Journal of Clinical & Experimental OncologyISSN: 2324-9110

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Research Article, J Clin Exp Oncol Vol: 3 Issue: 2

Biomimetic Three Dimensional Cell Culturing: Colorectal Cancer Micro-Tissue Engineering

Yusra L Kassim1,2*, Elias AL Tawil1,2,3, Didier Lecerf1,3, Jérôme Couteau5, Thomas Simon1,2, Catherine Buquet1,2, Jean Pierre Vannier1,2and Elise Demange4
1Normandie Université, France
2Microenvironementet Renouvellement Cellulaire Intégré (M.E.R.C.I laboratory) E.A 3829, University of Rouen, 22 Boulevard Gambetta, F-76183 Rouen cedex, France.
3Université de Rouen, Laboratoire Polymères Biopolymères Surfaces, F-76821 Mont Saint Aignan, France, CNRS UMR 6270 & FR3038, F-76821 Mont Saint Aignan, France
4Celenys, Biopolis 75 route de Lyons la Forêt, 76000 Rouen, France.
5Toxem, 25 Rue Philippe Lebon, 76058 Le Havre, France.
Corresponding author : Dr Yusra Kassim
Faculty of medicine and pharmacy, M.E.R.C.I laboratory, 22 Boulevard Gambetta, F-76183 Rouen cedex, France
Tel: + 33235148350; Fax: +33235148340
E-mail: [email protected]
Received: November 13, 2013 Accepted: April 15, 2014 Published: April 18, 2014
Citation: Kassim YL, Tawil EAL, Lecerf D, Couteau J, Simon T, Buquet C, Vannier JP and Demange E (2014) Biomimetic Three Dimensional Cell Culturing: Colorectal Cancer Micro-Tissue Engineering. J Clin Exp Oncol 3:2. doi:10.4172/2324-9110.1000123

Abstract

Background: Spheroid cultures are known to mimic closely the properties of tumor tissue than monolayer cultures with regard to growth kinetics and metabolic rates. The aim of this paper is to confirm that tumor micro-tissue in a 3D biocompatible microenvironment maintain the cells natural behavior when compared to 2D monolayer culturing.

Method: In order to validate our 3D culture system, we compared the 3D culture within a cross-linked hydrogel of hyaluronic acid, one of the major components of the extracellular matrix and the conventional 2D culture system.

Results: Interestingly within our culture system, cells could be analyzed either after retrieval from the scaffold or even without being extracted in the 3D form rendering the HA hydrogel an ideal tool for biological applications. We observed the difference in the cell cycle, cell proliferation and behavior in both culture systems. Additionally drug testing was carried out using a chemotherapeutic agent (cis-platinium) that is already in clinical use to unequivocally prove the clinical predictive significance of the test strategy as compared with less complex assay systems and more complex in vivo models. We observed the presence of cell cycle heterogeneity very similar to the situation in vivo human tumors. Moreover, we have confirmed that resistance to chemotherapeutic reagents within this 3D culture system is much higher than those used in 2D cultures, since the tight assembly of cells in 3D culture systems render them more resistant requiring chemotherapeutic doses that recapitulate the drug sensitivity of tumor cells in vivo. Additionally we have observed the difference of apoptotic protein expression between 2D and 3D cell culture.

