3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing
- Paola Longati1, 2,
- Xiaohui Jia1, 2,
- Johannes Eimer1, 2,
- Annika Wagman1, 2,
- Michael-Robin Witt3,
- Stefan Rehnmark3,
- Caroline Verbeke4,
- Rune Toftgård2,
- Matthias Löhr†1, 2Email author and
- Rainer L Heuchel†1, 2
© Longati et al.; licensee BioMed Central Ltd. 2013
Received: 15 June 2012
Accepted: 24 February 2013
Published: 27 February 2013
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance.
Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine.
Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect.
We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.
Over the past decades pancreatic ductal adenocarcinoma (PDAC) has become the subject of increased research activity, however, the prognosis of this disease remains the worst amongst solid tumours. The 5-year survival rate is still below 5%, and this is at least partially due to an almost complete resistance against both conventional and targeted chemotherapy. With the present standard of care, conventional chemotherapy results in a median life expectancy of around 6 months . Recent evidence suggests that the molecular basis for this chemoresistance is multifaceted and reflects a wide range of genetic changes in a multitude of cellular pathways and response , including drug transportation  and microenvironmental alterations . A better understanding of the underlying mechanisms is key to the identification of novel therapeutic strategies capable of overcoming this chemoresistance.
Three-dimensional culture of tumour cells was introduced as early as the 1970s. Initially, investigations focused on the morphology of and interactions between tumour cells . Various PDAC cell lines were tested for their ability to grow as spheroids in 3D culture [6, 7]. Among these, the widely used Panc-1, which carries both KRAS and p53 mutations, was shown to form aggregates under appropriate culture conditions . It became apparent that 3D cultures are generally more resistant to chemo- and radiotherapy than their 2D counterparts [8, 9], however validated three-dimensional in vitro tumour cell models allowing for fast and standardized drug screening are not routinely employed. Based on these observations, a new hypothesis relating chemoresistance to the microenvironment, i.e. the stroma and extracellular matrix, was proposed. This novel concept, coined cell adhesion mediated drug resistance (CAM-DR), was proposed for bone-marrow derived malignancies , but has not been applied to solid tumours, including PDAC . In this study, we characterize a 3D tumour model in which the PDAC acquires a more stroma-rich phenotype, which simulates more closely the in vivo situation, and provides evidence for the CAM-DR concept.
The following well-characterized human pancreatic ductal adenocarcinoma cell lines (ATCC) were used: AsPC-1, BxPC-3, Capan-1, Panc-1 [6, 12]. A human immortalized pancreatic stellate cell (PSC) line  was used as a non-transformed control cell line. KPC cells were established from a mouse PDAC model, carrying pancreas-specific Kras and p53 mutations (KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre; hence KPC) . Cells were cultured under standard culture conditions (5% CO2, at 37°C) in DMEM/F12 or phenol red-free DMEM/F12 medium (Gibco) containing 10% fetal calf serum (FCS, Invitrogen).
Cells were trypsin-treated and counted using the Casy Cell Counter according to the manufacturer’s recommendations (Schärfe System GmbH, Reutlingen, Germany). Subsequently, they were seeded onto round bottom non-tissue culture treated 96 well-plates (Falcon, BD NJ, USA) at a concentration of 2500 cells/well in 100 μl DMEM-F12 or phenol red-free DMEM-F12 medium, containing 10% FCS and supplemented with 20% methyl cellulose stock solution. For preparation of methylcellulose stock solution we autoclaved 6 grams of methylcellulose powder (M0512, Sigma-Aldrich) in a 500 ml flask containing a magnetic stirrer (the methylcellulose powder is resistant to this procedure). The autoclaved methylcellulose was dissolved in preheated 250 ml basal medium (60°C) for 20 min (using the magnetic stirrer). Thereafter, 250 ml medium (room temperature) containing double amount of FCS (20%) was added to a final volume of 500 ml and the whole solution mixed overnight at 4°C. The final stock solution was aliquoted and cleared by centrifugation (5000 g, 2 h, room temperature). Only the clear highly viscous supernatant was used for the spheroid assay (about 90-95% of the stock solution). For spheroid generation we used 20% of the stock solution and 80% culture medium. corresponding to final 0.24% methylcellulose. Spheroids were grown under standard culture conditions (5% C O2, at 37°C) and harvested at different time points for RNA isolation or drug testing as stated below.
