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.
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.
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 reported2,3,4,5; and unfortunately one cell line no longer expresses NET markers6 and three other SBNET cell lines (KRJ-I, L-STS, H-STS) were determined to be derived from transformed lymphoblasts instead of NETs7. In order to accelerate the identification of drugs for targeting SBNETs, alternative methods for in vitro drug testing are needed.
Here, we take advantage of the availability of resected SBNETs and have established a way to culture these patient-derived SBNETs as spheroids growing in ECM. The overall goal of this manuscript is to describe a method to culture SBNET as a three-dimensional (3D) culture and outline procedures to characterize these spheroids for the retention of SBNET markers by immunofluorescence staining and immunohistochemistry.
In addition, we demonstrate how these SBNET spheroids can be used for testing the effect of rapamycin, an anti-cancer drug for NETs8. The rationale behind this protocol is to develop a new method to grow SBNET cells in vitro and use them for drug testing. The advantage of this technique over the traditional method of establishing an SBNET cell line is that 3D cultures of SBNETs can rapidly be obtained and drug testing can be done within 3 weeks. SBNET spheroids could potentially be used as a model for performing in vitro drug screens to identify new drugs for SBNET patients. Since SBNET cell lines are not widely available, 3D cultures of SBNET spheroids can serve as a new in vitro model for studying SBNETs and can be shared among scientists in the field.
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
2. Culture of SBNETs as tumor spheroids in ECM
3. Quantification of SBNET spheroid size using ImageJ
4. Characterization of SBNETS spheroids by immunofluorescence
5. SBNET spheroids characterization by immunohistochemistry (IHC)
6. Treatment of SBNET organoids with rapamycin
7. Splitting SBNET spheroids
NOTE: This is done for expansion and for sharing with other researchers.
8. Cryostorage and recovery of SBNET spheroids
There are currently only 2 SBNET cell lines established and published2,3,4,5 and they are not readily available to many researchers. Here, we propose to culture SBNET as spheroids in ECM and use this as an alternative model to study SBNET drug sensitivity. Patient-derived tumor from an SBNET that metastasized to the liver was collected, digested to release SBNET cells, and mixed with liquid ECM for establishing an SBNET spheroid culture (Figure 1A). The Ki-67 of this SBNET was 4.3%. Although SBNET spheroids have a slow growth rate, their growth can be monitored by microscopic imaging (Figure 1A). It takes approximately 14 days for SBNET spheroids to double in size when the culture media is changed once a week (Figure 1B). After 14 days in culture, SBNET spheroids do not increase in size. Instead, some SBNET cells will dissociate to a neighboring location and form new spheroids. To propagate the SBNET spheroids culture, harvest the ECM containing SBNET spheroids and reseed them in new culture plate with new culture medium (Step 7).
To confirm that the organoid cultures contain SBNET cells, we describe a simple and fast method to stain the spheroids for SBNET markers such as synaptophysin, chromogranin A, and the somatostatin receptor type 2 (SSTR2) using immunofluorescence (IF) microscopy (Step 4). Using antibodies specific against synaptophysin, chromogranin A and SSTR2, our IF data showed that these markers are localized in the cytoplasm and at the membrane of SBNET cells (Figure 2A, in green) after 1 or 9 months in culture (Figure 2B). To ensure the specificity of the SYP, CgA and SSTR2 antibodies, we performed the same staining procedures on an organoid line from pancreas tumor that does not express SYP, CgA or SSTR2 (Figure 2C) as no green signal was detected. The main advantage of this SBNET spheroid IF experiment is that it can be performed within 4 h and gives similar staining information as the immunohistochemistry (IHC; Figure 3). We provide a protocol for performing IHC of the SBNET spheroids in step 5.
Culturing SBNET as spheroids is a valuable technique for identifying drugs that can inhibit SBNET growth. As a proof of principle, we treated SBNET spheroids with rapamycin for 5 days, an mTOR inhibitor, a class of drugs commonly used to treat NETs8. In comparison to our control SBNET spheroid, the rapamycin-treated spheroid formed a grape-like structure and became apoptotic or necrotic (Figure 4A-D). Dying cells can be detected using Ethidium homodimer to stain DNA and RNA and generate a bright red signal14. This dye cannot penetrate the cell membrane of live cells.
Figure 1: Patient-derived small bowel neuroendocrine tumors (SBNETs) grown in extracellular matrix (ECM) as spheroids. (A) Isolation of tumor cells from a resected SBNET and put in culture by mixing with ECM. Scale bar represents 100 µm. (B) Surface area of SBNET spheroids with respect to the number of days in culture quantified using ImageJ. Data were obtained from SBNET spheroids of 1 patient and are represented as the mean area ± standard error of the mean. The surface area of 30 to 60 spheroids were measured for each time point. Please click here to view a larger version of this figure.
Figure 2: Immunofluorescence (IF) staining of SBNET spheroids. IF staining of SBNET spheroids after (A) 1 month in culture and (B) 9 months in culture. (C) IF staining of pancreas tumor organoids that do not express SBNET markers as negative controls. Tumor spheroids were fixed in 4% paraformaldehyde and stained using antibodies against synaptophysin (SYP) at 1/600 dilution, chromogranin A (CgA) at 1/400 dilution, and somatostatin receptor 2 (SSTR2) at 1/400. IF images were taken at 100 ms, 200 ms and 400 ms exposure time for SYP, CgA and SSTR2 staining, respectively using the 10x, 20x or 40x objectives. Scale bar represents 50 µm. Please click here to view a larger version of this figure.
