Effect of Spheroidal Age on Sorafenib Diffusivity and Toxicity in a 3D HepG2 Spheroid Model

The enhanced predictive power of 3D multi-cellular spheroids in comparison to conventional monolayer cultures makes them a promising drug screening tool. However, clinical translation for pharmacology and toxicology is lagging its technological progression. Even though spheroids show a biological complexity resembling native tissue, standardization and validation of drug screening protocols are influenced by continuously changing physiological parameters during spheroid formation. Such cellular heterogeneities impede the comparability of drug efficacy studies and toxicological screenings. In this paper, we demonstrated that aside from already well-established physiological parameters, spheroidal age is an additional critical parameter that impacts drug diffusivity and toxicity in 3D cell culture models. HepG2 spheroids were generated and maintained on a self-assembled ultra-low attachment nanobiointerface and characterized regarding time-dependent changes in morphology, functionality as well as anti-cancer drug resistance. We demonstrated that spheroidal aging directly influences drug response due to the evolution of spheroid micro-structure and organo-typic functions, that alter inward diffusion, thus drug uptake.

www.nature.com/scientificreports www.nature.com/scientificreports/ candidate to replace animal testing due to their improved predictive capability 18 . Any reduction of animal tests is not only ethically desirable but would also reduce one of the main cost-drivers in drug development process 19 . For these reasons, multi-cellular spheroids are extensively used as promising in vitro models for evaluating therapeutic anti-cancer strategies including chemotherapy 20,21 , antibody-based immunotherapy 22 , gene therapy 23 and combinatorial therapies 24 .
Despite the many advantages of multi-cellular spheroids over monolayer cultures [25][26][27][28] , some limitations still prevent the integration of 3D cell culture models into mainstream drug discovery pipelines. For instance, the lack of standardization in cell culture protocols often leads to variations in structure and composition of the established multi-cellular spheroids, all known to heavily affect the outcome of drug delivery and efficacy studies 29 . It is important to note that the selected culturing method significantly influences spheroid size, shape, density, surface topography and microstructure that may alter their behavior 30 . In addition to variations in structure, multi-cellular spheroids also comprise of cell populations in different proliferative stages including proliferation, quiescence and apoptosis, which leads to heterogeneous cell responses during chemical and physical treatments, thus making the comparability of drug exposure studies a difficult task 31,32 .
To ensure reproducible generation of multi-cellular spheroids and to increase the reliability of in vitro 3D-cell based assays, a set of quality parameters including area, perimeter, solidity and roundness have been introduced to increase the reproducibility of toxicity tests, efficacy studies and drug penetration assessments 33 . Although the benefits of these quality parameters in multi-cellular spheroid cultures are well established, the influence of spheroid cultivation time, also referred to as spheroidal age, on dose-response relationships in drug screening studies still remains an underestimated factor. The present work sets out to provide a better understanding how spheroidal age influences the outcome of drug screening studies using multi-cellular spheroids. In the present work, we specifically investigate how spheroidal age modulates diffusivity, resistance and toxicity of sorafenib, an FDA-approved multi-kinase inhibitor against liver cancer. Our 3D hepatocellular carcinoma spheroid model is generated using a novel protein-based nanobiointerface that reliably eliminates cell-surface interactions over long periods of time. Our self-assembled nanobiointerface is based on the S-layer protein SbpA derived from Lysinibacillus sphearicus CCM 2177 and exhibits outstanding cell-repulsive and anti-fouling properties [34][35][36] , thus effectively promoting the formation 3D HepG2 spheroids in microwells without the need of any external forces. We show that spheroid quality remains constant over 18 days in culture in the presence of SbpA-coated protein surfaces, while ultrastructural morphology and organo-specific metabolic evaluations are used to differentiate between early-stage, mid-stage and late-stage spheroids. Following the identification of spheroidal ages, drug diffusivity, toxicity and resistance are determined in an attempt to describe the interplay between spheroidal age and efficacy of drugs when employing in vitro 3D cell culture models.