Keywords: 3D cell culture; Hydrogel; Hyaluronan; Colorectal cancer; Micro-tissue

Keywords

3D cell culture; Hydrogel; Hyaluronan; Colorectal cancer; Micro-tissue

Introduction

Traditional monolayer cell culture or two dimensional (2D) cell cultures have provided the nascent interpretation of complex biological phenomena. However, recent work has shown that cells often exhibit unnatural behavior when they are excised from native three dimensional (3D) tissues and confined to a monolayer. A 3D scaffold induces changes in cell shape and cell cluster arrangement not observed in cells grown in 2D on plastic or isolated individual components of the extracellular matrix (ECM) [1]. Furthermore, cell function difference between 2D and 3D cultures has been demonstrated by Bissell and coworkers in human breast epithelial cells which develop like tumor cells when cultured in two dimensions, but revert to normal growth behavior when cultured in 3D analogs of their native microenvironment [2]. These findings in oncogenesis elucidate acute disparities in cell morphology and function between 2D and 3D cultures and suggest that examining hierarchical biology in just two dimensions is insufficient.
For all the previously mentioned reasons and more, scientists have been encouraged to develop both biologically derived and synthetic matrices for 3D cell culturing. Among these matrices, hydrogels composed of cross linked polymer networks with high water content were fabricated. The chemistry of the scaffold has been observed of importance in the cellular phenotypic regulation [3]. For which biologically derived hydrogels have attracted great interest in scaffold tissue engineering because of their high water content in addition to their biocompatibility and mechanical properties that mimic aspects of the native biological microenvironment [1,4]. The importance of these biocompatible scaffolds does not reside to the similitude to the native microenvironment, it rather extends to the fact that they generate similar signals to those induced in vivo.
Among the desirable mechanical properties required in scaffold tissue engineering is adequate pore size and a high surface area to volume ratio through the presence of a network channel and interconnected pores, this facilitates cell penetration and formation of cellular associations [5,6]. The importance of this porous network also resides in the capacity to allow exchange of oxygen, nutrients and waste as well as realistic transport of soluble factors. This may lead to the formation of soluble gradients of oxygen, glucose as well as waste products within each spheroid, thus each spheroid may be considered as a non-vascular micro tissue. Hyaluronic acid (HA), a glycosaminoglycan (GAG), is one of the principle elements which contribute to the structuring of the ECM in addition to other molecules [7]. The 3D cell culture system developed in our laboratory is composed of a hyaluronic acid (HA) hydrogel. Ourreticulated HAhydrogel used for 3D tumor micro-tissue formation has been shown to possess a mean pore size of 227 μm x 234 μm suggesting that the scaffold structure isoptimal for cell proliferation [8].The invasiveness, distribution and infiltration of various malignant cell types and their sensitivity to chemotherapeutic agents have been previously tested using this hydrogel with or without the addition of biological cues as elastine [9-12].
The aim of this paper is to confirm that tumor micro-tissue in a 3D biocompatible microenvironment maintain the cells natural behavior when compared to 2D monolayer culturing; in terms of cell proliferation, persistence in the cell cycle and reaction towards chemotherapeutic agents. Interestingly cells could be analyzed either after retrieval from the scaffold or even without being extracted in the 3D form rendering the HA hydrogel an ideal tool for biological applications. The importance of 3D cell culture systems is that they allow better insight into therapeutic problems associated with proliferative gradients, such as altered responsiveness, effect of chronic hypoxic cells and importance of the 3D cell-cell and cell-matrix interactions [13]. In fact tumor cells cultured in 3D structures adopt the resistance towards apoptosis similar to that found in solid tumors. Additionally, the application of such 3D culture systems is increasingly discussed as to its potential to economically optimize preclinical selection of the most active effectors from a large pool of drug candidates and to replace some animal test modules. Although this has not yet fully been incorporated as a routine drug development and testing tool, encouraging the optimization of this tool for advanced cell based in vitro screening strategies is required.

