Multikinase activity of fibroblast growth factor receptor (FGFR) inhibitors SU5402, PD173074, AZD1480, AZD4547 and BGJ398 compromises the use of small chemicals targeting FGFR catalytic activity for therapy of short stature syndromes

ABSTRACT

Activating mutations in FGFR3 are responsible for achondroplasia, the most common genetic form of human dwarfism. Although small molecule inhibitors targeting FGFR tyrosine kinase activity have been proposed as a potential treatment, experimental support for this approach remains limited. In this study, five FGFR tyrosine kinase inhibitors (SU5402, PD173074, AZD1480, AZD4547, and BGJ398) were assessed for their ability to inhibit FGFR signaling in chondrocytes. All five inhibitors effectively suppressed FGFR activation in cultured chondrocytes and developing limb tissues, restoring chondrocyte proliferation and maturation disrupted by FGFR signaling. However, when administered to newborn mice, these inhibitors did not improve bone growth and instead caused severe toxic effects in the liver, lungs, and kidneys.

Further analysis showed that none of the inhibitors exhibited selectivity for FGFR3 over other FGFR family members, either in cell-free kinase assays or in cellular contexts. Moreover, all inhibitors demonstrated substantial off-target activity against a panel of 14 unrelated tyrosine kinases. This off-target activity was especially pronounced in SU5402 and AZD1480, which inhibited several other kinases including DDR2, IGF1R, FLT3, TRKA, FLT4, ABL, and JAK3 with potencies equal to or greater than their effects on FGFRs. The findings highlight significant limitations in specificity and safety of current FGFR tyrosine kinase inhibitors, making them unsuitable for treating achondroplasia. Alternative therapeutic strategies that more precisely target FGFR3 are needed.

INTRODUCTION

In mammals, long bone growth occurs at the epiphyseal growth plate, where chondrocytes follow a defined process of proliferation, hypertrophic differentiation, extracellular matrix mineralization, and eventually apoptosis. The regulation of cartilage growth involves multiple signaling pathways, including those driven by parathyroid hormone-related protein, C-type natriuretic peptide, bone morphogenetic proteins, and leukemia inhibitory factor. Equally essential are growth-inhibitory systems that help determine final skeletal size in terrestrial vertebrates.

One key negative regulator of cartilage growth is the receptor tyrosine kinase FGFR3. This is supported by evidence showing skeletal overgrowth in mice lacking FGFR3 and the presence of tall stature and other phenotypes in humans with FGFR3 loss-of-function mutations. Conversely, constitutive activation of FGFR3 in mice results in dwarfism, and duplications involving the FGFR3 ligand FGF4 are associated with short-limbed phenotypes in certain dog breeds. In humans, activating mutations in FGFR3 are linked to four related skeletal disorders: achondroplasia, hypochondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans, and thanatophoric dysplasia.

Achondroplasia is the most common form of dwarfism and is marked by disproportionately short stature, shortened proximal limbs, a narrow trunk, enlarged head with frontal bossing, midface hypoplasia, and trident hand configuration. Affected individuals may experience a range of complications including developmental delays, hydrocephalus, spinal stenosis, joint issues, hearing impairment, respiratory and sleep disturbances, obesity, and reproductive challenges.

Targeting FGFR3 after birth holds promise for correcting skeletal abnormalities in achondroplasia and may have applications in other skeletal disorders. Given FGFR3’s role as a physiological inhibitor of bone growth, a safe and effective inhibitor could significantly impact the treatment of short-stature syndromes more broadly.

Tyrosine kinases, including FGFRs, are appealing drug targets due to their well-defined enzymatic roles and the success of kinase inhibitors in cancer therapy. The FGFR family (FGFR1–4) is of particular interest because various cancers harbor activating mutations, amplifications, or gene fusions involving FGFRs. The first FGFR-targeting tyrosine kinase inhibitors were developed in the late 1990s and include compounds like SU5402 and PD173074. More recently, newer inhibitors such as AZD4547, AZD1480, and BGJ398 have been developed and are currently undergoing clinical evaluation for cancer.

While the idea that FGFR TKIs could be used to treat achondroplasia has gained attention, experimental validation remains limited. The current study was undertaken to rigorously assess the effectiveness of these inhibitors in targeting FGFR signaling within cartilage.

