDC tolerated cryopreservation and proliferated either as spheroids or adherent cells, and developed xenografts in immunodeficient mice, ensuring robust utility as a cell line. Intriguingly, the resected tumor focally contained serous carcinoma (SC) in a tiny protruding lesion. Both organoids and derivative xenografts resembled the CCC component of the original tumor in histology, immunostaining profile, and genome‐wide copy number changes, including focal gain of MET. Genomic analysis revealed that both organoids and the CCC component harbored only a few mutations, of which 2 mutations were shared in common. In contrast, the SC component showed a mutator‐phenotype and prominent genome instability along with biallelic inactivation of TP53, but none of them were found in organoids or the CCC component. The PDC proved sensitive to major chemotherapeutic agents and MET inhibitors. These observations clearly indicated that the PDC, designated as YMC7, can be used as a novel cCCC cell line and provide novel insights into the pathogenesis of mixed cervical adenocarcinoma. As a valuable resource for rare cancer, it will likely contribute to investigations in many fields.     

Establishment and characterization of a tumor cell line from human nasopharyngeal carcinoma tissue

An epithelial tumor cell line, CG1, was established from human nasopharyngeal carcinoma tissues. The CG1 cells are of an epithelial origin as shown by their reactivities with the epithelial-specific antikeratin antibodies and by the presence of the desmosome structure at cell-cell junctions. CG1 cells possess characteristics of tumor cells because these cells are tumorigenic in nude mice and also have reduced serum requirements for in vitro cultivation. The doubling time of CG1 cells is 20 h and these cells have been successfully cultured in vitro for more than 200 generations. The average chromosome number of these cells is 60. Slot and Southern blot hybridizations showed the presence of Epstein-Barr virus-DNA sequences in CG1 cells. This cell line provides us an in vitro system for the study of the role of Epstein-Barr virus in nasopharyngeal carcinoma.     

A new human chromogranin 'A' immunoradiometric assay for the diagnosis of neuroendocrine tumours

We investigated whether plasma chromogranin A (CgA), measured by a new immunoradiometric assay, may be a sensitive and specific marker of phaeochromocytoma and of other neuroendocrine tumours. This study involved 121 patients of whom 20 with phaeochromocytoma, 28 with other neuroendocrine tumours (19 gastroenteropancreatic tumors, 3 medullary thyroid and 6 small cell lung carcinomas), 25 with solid nonfunctioning adrenocortical tumours and 48 with essential hypertension. In addition, 130 normal subjects were taken as controls. Plasma catecholamines were measured by using high-performance liquid chromatography, and CgA by a two-site sandwich immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245) of human CgA. Plasma CgA in controls (49.0 +/- 3.1 ng ml(-1), mean +/- SE) and in essential hypertensives (50.8 +/- 3.5 ng ml(-1)) was lower (P< 0.0001) than in adrenocortical tumours (91.8 +/- 13.2 ng ml(-1)), in phaeochromocytomas (254 +/- 49 ng ml(-1)) and in patients with other neuroendocrine tumours (469 +/- 84 ng ml(-1)). Plasma CgA and catecholamines identified 13 and 18 out of 20 phaeochromocytomas with sensitivity of 65% and 90%, respectively. Combined measurement of both markers improved sensitivity up to 100%. In the other neuroendocrine tumours, CgA was abnormal in 23/28 cases (sensitivity 82%) and in 6 it was the only circulating marker of disease. In gastroenteropancreatic tumours, CgA measurement identified all cases (sensitivity 100%). Specificity of CgA in patients with essential hypertension was 98%. In conclusion, CgA determination showed high sensitivity in identifying gastroenteropancreatic tumours and, in association with catecholamines, in detecting patients with phaeochromocytoma. CgA sometimes appeared to be the only circulating marker of disease. Since the specificity of CgA proved to be excellent, this assay may be useful for diagnosis both of functioning and non-functioning neuroendocrine tumours.     

Establishment and characterization of a human urinary bladder carcinoma cell line (TSGH-8301)

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.     

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.     

Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma

Background:

Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear.

Methods:

We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen.

Results:

Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O₂, whereas that of OCUM-12/Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells.

Conclusion:

OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.     

Derivation of pancreatic acinar cell carcinoma cell line HS‐1 as a patient‐derived tumor organoid

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long‐term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy‐derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS‐1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS‐1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.     

Primary neuroendocrine carcinoma of the skin (Merkel's cell carcinoma)

Primary neuroendocrine carcinoma of the skin (Merkel's cell carcinoma) is a rare tumor. Until recently 86 patients with this tumor have been described. Two cases of this tumor are presented. This neoplasm has a high propensity for lymphatic as well as hematogenous metastases. It presents as a dermal or subcutaneous nodule. Awareness of this condition may lead to earlier diagnosis and improved survival.