Materials and Methods

Synthesis of cross-linked hyaluronan hydrogels: scaffold fabrication
Hyaluronan hydrogels consisted of a long-chain of hyaluronan cross-linked with adipic dihydrazide (ADH; Sigma-Aldrich, France) and 1-ethyl-3 [3-(dimethylamino)-propyl] carbodiimide (EDCI; Sigma-Aldrich). All the hydrogels were prepared from high molecular weight hyaluronan (>106 Da; Sigma-Aldrich) according to the procedure described by Prestwich et al. [14]. Briefly, the ratios ADH : hyaluronan and hyaluronan : EDCI were adjusted to obtain hydrogels optimized for cell adhesion and culture. The optimal conditions for cell proliferation and adhesion were obtained with a ADH : hyaluronan ratio of 10 : 1 and a hyaluronan : EDCI ratio of 1 : 1. Hyaluronan and hydrazide cross-linker (ADH) were dissolved in milliQ-water and the pH was adjusted to 4.6 by adding 0.1 N HCl. The carbodiimide reagent (EDCI) was dissolved in milliQ-water, added to the reaction mixture and allowed to gel for 2 h with gentle agitation. Hyaluronan hydrogels were dialysed against 0.1 N NaCl for 2 days, then in a water : ethanol mixture (3 : 1 v/v) for 2 days, and in milliQwater for 2 days to remove unreacted ADH and EDCI. Each dialysed hydrogel is placed in a plastic container and frozen. Following freezing, the hydrogels are placed in a lyophilizer (Alpha 1–2, Christ, Germany; performances, 2 kg ice/24 h, T= -55°C). Depending on the volume of water to be eliminated, the lyophilisation was carried out for 4–5 days. The lyophilized hydrogels were then stored at -20°C. Lyophilized hydrogels were cut into rectangular parallelepipeds (about 1x1x1 mm) prior to use. The cut hydrogels were sterilized at 100°C and then rehydrated in culture medium [Roswell Park Memorial Institute 1640 (RPMI1640), Eurobio, France]. The gel pH postrehydration was approximately 8.4 and the swelling ratio of hyaluronan hydrogels at room temperature in RPMI medium was 37 g/g.
Cell culture
The human CRC cell line HT-29 was obtained from ATCC Germany. HT-29 cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) heat-inactivated FBS, and 1% (2 mM)L-glutamine (Gibco, Invitrogen, France), 1% (2 mM) sodium pyruvate (Gibco, Invitrogen, France) and 0.1% of Penicillin/ streptomycin antibiotics incubated in a humidified atmosphere of 5% CO2 at 37°C.
Cell cycle analysis
2D HT-29 cells were harvested by Trypsin-EDTA. Cells cultured in the hyaluronan hydrogel during 5 and 8 days were harvested by hyaluronidase (Sigma, France); the cell suspension was washed and fixed in chilled absolute ethanol solution during 24 hours. Cells were then washed and incubated for 30 minutes with 50 μg/ml Propidium Iodide (PI) and 100 μg/ml RNAse A. For double labeling cells were incubated with 5 μl FITC conjugated anti Ki-67 antibody (Ki67-FITC) for 2 hours at room temperature, washed and incubated with the PI/ RNAse A mixture. For each sample, 104 cells were analyzed on Epics XL/MCL flow cytometer (Beckman Coulter, France). PI and FITC fluorescence were collected through a 630 and 520 nm bandpass filters, respectively.
Hyaluronan hydrogel culture of colorectal cancer (CRC) cells
