Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Summary

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Abstract

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Introduction

Small bowel neuroendocrine tumors (SBNETs) originate from enterochromaffin cells of the small intestine. Although SBNETs are generally known to grow slowly, they commonly metastasize to the liver¹. While the surgical removal or tumor ablation can be considered in many cases, recurrence is nearly universal, and, therefore, medical therapy plays an important role in management. Tremendous efforts have been invested to generate new SBNET cell lines for drug testing. However, there has been very little success. Only 6 SBNET cell lines (KRJ-I, CND2, GOT1, P-STS, L-STS, H-STS) have been reported^2,3....

Protocol

All experiments using human neuroendocrine tumor samples have been approved by the University of Iowa Hospital and Clinics IRB committee (Protocol number 199911057). A list of all materials and equipment is described in the Table of Materials. A list of growth media and key solutions is found in Table 1.

1. Small bowel neuroendocrine tumor (SBNET) collection and cell dissociation

1. Obtain resected patient SBNET samples after tumor tissues confirmatio.......

Discussion

Tumor 3D cultures have become a valuable resource for preclinical drug testing¹⁵. Various tumor organoid biobanks have recently been established from breast cancer and prostate cancer tumors^16,17. In this study, we provide a detailed protocol to culture SBNET as spheroids and a simple and fast method to validate the spheroid cultures for NET markers by immunofluorescence and test drug sensitivity. From our experience, SBNET spheroids can g.......

Materials

Name	Company	Catalog Number	Comments
Anti-rabbit FITC	Jackson ImmunoResearch	11-095-152	Secondary antibody couple to a green fluorophore
Antigen Retrieval Solution	Agilent Dako	S2367	Solution at pH 9 for preparing slides for IHC
Autostainer Link 48	Agilent Dako	Not Available	Automated system for antibody staining
Cell freezing container	Thermo Scientific	5100-0001	Container to for freezing cells
CellSence	Olympus	Version 1.18	Computer software for using fluorescent microscope
Chromogranin A antibody	Abcam-45179	RB-9003-PO	Antibodies for IF
Chromogranin A antibody (clone LK2H10)	Thermo Scientific	MA5-13096	Antibodies for IHC
Collagenase	Sigma	C0130	Enzyme for digesting tumor tissue
DMEM	Gibco	11965-092	Medium for tissue preparation
DMEM/F12	Gibco	11320-033	Medium for organoid cultures
DMSO	Sigma	D8418	Solvent for dissolving drug
DNAse	Sigma	DN25	Enzyme for digesting tumor tissue
Ethidium Homodimer	Chemodex	CDX-E0012-T1E	DNA and RNA binding dye
FBS	Gibco	16000044	Reagent for culture media
Fluorescent microscope	Olympus	CKX35	Microscope for taking pictures of SBENT spheroids
Glutamine	Gibco	A2916801	Reagent for culture media
ImageJ	National Institutes of Health	Version 1.51	Computer software for image analysis
Insulin	Sigma	I0516	Reagent for culture media
Matrigel	Corning	356235	Matrix to embed and anchore organoids
Mounting medium (VECTASHIELD)	Vector Laboratories	H-1200	Fixative for labelled-cells with a nuclear stain
Nicotinamide	Sigma	72340	Reagent for culture media
Paraformaldehyde	Electron Microscopy Sciences	15710	Reagent to fix cells
PEN/STREP	Gibco	15140-122	Reagent for culture media
PT Link	Agilent Dako	Not Available	Automated system to prepare slides for IHC staining
Rapamycin	Alfa Aesar	J62473	Drug that can inhibit NET growth
Secondary antibodies for IHC	Agilent Dako	K8000	Secondary antibodies for IHC using Polymer-based EnVision FLEX system
SSTR2 antibody	GeneScritp	A01591	Antibodies for IF
SSTR2 antibody (clone UMB1)	Abcam	ab134152	Antibodies for IHC
Synaptophysin antibody	Abcam	32127	Antibodies for IF
Synaptophysin antibody (clone DAK-SYNAP)	Agilent Dako	M7315	Antibodies for IHC
TritonX	Mallinckrodt	3555 KBGE	Reagent to permeablize cells
Y-2763 ROCK inhibitor	Adipogen	AG-CR1-3564-M005	To improve SBNET spheroid viability after freeze thaw