Human bronchial carcinoid tumor initiating cells are targeted by the combination of acetazolamide and sulforaphane

Abstract

Background Bronchial 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. Methods Bronchial 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. Results Data 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. Conclusions Human 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.

3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model

“…Human breast cancer cell lines were cultured in non-adherent conditions at a density of 5 × 10 6 cells/mL in 75 cm 2 3% agarose-coated cell culture flasks in serum-free RPMI-1640 medium (R8758; Sigma Aldrich Canada) supplemented with 30 ng//mL of basic fibroblast growth factor (bFGF; 100-18B; PeproTech Canada, Office Servicing Canada, 5 Cedarbrook Drive, Cranbury, NJ, USA), 30 ng/mL of epidermal growth factor (EGF; AF-100-15; PeproTech Canada) 4 ng/mL of insulin (91077C; Sigma-Aldrich Canada), 0.5% bovine serum albumin (BSA; 15561020; Thermo Fisher Scientific), and 0.03% B27 (17504044; Thermo Fisher Scientific, Fisher Scientific Company, Ottawa, ON, Canada), as previously reported [ 23 ]. Once spheroids started to form, the growth factor-supplemented culture medium was replaced every other day.…”

“…Drug-induced cytotoxicity was determined using the AlamarBlue assay, as previously described [ 23 ]. 3 × 10 3 monolayer breast cancer cells and 3rd generation spheroids were plated in 96-well plates and treated with cisplatin (0–40 μM) or DMSO control (0.2 × 10 −5 μM) for 72 h. The AlamarBlue agent (BUF012B; Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON, Canada) was added as 10% of the total volume to each well for 4 h. Fluorescence was measured using the SPECTRAmax Gemini Spectrophotometer (λex/λem = 540/590 nm).…”

“…Recently, we reported that bronchial carcinoid spheroids grown under stem cell factor-enriched medium in spheroid-promoting conditions compared to 2D monolayer cultures were enriched for the expression of the well-characterized stem cell markers ALDH1, CD44, Oct-4, Sox-2, and Nanog [ 23 ]. These 3D multicellular stem-like spheroids also had a tumor-initiating cell population with significantly increased tumorigenicity as xenografts.…”

Perspective on dietary isothiocyanates in the prevention, development and treatment of cancer

“…SFN can synergize with cisplatin to suppress human ovarian cancer [41] , and as a component with other anti-tumor phytochemicals [42] . Studies from our lab have noted the potent anticancer effects of SFN for both pediatric and adult cancers [35,43,44] . Our approach for therapeutic targeting of several cancers (bronchial carcinoids, bladder cancer, and neuroblastoma) was to exploit the anti-tumor properties of SFN in combination with targeting of carbonic anhydrase (CA) with acetazolamide, a pan CA inhibitor to perturb the pH regulation necessary for metastatic progression.…”

“…Elimination of CSCs or tumor initiating cells would therefore constitute the ideal targets in chemoprevention and anti-tumor targeting during progression. There are documented studies showing targeting of tumor stem cells with good evidence [38,40,43,53] . Furthermore, considering all the hallmark features of cancer originally elaborated by Hanahan and Weinberg [54] and now reviewed and expanded on by Girotti et al [55] , who describe how galectins can affect tumor progression, it is obvious that ITCs can have a significant impact on all of these, including galectins [56] .…”