The Development and Characterization of a Human Midgut Carcinoid Cell Line

Abstract

Purpose: Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents.We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2). Experimental Design: Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets.Results: CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors inmice. Electronmicroscopy revealed round, pleomorphic, electron-denseneurosecretorygranules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small ''salt-andpepper''nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-h, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors1to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations. Conclusion: The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.

MicroRNAs associated with small bowel neuroendocrine tumours and their metastases

“…Whilst KRJ-I cells have been used as a model for the in vitro investigation of SBNETs, it may be that other cells, especially those derived from metastatic disease, would have been better models for examining these miRNA-mRNA interactions. Unfortunately, we did not have access to other SBNET cells, such as P-STS (derived from primary tumour), L-STS (from LN metastasis), H-STS (from liver metastasis) or CNDT2 (from liver metastasis) to validate these miRNA-mRNA interactions (Van Buren et al 2007, Pfragner et al 2009). …”

TCEB3C a putative tumor suppressor gene of small intestinal neuroendocrine tumors

“…Primary tumors and metastases were snap-frozen in liquid nitrogen and kept at K70 8C. A human SI-NET cell line, CNDT2.5, developed from a liver metastasis from a patient diagnosed with primary ileal SI-NET (Van Buren et al 2007), was used in the experiments reported here at cell passages 10-30. These cells expressed neuroendocrine markers and somatostatin receptor 2 and responded to synthetic somatostatin analog (octreotide) treatment (Van Buren et al 2007, Li et al 2012, although skepticism regarding the neuroendocrine authenticity of this cell line has also been raised (Ellis et al 2010).…”

“…A human SI-NET cell line, CNDT2.5, developed from a liver metastasis from a patient diagnosed with primary ileal SI-NET (Van Buren et al 2007), was used in the experiments reported here at cell passages 10-30. These cells expressed neuroendocrine markers and somatostatin receptor 2 and responded to synthetic somatostatin analog (octreotide) treatment (Van Buren et al 2007, Li et al 2012, although skepticism regarding the neuroendocrine authenticity of this cell line has also been raised (Ellis et al 2010). The growth medium for CNDT2.5 was DMEM-F12 complemented with 10% fetal bovine serum (Sigma Aldrich), 1% vitamins, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids and 1% penicillin-streptomycin (PEST), and the cells were cultured at 37 8C in 5% CO 2 .…”

Varying Opinions on the Authenticity of a Human Midgut Carcinoid Cell Line – Letter

“…In 2007, our laboratory published an article in Clinical Cancer Research reporting on the development of a human midgut carcinoid tumor cell line (1). This cell line has now been shared with over 20 laboratories around the world.…”

“…At the time of publication of this article, we believed that we had developed the first human midgut carcinoid cell line in North America. These cells expressed dense neurosecretory granules on electron microscopy, VMAT-1/2, and somatostatin receptors (1). Recently, we performed short tandem repeat genotyping of the CNDT cell line, and we were unable to match this cell line to any other cell line in our own database at M.D.…”