RESULTS AND DISCUSSION

TKIs inhibit FGFR signaling in cultured chondrocytes

We assessed the effectiveness of two early-generation FGFR tyrosine kinase inhibitors (TKIs), SU5402 and PD173074, and three more recent inhibitors, AZD1480, AZD4547, and BGJ398, to determine whether significant progress in anti-FGFR activity had been achieved over the decade separating these groups. AZD1480, AZD4547, and BGJ398 are currently in clinical trials for cancer and are among the most promising candidates for treating achondroplasia. Rat chondrosarcoma (RCS) chondrocytes, a well-established model of chondrocyte biology, were used to initially test the inhibitors in a chondrocyte-specific environment.

RCS cells typically undergo strong growth arrest in response to FGFR activation through treatment with external FGF ligands. This process involves activation of the ERK MAP kinase pathway and is marked by premature senescence, reduction in extracellular matrix components, and changes in cell morphology. We first tested the TKIs’ ability to block FGF2-induced growth arrest in RCS cells, a sensitive measure of FGFR activity. The inhibitors were applied across a wide range of concentrations, spanning several orders of magnitude, to determine their baseline activity profiles. All of the TKIs successfully prevented FGF2-induced proliferation arrest at concentrations ranging from as low as 5 nanomolar for BGJ398 to 5 micromolar for SU5402. However, toxic effects were observed at certain concentrations, where the inhibitors also suppressed proliferation in the absence of FGF2. These effects were most pronounced for SU5402 and AZD1480 and occurred at concentrations lower than those required to counteract FGF2-mediated arrest.

The concentrations at which each TKI completely restored RCS cell proliferation without toxicity were approximately 15–20 nanomolar for PD173074, 10–50 nanomolar for AZD4547, and 5–10 nanomolar for BGJ398. Each of the TKIs maintained their ability to restore cell growth even when exposed to high concentrations of FGF2, far exceeding the level typically needed to produce strong growth arrest. Additional characteristics of FGF2-induced arrest, including altered cell shape and premature senescence, were also fully reversed by the inhibitors.

We further examined the inhibitors’ potential to reverse growth arrest that had already been established, an important consideration for future treatments of achondroplasia, where FGFR3-related growth suppression may have persisted for months before therapy begins. The inhibitors successfully reversed this prolonged FGF2-induced arrest in RCS chondrocytes.

In addition to restoring proliferation, the TKIs also blocked activation of key downstream signaling molecules in the FGFR pathway. This included preventing FGF2-induced phosphorylation of FRS2, MEK, and ERK kinases, all of which are part of the FGFR-RAS-ERK signaling cascade. The inhibitors also suppressed phosphorylation of LRP6, a protein involved in interactions between FGFR and WNT/β-catenin signaling in chondrocytes. Similar levels of pathway suppression were seen even when FGFR activation was amplified by adding heparin. Collectively, these findings demonstrate that all the TKIs tested effectively inhibited FGFR signaling in cultured chondrocytes.

TKIs inhibit FGFR signaling in limb rudiment cultures

We then turned to limb rudiment cultures, a well-established system for studying FGFR signaling in cartilage. Tibias extracted from embryonic mouse limbs continue to grow in culture, and this growth can be inhibited by external FGF2 exposure. Tibias from embryonic day 18 mice were cultured for eight days, after which their growth plates were examined histologically. FGF2 treatment significantly reduced growth and led to the disappearance of cells displaying the characteristic appearance of hypertrophic chondrocytes. This effect was further confirmed by the absence of collagen type 10 (Col10a1) expression, a marker specific to hypertrophic chondrocytes, in the FGF2-treated tibias. In contrast, untreated tibias showed normal Col10a1 expression.

The limb rudiment model replicates cartilage growth but not the development of bone, since cartilage can grow independently of blood supply, while bone formation requires vascularization, which is disrupted in culture. The growth plates of cultured tibias developed a band of hypertrophic cartilage at their ends that did not express Col10a1 mRNA. These areas likely represent matrix spaces containing apoptotic chondrocytes that no longer produce Col10a1 and would normally be cleared by invading bone, a process that is halted in culture due to the lack of bone growth. The specificity of the Col10a1 in situ signal was confirmed using freshly isolated tibias.

When tibias were treated with 0.5 micromolar AZD4547, the FGF2-induced growth suppression was reversed, and normal growth plate structure and Col10a1 expression were restored. Similar results were observed with 1 micromolar AZD4547 and with 0.5 micromolar BGJ398. Notably, tibias treated with AZD4547 or BGJ398, either alone or in combination with FGF2, grew longer than untreated controls. This suggests that these TKIs may block endogenous FGFR3 signaling, which naturally limits growth. Previous studies have shown that blocking FGFR3 activity using a C-natriuretic peptide analog, BMN111, enhances growth in mice and primates, supporting the idea that inhibiting normal FGFR3 signaling can stimulate growth in otherwise healthy animals.