Materials and Methods
S-layer Coating of Microwell Plates. Proteins were isolated from Lysinibacillus sphaericus CCM 2177 (SbpA) and subsequently purified as reported elsewhere 37 . To reconstitute the S-layer protein solution, 5 mg of lyophilized protein was dissolved in guanidine hydrochloride (5 M in 50 mM Tris-(hydroxymethyl)aminomethane x HCl buffer, pH 7.2) and dialyzed against Milli-Q-water (Millipore, Austria) for 1 h at 4 °C. Afterward, the protein solution was centrifuged at 1300 rpm for 15 min at 4 °C to remove self-assembly products. The concentration was adjusted to 100 μg/mL in a recrystallization buffer (0.5 mM Tris-(hydroxymethyl)aminomethane, 10 mM CaCl 2 , pH 9), 250 μL of the solution were added to U-bottom 96-well tissue culture plates (Greiner-Bio-One, Austria) and incubated over night at room temperature.
Cell Culture and Spheroid Generation. Hepatocellular carcinoma cells (HepG2, HB-8065, ATCC, USA) were cultivated in minimal essential medium (MEM, Sigma-Aldrich, Austria) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% GlutaMax TM (Life Technologies, Thermo Fisher Scientific, USA) and 1% antibiotic/antimycotic solution (Sigma-Aldrich, Austria). The cells were cultivated in 75 cm 2 cell culture flasks at 37 °C in 5% CO 2 humidified atmosphere as adherent monolayers. For spheroid generation, trypsinized cells were pelleted at 1250 rpm for 5 min (Megastar 1.6R, VWR) prior homogenization through a syringe to separate larger cell clusters into individual cells. Cells were seeded at an initial cell concentration of 10.000, 5.000 and 3.000 cells per well in SbpA-coated 96-well microtiter plates (Greiner Bio-one, Germany) and centrifuged at 1250 rpm for 10 minutes.
Albumin and Urea Secretion. Spheroid medium supernatants of day 1, 3, 6, 9, 12, 15 and 18 were collected, centrifuged at 1250 rpm for 10 minutes to remove cell debris and stored at −20 °C until sample analysis. The amount of human serum albumin (HSA) secreted into the culture medium was determined by Human Albumin ELISA Kit (Abcam, UK) according to the manufacturer's protocol. For ELISA, the supernatant was diluted 1:100 in dilution buffer. Urea was measured using a colorimetric assay kit from BioVision (Germany) according to the manufacturer's protocol.
Transmission Electron Microscopy (TEM). Spheroids were fixed in 4% formaldehyde solution (Carl Roth, Austria) at 4 °C and embedded in agarose (2% in Caco buffer) as previously reported 38 . The ultrathin www.nature.com/scientificreports www.nature.com/scientificreports/ sections were observed with a transmission electron microscope (FEI Tecnai G2 20, FEI, Netherlands) operating at 120 kV and images were acquired with an FEI Eagle 4 K camera (Nikon, Japan).
Histology. HepG2 spheroids were washed with 1X PBS (Sigma-Aldrich, Austria) after 3, 6, 12 and 18 days of incubation fixed with 4% paraformaldehyde in PBS (pH 7.6) at 4 °C and stored in PBS. For histological analysis, the HepG2 spheroids were cut in 3-μm serial sections, deparaffinized in xylene and rehydrated in a graded alcohol series. Histological staining was performed with hematoxylin and eosin (H&E).
Drug Screening. For monolayer culture, HepG2 cells were seeded at 3.000 cells per well in 96-well flat bottom plates and incubated for 6 days. Prior drug exposure, the monolayer reached a confluency of approximately 80%. For 3D culture, S-layer coated 96-well U-bottom plates were seeded at a cell density of 3.000 cells per well and incubated for 6 days. Drug exposure was carried out with different concentrations of sorafenib diluted in DMSO. Cells were treated with different concentrations of sorafenib (0-100 µM) in triplicates and incubated for 24 and 48 hours at 37 °C and 5% CO 2 . For analysis, AlamarBlue ® reagent (Invitrogen, Austria) was added directly to medium (10% v/v) and incubated overnight at 37 °C. Fluorescence was measured at ex/em 560/595 nm and absorbance was measured at 565 nm and 595 nm (Infinite F200, Tecan, Austria). The cytotoxicity index was determined using the untreated cells as a negative control and the IC 50 was extrapolated from the dose-response graph. For dose-age responses, HepG2 spheroids were treated with 100 µM sorafenib for 24 h up to 12 days of incubation.
Compound Diffusion. HepG2 spheroids were incubated for 3 and 12 days and treated with rhodamine B (Roth, Germany) diluted to a final concentration of 100 µM in cell culture media. Fluorescence micrographs were taken every minute to monitor diffusion towards the spheroid core until fluorescence intensity profile of the spheroid reached the same level as fluorescence background. Intensity values of spheroid at 3 different depths were taken using ImageJ (NIH, USA) and monitored over time.