mRNA isolation and RT-PCR analysis
Cells or spheroids were collected, washed once with cold PBS, and processed for total RNA isolation using the RNeasy or the miRNeasy Mini Kit (Qiagen). RNA integrity and concentration were analyzed using agarose gel electrophoresis and Nanodrop Spectrophotometer. One μg of total RNA was retrotranscribed (First Strand cDNASynthesis kit, Roche). In the case of microRNA analysis, the NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen) was used for retrotranscription.
SYBR-Green Technology (Fermentas) was used for all qRT-PCR experiments. Further detailed information regarding qPCR reactions and oligonucleotide primers sequences is included in Additional file 1: S1.
SDS-PAGE and western blotting
Whole cell lysates from 2D or 3D cultured cells were prepared using M-PER® Mammalian Protein Extraction Reagent lysis buffer (Pierce Biotechnology, Thermo Scientific, Rockford, USA). The protein concentrations were measured using a BCA Protein Assay kit (Pierce). Cell lysates (50 μg) were resolved on 8% SDS-PAGE and analysed by immunoblotting. Anti-E-cadherin antibody was from BD transduction laboratories (BD610182, dilution 1: 2500). Anti-HIF1α antibody was from NOVUS Biologicals (NB100-449, dilution 1:500. Anti-Glut-1 and Anti-GAPDH (used as loading control) antibodies were from Abcam, Cambridge, UK (ab40084, dilution 1:2000 and ab9483, dilution 1:5000, respectively). Primary antibodies were detected with peroxidase-conjugated donkey Anti-rabbit immunoglobulin antibody (Amersham) and visualized with Immun-Star WesternC Chemiluminescence Kit (BIO-RAD) by a cooled CCD camera system (molecular Imager Chemo DocTM XRS System, BIO-RAD).
Immunofluorescence and electron microscopy
Spheroids were harvested at fixed time points and washed twice with PBS. For immunohistochemistry, spheroids were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned. Seven μm sections were stained as described below. Prior to blocking (PBS-tween 1% BSA), 0.01 M Sodium Citrate Buffer, pH 6.0, was used as an antigen retrieval solution. Anti-collagen I (rabbit polyclonal, ab292, Abcam, dilution 1:500) and Anti-fibronectin (mouse monoclonal, ab6328, Abcam, dilution 1:200) were used as primary antibodies. Biotinylated Anti-rabbit or Anti-mouse secondary antibodies from Vector Laboratories (Bulingame, CA, USA) were used in combination with streptavidin-coupled DyLight 549 from Jackson ImmunoResearch for fluorescence detection.
For electron microscopy, spheroids were fixed in phosphate buffer pH 7.4 containing 4% glutaraldehyde and 1% paraformaldehyde, and subsequently embedded and processed. Imaging was performed on a Tecnai 12 Spirit Bio TWIN transmission electron microscope (Fei Company, Eindhoven, The Netherlands) at the Central Electron Microscopy Unit of Karolinska Institutet.
Lactate accumulation measurement
Cells were grown both in 2D and 3D culture (2500 cells/well in 96 well plates) without medium change for the whole experiment time (from day 1 to day 10). Lactate accumulation was measured in the medium of four different wells at each time point using the YSI 2700 SELECT™ Biochemistry Analyzer (YSI life sciences, Yellow Springs, Ohio, USA) according to manufacturer’s recommendations. Cell-free medium was used as a control. Mean concentrations of lactate were calculated after subtracting lactate levels measured in the cell-free medium. Cells in corresponding wells (2D or 3D cultures) were lysed with M-PER® Mammalian Protein Extraction Reagent (#78501, Pierce). Protein quantification was performed using Pierce BCA protein Assay Reagent kit (#23225) and quantified with the ELISA reader (Molecular Devices Spectra MAX 250). The number of lactate moles per well was calculated from the measured lactate molar concentration, normalized for the total protein content of the cells/spheroid from the same well. The metabolite concentration was then expressed as mol/g total protein.