These findings suggest that anti-FGFR3 therapies may have potential applications beyond FGFR3-related skeletal disorders, possibly extending to other forms of short stature. Our results demonstrate that TKIs effectively target FGFR3 signaling within growth plate cartilage, even under conditions where the tissue is embedded in a dense extracellular matrix and lacks a vascular supply. Similar outcomes have been reported by others, who showed that a derivative of PD173074 reversed growth inhibition in tibias from mice carrying the FGFR3-Y367C mutation, a model for a human skeletal dysplasia.

AZD4547 suppresses skeletal growth and causes lethality in vivo

We aimed to determine whether the bone growth-promoting effects of TKI treatment observed in vitro could also be achieved in vivo. AZD4547 was selected for this purpose due to its favorable performance in earlier in vitro studies. Various doses of AZD4547 (ranging from 0.5 to 2000 micromolar) were administered intraperitoneally to newborn wild-type CD1 mice. These treated animals were compared to littermate controls injected with the vehicle, DMSO. Injections were given weekly over a 28-day period, during which body weight, body length, and tail length were monitored.

Doses ranging from 0.5 to 250 micromolar produced no significant changes in growth parameters, although a slight increase in body weight was observed at 50 micromolar. Higher doses of 1000 and 2000 micromolar led to weight loss, growth impairment, and lethality. Mortality occurred within 2 to 3 days following the first injection. Mice that survived this initial period remained alive for the remainder of the 28-day study. Lethal toxicity was marked by progressive muscle paralysis, abnormal breathing patterns, cyanosis, and respiratory failure.

Histological examination showed tissue damage in several organs in lethally affected animals. The liver exhibited infiltration by inflammatory cells and loss of normal hepatocyte structure. The kidneys showed signs of cellular shrinkage and reduced convoluted tubules. Lung tissue displayed hemorrhage and structural disruption of the interalveolar septa. In animals that survived high-dose AZD4547 treatment, liver tissue showed milder changes such as fat accumulation and the presence of lipid droplets. No changes were observed in the lungs or kidneys of these survivors.

Skeletal analysis revealed no alterations in bone development in animals treated with 0.5 to 250 micromolar AZD4547. However, treatment with 1000 and 2000 micromolar significantly reduced the length of long bones and skull bones. Despite the reduction in bone size, the overall shapes and anatomical features of the bones remained similar to those of the control group, suggesting that the observed growth inhibition was likely a result of systemic toxicity rather than a specific effect on bone tissue.

Biochemical analysis showed that AZD4547 treatment did not alter levels of blood glucose, bile acids, vitamin D, phosphate, calcium, HDL, cholesterol, or triglycerides.

FGFR TKIs inhibit other tyrosine kinases

In summary, our findings show that although FGFR-targeting TKIs are strong inhibitors of FGFR signaling in cultured chondrocytes, they do not promote skeletal growth in vivo and instead produce significant toxicity and lethality. This harmful effect may result from either widespread inhibition of FGFR signaling throughout the body or from the inhibition of other kinases unrelated to FGFR.

To better understand the specificity of these TKIs, we conducted cell-free kinase assays to evaluate their activity against all four FGFR isoforms (FGFR1–4) as well as 14 unrelated tyrosine kinases. The kinase activity was assessed using phosphorylation of STAT1 or autophosphorylation of the kinase itself, with TKIs added directly to the reaction mixtures. The TKIs inhibited FGFR1–4 to varying degrees. SU5402, for example, almost completely suppressed FGFR1 activity but had much less effect on FGFR3 and FGFR4. BGJ398 showed relatively low inhibition of FGFR2 activity in this assay.

Each TKI had a distinct pattern of FGFR isoform inhibition. SU5402 was most effective against FGFR1 and FGFR2, while PD173074 showed strong inhibition across FGFR1, FGFR2, and FGFR3. AZD1480 was more effective against FGFR1, followed by FGFR2 and FGFR3. AZD4547 strongly inhibited FGFR1 and moderately affected FGFR2 and FGFR3. BGJ398 was most active against FGFR3 and FGFR4, with lower effects on FGFR1 and FGFR2.