Statistical and image analysis. Data were expressed as mean ± standard deviation (SD). All experiments were done independently in triplicates. Statistical significance among the experimental groups was determined with Student's t-test. A P value < 0.05 was considered statistically significant (*). Graphs were plotted using Prism 6 (GraphPad software, USA). Micrographs were analyzed using ImageJ (NIH, USA). Spheroid cultivation was monitored over 18 days with an inverted fluorescence optical microscope (TE2000, Nikon, Japan) equipped with a digital camera (DS-Qi1MC, Nikon, Japan). Spheroid area, perimeter, solidity roundness and diameter were measured by using the BioVoxxel Image Processing and Analysis Toolbox for ImageJ (Biovoxxel, Germany). Drug penetration distances were determined by analyzing the RGB profile of fluorescence micrographs using ImageJ (see also Fig. S3).

Results and Discussion
HepG2 spheroid formation and cultivation. Spheroid uniformity and quality including morphology, ultrastructure and organo-typic functionality are all crucial parameters and need to be optimized when using a novel protein-based cell-repulsive nanobiointerface for spheroid generation and long-term cultivation. Figure 1a shows the workflow used to generate the nanobiointerface inside the microtiter plate wells and generation of multi-cellular spheroids for drug toxicity measurements. Initial optimization investigates spheroid shape after 6 days in culture using increasing cell-seeding densities ranging from 3.000, 5.000 and 10.000 HepG2 cells per well. The dependence of spheroid shape on seeding density is illustrated in Fig. 1b where highest spheroid solidity and roundness (>0.9) is observed at a seeding density of 3.000 cells/well. Higher seeding densities resulted in inhomogeneous cell aggregates and unfavorable shape and size variations. Another important aspect of our dose-age relationship study is the long-term stability of HepG2 spheroids when cultivated over a period of 18 days on top of the self-assembled nanobiointerface. Results shown in Fig. 1c demonstrate that only in the presence of an initial seeding density of 3.000 cells per well no deformations in spheroid shape are obtained in long-term cultivations. As a result, the seeding density of 3.000 cells/well is chosen in the presence of our cell-repulsive protein nanobiointerface for all subsequent experiments.
Evaluation of HepG2 spheroids based on morphology. In a next set of experiments, additional key parameters of spheroid morphology including area, perimeter, solidity, roundness and diameter are monitored over a period of 18 days, as shown in Fig. 2.
At day 1 post-seeding individual HepG2 cells spontaneously self-assembled to an irregular shaped cell aggregate (see also Fig. 1c), exhibiting an area of 0.43 ± 0.027 mm 2 , a perimeter of 6.31 ± 3.18 mm, a solidity of www.nature.com/scientificreports www.nature.com/scientificreports/ 0.75 ± 0.25 and a roundness of 0.80 ± 0.01. At day 3 post-seeding spheroid area, solidity and roundness showed a slight increase, whereas a significant reduction in perimeter at the same time is observed (p < 0.05). The perimeter as an indicator for surface roughness decreased significantly within the first 3 days but remained constant over the remaining cultivation period. At day 6, a significant increase in spheroid solidity and roundness was noted, while perimeter and area remained at similar values (see Table S1). After day 6 no significant change in morphological parameters are evident (p > 0.05). Interestingly spheroid area did not increase significantly over time and remained in the range of 0.42 to 0.5 mm 2 . These results reveal that proper spheroid shape is reached at day 6 post-seeding and remained constant for a period of 3 weeks. This initial decrease in spheroid perimeter to 3.29 mm ± 0.5 mm and simultaneous increase of solidity to 0.95 ± 0.02 and a roundness of 0.93 ± 0.02 is consistent with literature on well-shaped spheroids exhibiting a roundness and a solidity above 0.9 31 . Spheroid diameter increased for the first 9 days from 746 ± 12 µm to 857 ± 19 µm and stayed stable for the following days of culture  www.nature.com/scientificreports www.nature.com/scientificreports/ with 810 ± 30 µm for day 12, respectively. At day 15 and 18 spheroid diameter decreased to 766 ± 51 µm and 743 ± 10 µm indicating the start of spheroid disintegration. In the context of our dose-age relationship study, we therefore defined early-stage, mid-stage and late-stage HepG2 spheroids based on obtained differences in perimeter, solidity and roundness between days 3 to 5, 6 to 12 and 15 to 18, respectively.