In each well, 1x106 HT-29 cells were cultured in 2 ml of culture medium. Ten hyaluronan hydrogels, prepared as described above, were placed in each culture well. The cultures were incubated for one day in contact with cells, and then incubated in a new well containing fresh culture medium during 5 days allowing spheroid formation. After which, HA hydrogels are incubated in a new well containing fresh culture medium and supplemented or not with various concentrations ofcis-platinium (CDDP) (Sigma, France) at 37°C; 5% CO2 during 6 days.
Cell viability of spheroids cultured in hyaluronan hydrogel
HT-29 spheroids formed in the hyaluronan hydrogel were stained using live/dead cytotoxicity/viability kit for mammalian cells (Life Technologies, France). The live cells are stained with calcein AM, which emit green fluorescence light (517 nm); while the dead cells are stained with ethidium homodimer-1 (Eth-1), which emits red fluorescence light (617 nm). The two components were added to PBS (Gibco, France), each at a concentration of 2.5μg/ml and 0.75μg/ml respectively. Hydrogels with spheroids were incubated in the solution for 30 minutes at 4°C. After rinsing with PBS, spheroids were observed using an M200 microscope (Zeiss, Germany).
Volume of spheroids during Cis-platinium treatment
Phase contrast imaging and morphological analyses of spheroids in each hydrogel were carried out manually onAxiovert 135 microscope (Zeiss, Germany) equipped with an AxioCam (Sony, Germany). Spheroid diameters and volumes were determined from images taken with a 2.5x objective. The two-way ANOVA test was used for statistical analysis with the Bonferroni post-test to compare each point to its corresponding control.
Analysis of drug sensitivity by WST-1 assay
Cells were cultured either in a monolayer and a 3D system separately, as mentioned above. After which cell proliferation and drug sensitivity analysis were carried out by adding WST-1 reagent (Roche Applied Sciences, France) at a dilution of 10:1 to the seeding well. Hydrogels were incubated at 37°C for up to 4 h, and 70 μl WST-1/ medium solution were transferred to 96-well plate. To establish a standard curve, optical densities were measured at 420nm and 620nm using a micro plate spectrophotometer (Powerwave X, Bio-tek instruments, France). The absorbancedirectly correlates with the number of viable cells. All experiments were done in triplicate, and the 50% inhibition concentration (IC50) (μM) values were calculated using Sigma plot software and the results expressed as mean ±SD.
Protein extraction from cells cultured in hyaluronan hydrogel
Proteins were extracted from the cells cultured in the hyaluronan hydrogel using chilled lysis buffer containing 1% (v/v) Triton (Sigma, France), 50 mM Tris-HCl (Sigma, France) pH 8.0, 150 mM NaCl, 1 mM MgCl2, and complete mini protease inhibitor cocktail with EDTA (Roche Diagnostics, France). Lysates were clarified by centrifugation at 15,000 rpm for 3 minutes; supernatants were stored at -80°C before use. Total protein content determination was done by Bradford technique. The same method of protein extraction was used for cells cultivated in 2D after cell detachment.
Caspase-8 dosage
Cell lysates were obtained directly from cell spheroids cultured in hyaluronan hydrogel using the lysis buffer provided with the Caspase-8 human Elisa kit (Abcam, France). Caspase-8 was dosed using the previously mentioned kitaccording to manufacturer’s instructions. The experimental series was done in triplicate and oneway ANOVA test was used for statistical analysis with the Dunnett post-test to compare all columns versus the control.

