Abstract
BackgroundBronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior.MethodsBronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids.ResultsData demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice.ConclusionsHuman bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.
Highlights
Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic
Bronchial carcinoid cell lines form spheroids in 3D culture and show evidence of a stem cell-like tumor initiating cell (TIC) progenitor population To first evaluate bronchial carcinoids for presence of TIC characteristics the H727 and H720 cell lines were subjected to spheroid culture under stem cell culture conditions, and growth observed for 20 days
H727 and H720 cell lines were able to form spheroids (SP) that grew expansively in stem cell culture conditions when plated from monolayer culture (Fig. 1a)
Summary
Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Bronchial carcinoids are a more indolent subgroup of neuroendocrine tumors (NETs) that arise in the lateral region of the bronchus. The slower growth of bronchial carcinoids generally portends a better prognosis but is dependent on the degree of differentiation. Bronchial carcinoids present as typical carcinoids, TC, or a more aggressive form, atypical carcinoids, AT. TC tumors are well-differentiated, rarely metastasize, and have a good prognosis with a survival rate of 87 to 100% [1]. AT, have a substantially lower 5-year survival rate of 25 to 69%, due to their greater metastatic potential. The malignant characteristics of bronchial carcinoids are likely due to its invasiveness and the intrinsic tumor stem cell population [1]
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