Histological and ultrastructural evaluation of HepG2 spheroid morphology. A known drawback of aged spheroids is necrotic core formation, which is an unwanted phenomenon resulting from the accumulation of metabolic waste products and insufficient diffusion of oxygen/nutrients starting at a spheroid diameter above 200 to 500 µm 39,40 . To assess the overall structural architecture, HepG2 spheroid solidity and compactness was investigated in more detail using histochemistry and transmission electron microscopy (TEM). Histological and ultra-structural analysis as shown Fig. 3a,b demonstrates that early-stage HepG2 spheroids are loosely packed cell clusters interstitial spaces between individual cells, while mid-stage spheroids (day 6) display tight cellular junctions and an overall condensed spherical morphology with intact and smooth outer spheroid surfaces. Importantly, both early-stage and mid-stage spheroids revealed equally distributed chromatin in the nucleus as well as intact cytoplasm, thus indicating viable HepG2 cells. In contrast, late-stage spheroids at day 18 displayed typical apoptotic characteristics with loss of integrity of the outermost lining layer, specialized inter-and intra-cellular structures such as cell-cell contacts, shrinking of cytoplasm, membrane blebbing and formation of membrane-bound apoptotic bodies 41 . Nuclear shrinking and chromatin condensation, also referred as pyknosis, is observed in the cell nucleus and represents a hallmark of apoptosis 42 . Additionally, disintegration of the outmost surface was observable for late-stage spheroids. These results further confirm our above definition of late-staged spheroids between days 12 and 18 in culture where the formation of necrotic cores in late-stage apoptotic spheroids takes place (see also Fig. S1).

Evaluation of secretion of organo-specific metabolites.
In addition to spheroid morphology, their organo-specific functionality is also an essential indicator for physiologically relevant 3D cell culture models. It is important to highlight that native liver tissue shows a highly specialized architecture and unique organization on a cellular level with highly specialized intercellular structures, so-called bile canaliculi, which are formed by plasma membranes of adjoining cells accounting for 15% of hepatocyte's total plasma membrane surface 43 . To verify liver-specific ultrastructural intercellular morphology and metabolic functions of our HepG2 spheroids over an 18-day cultivation period, additional TEM measurements and metabolic assays were performed to assess the formation of bile canaliculi, albumin and urea secretion. Electron transmission microscopy results revealed that after day 6 post-seeding bile canaliculi with integrated luminal microvilli are present in mid and late-stage HepG2 spheroids as seen in Fig. 4a,b, while in early-stage spheroids only loosely associated cells are found that form softly packed aggregates lacking bile canaliculi. Both, mid-stage and late-stage spheroids displayed proper structural liver-specific phenotypes containing lumenized bile canaliculi that increase in diameter over time. In addition to the formation of specialized cellular structures, liver-specific metabolic activity including albumin secretion and urea excretion is evaluated in subsequent experiments. Since human CYP 3A4 has a major role in www.nature.com/scientificreports www.nature.com/scientificreports/ biotransformation and oxidation of bioactive compounds such as sorafenib 44 , the liver-specific enzymatic activity of CYP 3A4 was additionally monitored over incubation time of 18 days. As shown in Fig. 4c, CYP 3A4 activity remained constant for HepG2 spheroids until day 6 around 69 ± 5 RLU per day and 4500 cells. An increase of enzymatic activity was observable between day 6 to day 15 by 3-fold whereas values for HepG2 spheroids for day 18 did not further increase. In addition, Fig. 4d shows that initially, HepG2 spheroids secreted very low levels of albumin with 0.08 ± 0.1 µg/mL, 0.23 ± 0.07 µg/mL, 0.28 ± 0.06 µg/mL and 0.23 ± 0.05 µg/mL per day at day 1 to day 9, while gradual increase of albumin secretion is obtained with mid-stage and late-stage spheroids peaking at 1.15 ± 0.10 µg/mL per day at day 18. In contrast to albumin, urea concentration as shown in Fig. 4e in the collected supernatant samples decreased gradually over 18 days exhibiting an average excretion rate between 0.84 and 0.94 µg/mL per day and 4500 cells for mid-stage and late-stage spheroids. Even though tissue architecture may be changing for central zones of late-stage spheroids, these functional liver-specific evaluations suggest that between day 9 and 18 HepG2 spheroids display increased liver-specific activity compared to early stages.