PD173074 and AZD4547 demonstrated moderate selectivity for FGFRs, but they also inhibited other kinases to a degree similar to their inhibition of FGFRs. PD173074 affected INSR and FLT4, while AZD4547 impacted FMS, TRKA, FLT4, and ABL. In contrast, SU5402 and AZD1480 were highly non-selective and inhibited multiple non-FGFR kinases, in some cases more potently than FGFRs. This includes FLT3, TRKA, FLT4, and JAK3 for SU5402, and DDR2, IGF1R, FLT3, TRKA, FLT4, ABL, and JAK3 for AZD1480. These two TKIs also caused the most toxicity in RCS cell growth assays.

Despite its weak performance in kinase assays, BGJ398 showed strong effects in RCS cell growth arrest experiments. To investigate this discrepancy, we tested its activity in 293T cells transfected with FGFR1–4. BGJ398 effectively inhibited wild-type FGFR1 and FGFR2, as well as disease-related FGFR1-Y374C and FGFR2-C342R mutants. However, FGFR3 and FGFR4 activity could not be assessed in this system because the wild-type versions of these receptors were not activated after transfection. The FGFR3-K650M mutant, linked to skeletal and cancer disorders, showed only partial sensitivity to BGJ398, while the FGFR4-V550E mutant was completely resistant. These mutations are known to enhance activation and restrict TKI access, respectively, so their resistance does not reflect general TKI activity against FGFR3 and FGFR4. Therefore, based on the in vitro transfection data, BGJ398 can be confirmed as an effective inhibitor of FGFR1 and FGFR2, and its weak performance in cell-free assays likely results from limitations in the assay method itself.

TKIs are not selective for FGFR1-4 in vitro or in vivo

We further investigated the effects of TKIs on FGFR signaling using two additional models relevant to bone development: primary cultures of mouse limb bud mesenchymal cells undergoing chondrocyte differentiation in micromass culture, and chicken wing buds treated with TKIs and allowed to develop in vivo. Micromass culture replicates early stages of limb development, including mesenchymal condensation, chondrocyte differentiation, and the formation of cartilage nodules, which are typically observed after seven days. FGF signaling plays a key role in this process due to its influence on mesenchymal cell proliferation. Treatment with AZD4547 significantly reduced cartilage nodule formation in E12 mouse limb buds after seven days of differentiation.

In the second model, AZD4547 was injected into the right wing bud of chicken embryos at developmental stage HH20-21. The wings were then allowed to develop for 10 to 12 days. Wings treated with DMSO or left untreated developed normally. Injection of 50 or 250 micromolar AZD4547 did not result in visible external abnormalities. However, treatment with 1 millimolar AZD4547 led to shortened wings, stump-like structures, or complete suppression of wing development. Skeletal analysis revealed structural abnormalities in the middle and distal limb regions, including the absence of specific bones such as the radius and digitus allulae and reductions in the size of distal skeletal components. At 10 millimolar, AZD4547 caused more severe defects, including complete loss of distal bones and major abnormalities in proximal elements like the scapula and coracoid.

Each of the experimental models used in this study exhibited distinct patterns of FGFR expression. RCS chondrocytes expressed FGFR3 and FGFR2. Limb growth plate cartilage mainly expressed FGFR3, with some FGFR1 expression. Mouse micromass cultures primarily expressed FGFR1 and FGFR2. In chicken limb buds, FGFR1 was present in mesenchymal cells, while FGFR2 was expressed in the overlying ectoderm; FGFR3 and FGFR4 were only weakly expressed during early development. Other cell lines used included MCF7, which expressed FGFR1 and FGFR2; 293T, which expressed all four FGFRs; and NIH3T3, which expressed FGFR1 and FGFR2.

Despite differences in FGFR expression across these systems, all seven models responded similarly to treatment with AZD4547 and/or BGJ398, showing full suppression of FGFR signaling. The only exception was in NIH3T3 cells, where FGF2-induced activation of ERK MAP kinase was less effectively inhibited by AZD4547 compared to BGJ398. These results indicate that although TKIs may display some selectivity among FGFRs in cell-free assays, they broadly inhibit FGFR signaling across different cell types and developmental systems when tested in cellular contexts.

FGFR TKIs represent poor therapeutic options for ACH and other short-stature syndromes

Effective therapy for achondroplasia (ACH) must meet several demanding criteria. First, it needs to be highly specific to FGFR3 signaling within cartilage. Because stimulating chondrocyte proliferation and restoring proper hypertrophic differentiation are central goals of ACH treatment, any drug must target FGFR3 or its downstream signaling components specifically in cartilage, even in the presence of similar pro-growth pathways like those mediated by IGFR. Disruption of other processes essential to chondrocyte function would undermine the therapeutic outcome.