Drug test, acidic phosphatase (APH) assay
Experimental drugs used in 2D and 3D cultures with respective viabilities
Viability after treatment,%
Results and discussion
Formation of compact 3D spheroids
The growth kinetic of Panc-1 spheroid formation was assessed longitudinally (Figure 1B). Loose cell clustering occurred on day 2, and was followed by a gradually more compact growth, until on day 4, a spheroid with a diameter of 450–500 μm had developed and remained relatively stable until day 8. Cell viability, evaluated by trypan blue staining, was approximately 90% in both 2D and 3D cultures (data not shown). The increase in cell numbers over time indicated that proliferation was reduced in 3D compared to conventional 2D culture, especially after day 4 (Figure 1C).
Altered energy metabolism and lactate accumulation in 3D spheroids
As non-vascularized 3D tissue culture may develop hypoxic regions, the expression of HIF-1α and downstream target genes was investigated in both 2D and 3D Panc-1 cultures on days 4 and 7. The total HIF-1α protein level was similar in 2D and 3D cultures at day 4 but was lower at day 7 in 2D culture, whereas it was maintained at the same level in 3D culture (Figure 3B). This indicates that HIF-1α protein stability is higher in cells growing in 3D compared to 2D culture.
In order to corroborate this finding, the expression of genes downstream of HIF-1α, ie. GLUT1, GLUT12, PTGS2, VEGFA, HK2 and PDGFB, was assessed by RT-PCR at various time points (Figure 3C). RNA expression of GLUT1, VEGF and HK2 was found to be higher in 3D compared to 2D culture particularly from day 4 onward, whereas GLUT12 expression was decreased over time. For GLUT1 this was also verified at the protein level (Figure 3D).
Increased extracellular matrix (ECM) in 3D culture
Furthermore, we searched for additional modulators of ECM. miRNAs have been described recently as a new class of gene regulators, also in PDAC , where some were reported to regulate stromal molecules. mir-146a suppresses invasive cell properties and is under-expressed in Panc-1 cells compared to normal human pancreatic ductal cells . We found a strong up-regulation of mir-146a when Panc-1 cells were grown in 3D (Figure 5C). This may possibly reflect the forced immobilization of cancer cells in the spheroid .
Increased expression of chemoresistance-related genes
Chemoresistance in solid tumors is conveyed by different mechanisms. The classical are based on MDR genes and transporter proteins, all reported to contribute to chemoresistance in PDAC [3, 4, 41]. We therefore evaluated the mRNA expression of genes involved in drug resistance by RT-PCR in 2D and 3D Panc-1 cultures. The ATP binding cassette ABCC1 was up-regulated during the initial sphere formation period (Figure 4A). Furthermore, expression of miRNAs miR-21 and miR-335 associated with elevated chemoresistance [42–44] was increasing in 2D culture until day 4 and then constantly decreasing until day 10. In contrast, in 3D culture the expression of miR-21 and miR-335 peaked later on day 8, decreasing slightly thereafter, resulting in higher expression (Figure 4B). There are other molecules described more recently. PPP1R1B (protein phosphatase1, regulatory subunit1B) formerly called DARPP-32, is a bifunctional signal transduction molecule acting both as kinase and phosphatase inhibitor, that has been detected in several solid tumours including some carcinomas of the GI tract. The truncated form, t-DARPP-32, has been demonstrated to confer drug resistance, e.g. against trastuzumab in breast cancer via the AKT pathway, or against gefitinib in gastric cancer via EGFR/ERBB3  and by reducing drug-related apoptosis via CREB/PKA . T-DARPP is also responsible for the nuclear translocation of ß-catenin . We found it highly upregulated in the 3D culture system. SNED1, as described above, conveys drug resistance against platinum . Finally, PDAC cells become more resistant to drugs if cultivated on fibronectin or collagen I, both found upregulated (see above), indicating a role for these ECM proteins in protecting cells from chemotherapy [48, 49]. Due to increased extracellular matrix in vitro 3D systems provide mechanical properties that act as a barrier to drug diffusion [49, 50]. Collagen I, for example, a major component of ECM, is expressed at a higher level in 3D than in 2D breast cancer cell cultures . This observation is of particular interest, as collagen I is involved in gemcitabine resistance in pancreatic cancer . Fibronectin-1, which mediates cell and tissue cohesion, is also up-regulated in pancreatic and other cancers [52–54].