Figure 3: Immunohistochemistry (IHC) staining of SBNET spheroids. Formalin-fixed and paraffin-embedded SBNET spheroids sections were deparaffinized, rehydrated, blocked and stained with (A) SYP, (B) CgA, and (C) SSTR2 antibodies. Images were taken using the 400x objective. Scale bar represents 50 µm. Please click here to view a larger version of this figure.
Figure 4: Using SBNET spheroids for drug testing. (A) Bright field image of SBNET spheroids treated with DMSO for 5 days. (B) Image of SBNET spheroids treated with DMSO and stained with Ethidium homodimer (Ethidium H). (C) Bright field image of a dead SBNET spheroid forming grape-like structure after treatment with 10 µM of rapamycin for 5 days. (D) Dead SBNET spheroid stained with Ethidium H appears as red dots. Images of Ethidium H staining were taken using the red filter cube at 100 ms exposure time. Scale bar represents 10 µm. Please click here to view a larger version of this figure.
Growth media or solution | Composition |
Wash Medium | DMEM containing 1% FBS, 1% PEN/STREP, 1% Glutamine |
SBNET culture medium | DMEM/F12 + 10% FBS + 1% PEN/STREP + 1% Glutamine + 10 mM nicotinamide + 10 µg/mL insulin |
Antibody buffer | 2.5% bovine serum albumin, 0.1% sodium azide, 25 mM Tris pH 7.4, 150 mM sodium chloride |
Freezing medium | 90% FBS + 10% DMSO |
Human liver stem cell isolation medium | DMEM/F12 , 1% Pen/Strep, 1% GlutaMAX, 10 mM HEPES, 1/50 B27 Supplement, 1/100 N2 Supplement, 1 mM N-acetylcysteine, 200 ng/mL Rspo 1, 50ng/mL EGF, 100 ng/mL FGF10, 10 mM nicotinamide, 10 uM forskolin, 5uM A83-01 |
Human pancreatic stem cell isolation medium | DMEM/F12 , 1% Pen/Strep, 1% GlutaMAX, 10 mM HEPES, 1/50 B27 Supplement, 1/100 N2 Supplement, 1 mM N-acetylcysteine, 200 ng/mL Rspo 1, 25 ng/mL Noggin, 50ng/mL EGF, 100 ng/mL FGF10, 10 mM nicotinamide, 10 uM forskolin, 5uM A83-01, 3 uM PGE-2 |
Table 1. List of growth media and solution.
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 tumors16,17. 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 grow in various culture media. They grow slightly faster in stem cell media for human pancreas or liver isolation that we adapted from previously published protocols9. We chose to grow the SBNETs in DMEM/F12 medium supplemented with insulin and nicotinamide because this is less expensive than stem cell media. Increasing the percentage of fetal bovine serum is another strategy to promote the growth of SBNET organoid cultures; however, this would also increase the overall cost for culture maintenance.
Performing IF staining and imaging with a fluorescent microscopy using the 10x, 20x or 40x objectives is a quick and simple method to test for the expression SBNET markers. However, it does not give a well-defined localization in comparison to IHC (Figure 2, Figure 3). For example, the IF data showed that the membrane localization of SSTR2 is difficult to detect. We mainly detect the cytosolic SSTR2, which has previously been reported18. In order to obtain a better localization of the marker proteins, we recommend using IF and confocal microscopy. Overall, IF is a useful method to rapidly confirm SBNET markers. Our antibodies (anti-SYP, anti-CgA, anti-SSTR2) did not cross-react with the negative control organoids that do not express SYP, CgA, or SSTR2 (Figure 2C) in the IF experiment. This suggestion that the fluorescent signals that we detected are specific to SBNET spheroids.
To increase the yield of SBNET spheroids, the critical steps of this protocol are during the cell filtration step (step 2.2) and mixing the SBNET with the liquid ECM for aliquoting into tissue culture plates (step 2.8). Make sure to cover the cell strainer membrane and the collection tube with media in order to prevent the SBNET cells from sticking to the plastic. Liquid ECM will rapidly solidify if not placed on ice. Make sure to have a small ice container in the tissue culture hood in order to keep the ECM and SBNET cells cold.
The limitation of this technique is the slow growth rate of the SBNET spheroids. Experiments must be planned efficiently and avoid using an excess amount of SBNET spheroids. Another limitation is the slow recovery after freeze thaw. It takes over 1 month after thawing for the SBNET spheroids to start growing. To overcome this limitation, we suggest to continuously maintain SBNET spheroids in cultures and splitting them as needed (step 7). Even after 9 months in culture, the SBNET spheroids still maintain expression of SBNET markers (Figure 2B).
Although 3D culture of SBNET spheroids is more labor intensive than traditional 2D culture of other cancer cell lines, it is an extremely valuable model for in vitro culture of SBNET because many SBNET researchers do not have access to the existing cell lines. With this protocol, scientists and clinicians can establish SBNET spheroid cultures from resected tumors and share them with other laboratories. In addition, the SBNET spheroids could potentially be used to establish SBNET patient-derived xenograft mouse models. Overall, the techniques presented here can be adapted for culturing, characterizing and performing drug testing of other NETs such as pancreatic or lung NETs. Note that the growth rate of the organoids will vary between the different types of NETs and patient samples.
The authors have nothing to disclose.
This work was supported by NIH grants P50 CA174521 (to J.R. Howe and A.M. Bellizzi). P.H. Ear is a recipient of the P50 CA174521 Career Enhancement Program award.
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 |