Since our spheroid evaluation study also indicated that in the presence of an initial cell seeding density of 3.000 cells per well, HepG2 spheroids can be considered as organo-typic starting at day 6 to day 9 post-seeding, differences in dose-response relationships between mid-stage HepG2 spheroids and 2D monolayer culture are examined in follow-on experiments. Both 2D and 3D liver cell culture models were exposed to increasing concentrations of sorafenib ranging from 0 µM to 100 µM for a period of 24 h and 48 h. Figure 5a shows the concentration-dependent inhibitory effects of sorafenib after 24 hours of drug exposure resulting in IC 50 values for HepG2 spheroids and monolayer cultures of 47.77 ± 3.12 µM and 29.14 ± 1.14 µM, respectively. However, similar sorafenib dose-response curves are obtained for both HepG2 spheroids and 2D monocultures when exposure times are increased from 24 to 48 hours. Figure 5b shows cell viabilities in the presence of increasing sorafenib concentrations, exhibiting IC 50 values 8.40 µM and 7.66 µM (p > 0.05) for spheroid and 2D monocultures, respectively. In other words, the required drug concentration necessary to inhibit 50% of the cells in HepG2 spheroids decreases from approximately 60 µM after 24 hours to 15 µM after 48 hours exposure, which translates to an overall 75% decrease. Following the same trend, an extended exposure time of 24 hours leads to a 95% decrease from 100 µM to 5 µM inhibitory concentration in the presence of 2D monolayer culture. Obtained IC 50 values and p-values for both cell-culture methodologies are listed in Table 1 showing no statistical difference in growth inhibition after 48 hours. These results were also confirmed by Live/Dead viability assay based on Calcein AM and www.nature.com/scientificreports www.nature.com/scientificreports/ ethidium bromide as shown in Fig. 5c and compared to the dose-response behavior of HepG2 monolayer cultures (see Fig. S2 for Live/Dead images). This phenomenon can be attributed to several factors including (a) reduction of drug diffusivity into the spheroids, (b) the presence of quiescence cells and hypoxic areas within the core region of spheroids, (c) altered gene expression, (d) enhanced cell-cell contact and (e) the presence of ECM 45 .
Next, the influence of spheroidal aging on drug diffusivity and resistance was investigated to gain a better understanding of the impact of culture time on drug efficacy. In a final set of experiments, early, mid-stage and late-stage HepG2 spheroids were subjected to a 24-hour treatment of 100 µM sorafenib. Results based on time-resolved monitoring of cell viability are shown in Fig. 6a where sorafenib toxicity reduced the viability of our HepG2 spheroids to 65%, 77%, 86% at day 3, 4, and 5 post-seeding in comparison to untreated controls (overall relative standard deviation, RSD = 6%, n = 6). Surprisingly, already during the transition phase from early-stage spheroids (day 3) to mid-stage spheroids (day 5) showed an increase in drug resistance and reduced toxicity. After a cultivation period of 5 days, mid-stage HepG2 spheroids displayed no significant cytotoxic effect of sorafenib with viability values of 90.80%, 96.30% and 100.13% over the following 6 days (p > 0.05, overall RSD = 10%, n = 9). Interestingly, these results correlate well with the emergence of organo-typic microstructures and increased metabolic activities that start with the emergence of spheroid maturity around day 6. To verify the importance of spheroidal age on the outcome of drug toxicity studies, a comparative analysis of dose-response relationships of sorafenib between early (day 3) and late-stage (day 12) spheroids were conducted in a final set of experiments. Figure 6b shows spheroid age-related dose-response curves obtained after 3 days and 12 days in culture resulting in elevated drug resistance to 100 µM sorafenib in late-stage spheroids.  www.nature.com/scientificreports www.nature.com/scientificreports/ Additionally, IC 50 values listed in Table 2 show that late-stage HepG2 spheroids displayed elevated IC 50 values of 168.70 ± 1.26 µM in comparison to early HepG2 spheroids of 43.75 ± 1.34 µM. This means when using late-stage spheroid a 4-fold higher concentration of sorafenib needs to be applied to reach approximately 50% inhibition of the cells compared to an early-stage HepG2 spheroid.