In other tumor cell models, cellular stress caused MRP1 and P-gp overexpression leading to increased Gemcitabine sensitivity, which could be abolished by blocking these efflux pumps with verapramil .
Increased chemoresistance in 3D culture
The mode of action and molecular mechanisms of these two compounds are subject of further studies.
Testing drugs in a 3D culture model raises the issue of drug penetration, which may be impaired by structural features of the three dimensional culture, including the size of the spheroids . Drug penetration into the spheroid is also determined by diffusion through the ECM. The specific interactions between cancer cells and their microenvironment, both cell-cell and cell-matrix adhesion, are amongst the factors that determine the effect of chemotherapy , and are likely to vary from one cell type to another. PDAC cells express already endogenous ECM components such as collagen and fibronectin-1 . Higher drug resistance was shown in PDAC cells grown on fibronectin-1 or collagen coated culture dishes . In our study the acquisition of elevated drug resistance of cancer cells in the 3D culture model may be explained by the increased endogenous ECM protein expression within the microenvironment of the spheroids, thus supporting the proposed cell adhesion-mediated drug resistance (CAM-DR), and upregulation of other, more recently identified molecules described above, e.g. ABC transporters, PPP1R1B, SNED1. However, since we have only tested a limited number of transporters, we can not exclude that other transporters such as P-glycoprotein may play a role, as described in other solid tumor cells in vitro .
3D culture of pancreatic tumour cells from KRAS mouse model
For decades, conventional two-dimensional (2D) cell culture has been the cornerstone of screening of novel drugs for pancreatic cancer as much as for other solid tumours . It represents a convenient and high-throughput but rather artificial method of growing cells. Nonetheless, the predictive value was satisfactory, especially in non-solid malignancies.
As cellular response to drugs is profoundly affected by microenvironmental factors, the use of a 3D-culture seems more appropriate for drug testing. This applies in particular to tumours such as PDAC, which are chemoresistant in most patients, despite a good response in (2D) tissue culture and xenograft models . The newly described genetically engineered mouse models, namely the KP and KPC mouse, better recapitulate the impact that inflammatory and stromal cells have in the pathogenesis of PDAC .
Our results confirm the previously described increased chemoresistance in 3D; we further demonstrate a more matrix-rich phenotype in 3D culture that may be advantageous for drug testing as it simulates more closely the in vivo situation: in 3D culture the microenvironment acquires new features with altered ECM composition, which has a major role in protecting the cells from drug activity [10, 58]. Expression of several key matrix proteins and miRNAs related to stromal development is increased, as is glycolysis. These changes mirror features that are characteristic of PDAC, i.e. a high content in ECM components  and growth factors such as PDGFB and VEGF, which are responsible for tumour progression.
In addition to elucidating the mechanisms of chemoresistance and the role of CAM-DR in PDAC , the 3D model characterized in this study may serve as a high-throughput screening platform for chemotherapeutic drug testing that provides a more reliable prediction of the response to treatment of patients with pancreatic cancer.
This study was supported by the Swedish Research Council (Vetenskapsrådet 2007–4034) to JML and from Cancerfonden (CAN 2008/982) to RLH. Further support was received by an EU FP7 program, EPC-TM net (to JML, RLH, MRW and SR). We thank Stig Linder, Dept. of Onco-Pathology, Karolinska Institutet, for providing us with the microtubulin-inhibitors and Yehuda Miron and Talia Miron from the Weizman Institute, Rehova, Israel, for providing us with the allicin and MT drugs. We thank Kjell Hultenby from the Central Electron Microscopy Unit at Karolinska Institutet and Beth Hagman for help with immunohistochemistry.
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