Results

Spheroid formation, size and culture time
Hyaluronan hydrogels were incubated for 24h with 1x106 HT-29 cells allowing invasion of the hydrogels. During the first 4 hours, we observed that cells invading the hydrogel have a tendency to migrate towards each other; this cell aggregation permitted the spheroid formation (Figure 1).
Figure 1: HT-29 cells visualized in the hyaluronic hydrogel by timelapse microscopy. Successive photos were taken of HT-29 cells within the first 24h showing aggregation and primary spheroid formation. Scale bar represents 50μm.
After the first 24h culture medium was changed with fresh standard culture medium and left for 5 days allowing the formation of fully developed spheroids. During which we measured the diameter of spheroids every 3 days. With the standard medium (DMEM supplemented with 10% FCS), spheroid volume increases and reaches the maximum diameter within 6 days (Figure 2). Spheroids with an average diameter of 35 μm (ranging between 30-60 μm) were reached after 12 days of culture; the spheroid diameter doubling was about 72 h for HT-29 spheroids (Figure 2). Cell death in the spheroid core was noticed as the spheroids reached a diameter superior to 40 μm (Data not shown).
Figure 2: Spheroid size evolution at different culture time periods; CRC Cells were cultured in hyaluronan hydrogel treated or not with different doses of CDDP.The spheroid diameter (μm) were measured every 72h along the entire culture period (12days). Spheroid diameter was noticed to increase in time and reached its maximum limit after 6 days of culture. Therefore CRC spheroid cultures were treated or not with different doses of CDDP at day 6 of culture. In which spheroid diameters were found to decrease significantly upon treatment with 500μM of CDDP (*= P<0.05, ***= P<0.001)
Cell cycle analysis of cells cultured in 3D system
In order to confirm the fact that the 3D culturing system exhibits higher similarity to real tissues in many aspects than monolayer culturing, we analyzed the cell cycle of CRC spheroids cultured in hyaluronan hydrogel and compared it to the cell cycle of CRC cells cultured by the conventional 2D monolayer method. Cells harvested from both culture methods after 5 and 8 days were stained either with PI alone or in combination with FITC conjugated Ki-67 and analyzed by flow cytometry. It was observed that 75% of cells cultured in hyaluronan hydrogel were in the G0-G1 phase of the cell cycle after 5 and 8 days of culture, with a high percentage of cells in the sub-G1 phase after 8 days of culture (Figure 3A). This necessitated the double staining of PI associated with FITC conjugated Ki-67, which showed that 81% of the cells were proliferating cells after 5 days of culture, while only 67% of cells were shown to be positive for Ki-67 after 8 days (Figure 3B). This elaborated that the high percentage of cells in the sub-G1 phase represented debris or apoptotic cells emancipated from the core of the spheroids after 8 days of culture. These results confirm that 3D culture of cancer cells possess a layered structure composed of: a central necrotic area, deficient of nutrients and oxygen, an outer rim of proliferating cells and a layer of quiescent cells separating these two regions.
Figure 3: Flow cytometry cell cycle analysis of HT-29 cells cultured in a 3D system or (2D) monolayer. Cells were harvested after 5 and 8 days of culture in the hyaluronic acid hydrogel. A) Cells stained with PI alone. The majority of cells cultured in 2D were found to be proliferative while the percentages of cells in subG1 phase (quiescence) were found to increase during the time of 3D culture. B) Cells double stained with PI and FITC conjugated Ki-67. Percentage of non-proliferative cells were found to increase during the culture period.
Drug efficacy in monolayer versus 3D cell culture systems
As described above our hyaluronan hydrogel generated after the period of spheroid formation (6 days), a multiple spheroid culture that are heterogeneous in size ranging from 22 μm to 47 μm. This system could be used as a biological tool that may mimic the pathophysiological conditions found in vitro. In order to emphasize the difference of drug efficacy in conventional 2D monolayer culture and 3D spheroid cultures, we chose the well-known CDDP compound already used in the treatment of colorectal cancer. The cells cultured in either culture systems were treated with various concentrations of CDDP for 144 hours (6 days). At this time interval it was noticed that the tumor spheroids were completely dissociated at the highest concentration of CDDP (500 μM) while spheroids size diminished to range between 24 to 41 μm starting from 50μM of CDDP, i.e. at this concentration of the chemotherapeutic agent, the cell proliferation in 3D cultures is seized, thus no augmentation is observed in the spheroid size.