To investigate this age-related effect in more detail, drug penetration depth of sorafenib was analyzed using a fluorescent dye-exclusion assay (e.g. Live/Dead cytotoxicity assay). Results from our drug penetration study show the formation of an apoptotic outer rim at a sorafenib concentration of 100 µM for early and late-stage HepG2 spheroids as seen in Fig. 7a. The observed apoptotic edge is caused by the strong cytotoxic effect of the drug on the outer-most cell layers since fluorescent intensity profile analysis of spheroid cross-sections shows highest intensities of dead cells (red channel for necrotic cells) near the outer rim of the spheroid and decreases gradually towards the spheroid core. Late-stage spheroids exhibit an even higher intensity at the rim caused by the more compact outer cell layers of otherwise properly formed spherical structures. However, when calculating the drug diffusion distance as a ratio between living and dead cells a significant decrease in diffusion distance from around 30 ± 3 µm in mid-stage spheroids (up to day 9) to 18 ± 2 µm for late-stage spheroids (after day 12 post seeding) is evident as shown in Fig. 7b. To quantify inward diffusion distance in more detail, rhodamine B was chosen as a model molecule that features molecular weight like sorafenib. As shown in Fig. 7d, early stage HepG2 spheroids display a 4.2-fold higher diffusivity towards molecules of molecular weight around 470-480 g/  Table 2. Dose-age dependence of 3D HepG2 spheroids after 3 -and 12 days post-seeding.
www.nature.com/scientificreports www.nature.com/scientificreports/ mol already plateauing after 30 and 125 minutes in the central core region of HepG2 spheroids respectively. In addition, further histochemical evaluation of central spheroid regions confirmed that HIF-1 positive hypoxic cells were gradually increasing with 100% hypoxia-positive cells at day 18 post-seeding (see Fig 7e and S4). Overall these results point at different penetration depths of bioactive compounds between early, mid-stage and late-stage spheroids, due to changes of organotypic architecture and function of HepG2 spheroids. This limited drug diffusion into the inner regions in aged tumor models significantly decreases the efficacy of chemotherapeutic drugs.

Conclusion
In vitro multi-cellular spheroid models have become a promising tool in drug discovery and development, but comparability and reproducibility remain a pressing issue. Additionally, the lack of standardization, scalability, and compatibility with current screening systems has led to large lab-to-lab discrepancies and inconsistent data outcome in drug studies even when using the same tissue type 46 . As a consequence, the reproducible generation of multi-cellular spheroids including shape, size, cell density and morphology, is key in increasing the reliability of in vitro 3D-cell based assays. In the present work, we have investigated the impact of spheroidal age on drug efficacy to gain a deeper understanding how methodological inconstancies influence the outcome of drug screening studies.
Using a protein-based nanobiointerface, we were able to establish HepG2 spheroids of similar size, shape and morphologies, while structural evaluation showed the formation of liver-specific morphologies over an 18-day cultivation period. Based on our ultrastructural and organo-typic functional investigations, distinctly different spheroid phases were identified including an early (day 3 to 6), mid-stage (day 6 to 12) and late stage (day 15 to 18) development. The three spheroidal development stages show significant differences in cell-to-cell interactions, specialized microstructures such as bile canaliculi formation, and metabolic activists including albumin and urea secretion. Results from our initial spheroidal aging study revealed a decreased sorafenib toxicity of following a 24-hour exposure with early, mid-stage and late-stage HepG2 spheroids. In fact, a 4-times higher sorafenib doses are needed to exhibit similar toxic effects in late-stage spheroids when compared to early-stage spheroids. To investigate this age-related effect in more detail, drug penetration depths were analyzed resulting in a significant decrease in diffusion distance from 31 ± 3 µm in mid-stage spheroids (up to day 9) to 18 ± 3 µm for late-stage spheroids. This phenomenon can be explained through intercellular interactions and physical limitations such as higher interstitial fluid pressure, which is known to restrict drug transport into deeper regions of the spheroid and thus leads to enhanced resistance to chemotherapeutics 33 . Additionally, increasing cell densities as natural diffusion barriers as well as biological limitations of complex in vitro models (e.g. apoptosis due to limited membranes permeability, an increasing acidic microenvironment and hypoxia in the central spheroid core regions) 47 have been linked to reduced efficacy of radio -and chemotherapies 48,49 . Independent of the reasons for the observed drug transport limitations, our study has clearly demonstrated that spheroidal age needs to be considered as an important variable for future drug sensitivity tests using spheroid-based in vitro models.