Identification of cancer stem cells in human gastrointestinal carcinoid and neuroendocrine tumors

Background & Aims Metastatic gastrointestinal neuroendocrine tumors (NETs) frequently are refractory to chemotherapy. Chemoresistance in various malignancies has been attributed to cancer stem cells (CSCs). We sought to identify gastrointestinal neuroendocrine CSCs (N-CSCs) in surgical specimens and a NET cell line and to characterize novel N-CSC therapeutic targets. Methods Human gastrointestinal NETs were evaluated for CSCs using the Aldefluor (Stemcell Technologies, Vancouver, Canada) assay. An in vitro, sphere-forming assay was performed on primary NET cells. CNDT2.5, a human midgut carcinoid cell line, was used for in vitro (sphere-formation) and in vivo (tumorigenicity assays) CSC studies. N-CSC protein expression was characterized using Western blotting. In vivo, systemic short interfering RNA administration targeted Src. Results By using the Aldefluor assay, aldehyde dehydrogenase–positive (ALDH+) cells comprised 5.8% ± 1.4% (mean ± standard error of the mean) of cells from 19 patient samples. Although many primary cell lines failed to grow, CNDT96 ALDH+ cells formed spheres in anchorage-independent conditions, whereas ALDH- cells did not. CNDT2.5 ALDH+ cells formed spheres, whereas ALDH- cells did not. In vivo, ALDH+ CNDT2.5 cells generated more tumors, with shorter latency than ALDH- or sham-sorted cells. Compared with non-CSCs, ALDH+ cells demonstrated increased expression of activated Src, Erk, Akt, and mammalian target of rapamycin (mTOR). In vivo, anti-Src short interfering RNA treatment of ALDH+ tumors reduced tumor mass by 91%. Conclusions CSCs are present in NETs, as shown by in vitro sphere formation and in vivo tumorigenicity assays. Src was activated in N-CSCs and represents a potential therapeutic target in gastrointestinal NETs.