The correlation between cell proliferation and CDDP concentration was studied by using the WST-1 assay to calculate the IC50. Dose response curves were recorded on exponential HT-29 cultures and in spheroids sized 22-47 μm at day 6 (first day of treatment). In 2D monolayer cultures, IC50 value after 6 days of treatment with CDDP was 20 μM ± 1.52 μM, whereas for spheroid cultures IC50 value was found to be 48 μM ± 1.16 μM. Therefore the efficacy of CDDP tested was lower in HT-29 spheroids compared to monolayer culture (Figure 4).
Figure 4: Dose response curves for HT-29 cells cultured either in a monolayer (2D) or in the hyaluronic acid (3D) treated or not with different doses of cis-platinium (CDDP). The IC50 level is indicated by an interrupted line. The dose response curves were recorded in three independent experimental series showing high reproducibility.
Cell viability of spheroids cultured in hyaluronan hydrogel
In the aim of visualizing the cell viability, the spheroid containing hydrogels were stained using live/dead cytotoxicity/viability kit for mammalian cells and observed by fluorescence microscopy imaging at intervals of 3 and 6 days with or without CDDP treatment. The nontreated spheroids maintained high viability for up to at least 12 days of culture in which only <1% of cells revealed necrosis in the core of the spheroid. The successive augmentation of the chemotherapeutic dose induced more necrosis of the cells situated in the central core. Additionally it was observed that treatment with >100 μM of CDDP led to the complete destruction of the spheroid micro-tissue configuration (Figure 5).
Figure 5: Live and dead CRC cell spheroid imaging in a hyaluronan hydrogel either non-treated or treated with different doses of CDDP (ranging from 50 nM to 500 μM). Calcein AM stained live cells in green and EthD-1 stained dead cells in red.
Apoptotic/ Necrotic protein profiling in monolayer versus 3D cell culture
In the aim to emphasize the lack of resemblance of the monolayer culture system with the in vivo status, we performed antibody arrays to compare the expression of apoptotic proteins in the 2D as well as the 3D culturing system. Proteins were extracted from HT-29 cells cultured in either a monolayer or in hyaluronan hydrogel treated or not for 6 days with CDDP. Our results showed that, in comparison to the non-treated monolayer culture, the non-treated spheroids expressed a variety of apoptotic proteins including HSP60 and IGF-1sR, all of which were accentuated by the treatment with 50 μM of CDDP (dose equivalent to the IC50 value of CDDP as previously mentioned) on spheroid cultures (Figure 6). The expression of these apoptotic proteins are an indication of stressful condition of tumor micro-tissues as hypoxia and nutrient deficiency particularly to the central core cells in the spheroids. As Caspase 8 is considered to be the apex of the apoptotic cascade and in the aim to proceed in the confirmation of the most probably implicated signaling pathway in spheroid cell apoptosis, Caspase 8 was measured from spheroid cell lysis within the hydrogel i.e. without cell retrieval from the hydrogel. We observed that in non-treated spheroids, Caspase 8 concentrations were rather negligible; this concentration corresponded to the few apoptotic cells that are found in the central core of the spheroids which are deprived of nutrients and oxygen. Moreover it was observed that the Caspase 8 concentration significantly increased upon 50 μM of CDDP treatment (IC50 concentration) (Figure 7).
Figure 6: Apoptosis protein microarray profiling of HT-29 cells cultured in monolayer or in 3D spheroids treated or not with 50μM of CDDP (dose corresponding to the IC50 value). The expression of a variety of apoptotic proteins in non-treated 3D culturing conditions was observed in comparison to the non-treated 2D culturing condition. Additionally there is a remarkable over expression of the apoptotic proteins in spheroids when treated with 50μM CDDP.
Figure 7: Caspase 8 concentration measurement: HT-29 cells cultured in 3D spheroids treated or not with different doses of CDDP. The concentration of Caspase 8 significantly increased as 50 μM of CDDP was added to the spheroid culture (dose equivalent to the IC50) when compared to the control (*=P<0.05).
Taken together, these results confirm that the spheroid culture is much more realistic in comparison to the conventional 2D cell culturing system and considered to reflect the pathophysiological conditions found in vivo.

Discussion

Solid tumors often show hypoxia/necrosis, stem cell characteristics, slow proliferation and barriers to drug diffusion that contribute to drugresistance and these features are not properly reflected in monolayer cell cultures [15]. In 3D cell culture, cells are grown in a microenvironment that more closely mimics in vivo tissue architecture and function. This method of cell culturing has many applications in developmental cell biology, drug screening and regenerative medicine [16]. In order to validate our 3D culture system, we compared the 3D culture within a cross-linked hydrogel of hyaluronic acid, one of the major components of the extracellular matrix and the conventional 2D culture system. We observed the difference in the cell cycle, cell proliferation and behavior in both culture systems. Additionally drug testing was carried out using a chemotherapeutic agent (CDDP) that is already in clinical use to unequivocally prove the clinical predictive significance of the test strategy as compared with less complex assay systems and more complex in vivo models. As hyaluronic acid is known to be highly expressed in malignant tumors, the use of a HA hydrogel was shown to guide the cells to spontaneously self-assemble into 3D micro-tissues in form of spheroids through interactions with its receptors, CD44 and receptor for hyaluronan mediated motility (RHAMM) [17]. Spheroid diameter was found to be heterogeneous ranging between 30 and 60μm after 12 days of culture. Yet as in all micro-tissues limited access of nutrients leads to central necrosis which is related to the spheroid diameter. Spheroid diameters (and coefficient variants) are dependent on the behavior of different cell types [18]. Culture of immortalized glioma cell spheroids reaching up to 200μm with no central necrosis has already been used for drug and irradiation testing [19].These spheroids were generated in a scaffold free culture system; not only obliterating the cell-matrix interaction and its role in cell survival and tumor resistance, but also could not reflect drug and radio resistance due to the absence of the indirect effects of gene modulating patterns induced by hypoxia. In our culture system a central necrosis could be observed in spheroids with an average diameter of 40 μm. Cell cycle analysis showed that 8 day old spheroids possessed a high percentage of cells in the sub-G1 phase, with 75% of cells in the G0-G1 phase. Thus confirming the 3 layered structure of spheroids with a central necrotic area; an outer proliferating rim and layer of proliferating cells separating the two regions. Additionally, Ki-67 expression was found to be 1.2 folds lower in spheroids after 8 days of culture compared to monolayer culture and 5 day old spheroids. This could be due to the fact that in 8 day old spheroids, cells are more tightly attached limiting the diffusion of soluble factors including oxygen, thus rendering the central cells more hypoxic. Altogether, these factors are associated with drug resistance and must explain the difference between drug efficacy in spheroids and monolayer cultures. The IC50 of CDDP in monolayer culture (20μM ± 1.52) was lower than that of spheroids cultured in our system (48μM ± 1.16). We demonstrated that the chemotherapeutic agent acts on cells on the outside of the spheroids within the hydrogel in the initial stages of treatment before attacking the center by completely distorting the configuration of the spheroid. This could be due to the free diffusion of the chemotherapeutic agent through the hydrogel, thus promoting its action on the outer cells in order to reach inner cells. Similar results have been described in previous publications where the drug gradient action affects cancer cells from the outside of the cluster in [20,21]. This drug resistance found in our 3D culture system could be explained either by the fact that chemotherapeutic agents exert their activities on proliferative cells only thus the outer rim cells in our spheroids. But this also could be explained by the reduced chemotherapeutic activity in a hypoxic and nutrient deprived microenvironment [22].
In an attempt to emphasize the resemblance of our culture system to the pathophysiological status of non-vascularized solid tumors we performed an apoptotic/ necrotic protein profiling revealing that in monolayer cultures the cells show no apoptosis while even in nontreated spheroids the expression of these proteins could be detected. In our culture system, additional research is required to extend our protocol to the use of primary tumor material and to improve cell-line based screens by the combination of both the 3D organization and the multi-cellular complexity.

Conclusion

Limitations in drug penetration, contact-dependent multidrug resistance phenomena and oxygen deficiency are just three of the parameters influencing drug effects. Gene and protein expression modified by the 3D environment are further features that affect treatment outcome. Thus3D tumor micro-tissue spheroids formed can become a valid tool for negative and positive selection of the most promising candidates in drug development programs resulting in economical savings.

Acknowledgments

The authors would like to thank Dr. Fabienne Gravé, for kindly performing the flow cytometry analysis. This work was financially supported by the Agence National de Recherche (ANR-1- EMMA-040-02) and by Vie etEspoir association.

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