Second, the therapy must be selective for FGFR3 over other members of the FGFR family. FGFR3 activity is largely confined to growth plate cartilage, while other FGFRs play widespread roles in numerous physiological systems. To avoid harmful effects in non-skeletal tissues, an effective ACH therapy must suppress FGFR3 without interfering with the signaling of other FGFRs. This selectivity becomes even more critical given the likelihood that high drug concentrations will be required to penetrate the dense, avascular growth plate cartilage.

Third, the treatment must be effective over the extended period of postnatal development. Since the goal is to increase overall skeletal size, the therapy will need to function reliably for several years. While resistance mechanisms seen in cancer are not expected in ACH—since FGFR3 inhibition benefits rather than harms chondrocytes—other factors such as metabolic or hormonal shifts during growth may alter the drug’s efficacy over time.

Fourth, the therapy must be free from side effects. Given that the target population is children, any toxicity during treatment or delayed adverse effects later in life would be unacceptable.

Based on the results of this study, currently available FGFR-targeting TKIs are unsuitable for ACH therapy. Their inability to selectively inhibit FGFR3 over other FGFRs and their toxic effects in cells and animals render them poor candidates for treating skeletal dysplasias. This conclusion applies to both early and late generation TKIs, indicating that small molecules targeting the ATP-binding site of FGFRs are unlikely to be viable for ACH. Future efforts should focus on alternative strategies for modulating FGFR3 signaling in skeletal growth disorders.

MATERIALS AND METHODS

Cell, micromass and limb organ culture

Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (Invitrogen, Carlsbad, CA). For RCS growth assays, 2.5 × 10² cells per well were seeded into 96-well tissue culture plates and grown for 5 days. Cell numbers were determined using crystal violet staining, following a previously established protocol (27). The chemicals used in the experiments were sourced from the following manufacturers: FGF2 and FGF22 (R&D Systems, Minneapolis, MN); heparin (Sigma-Aldrich, St. Louis, MO); SU5402, PD173074 (Tocris Bioscience, Bristol, UK); AZD1480, AZD4547, and BGJ398 (Selleckchem, Houston, TX).

Primary mesenchymal cultures were derived from the forelimb buds of E12 mouse embryos through proteolytic digestion with dispase II (Sigma). Cells were spotted as 10 µl aliquots at a concentration of 2 × 10⁷ cells/ml and allowed to adhere for 1 hour before adding differentiation media (60% F12/40% DMEM, 10% FBS, 50 µg/ml ascorbic acid, 10 mM β-glycerol phosphate). The micromasses were cultured for 7 days at 37 °C in supplemented media, which was replaced every other day.

Tibias were dissected from E18 mouse embryos, placed on Millipore filters above a metal mesh, and cultured in micromass differentiation media for 8 days at 37 °C, with daily media changes. At the start and end of each experiment, tibias were photographed and measured. Their lengths were measured using Axio Vision (Zeiss, Germany). Statistical analyses were conducted using Statistica 8.0 (StatSoft, USA), with ANOVA and Tukey’s post hoc test applied to assess the significance of observed differences between individual treatments.

Western blotting (WB), kinase assays and transfection

Cells were lysed in a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, and 25 mM NaF, supplemented with proteinase inhibitors. Samples were resolved by SDS-PAGE, transferred onto a PVDF membrane, and visualized by chemiluminescence using Thermo Scientific reagents (Rockford, IL). The following antibodies were used for western blot (WB) detection: lamin A/C, caveolin, ID2, LRP6, pFRS2Y436, pMEKS217/221, MEK, pERKT202/Y204, ERK, FGFRY653/Y654, AXL, pcKITY703, DDR2, EGFR, FLT3, FMS, IGF1R, JAK3, STAT1, pSTAT1Y701, TRKA, FGFR4 (Cell Signaling, Beverly, MA); pLRP6T1572, pTyr (4G10) (Millipore, Billerica, MA); FRS2, actin, FGFR2, FGFR3 (Santa Cruz Biotechnology, Santa Cruz, CA); V5 (Invitrogen); FGFR1 (Sigma-Aldrich).

For kinase assays, 200 ng of recombinant kinases (FGFR1-4, AXL, TYRO3, DDR2, EGFR, IGF1R, INSR, MET, cKIT, FLT3, FMS, TRKA, FLT4, ABL, JAK3) were incubated with recombinant STAT1 (SignalChem, Richmond, CA) as a substrate in 50 μl of kinase buffer (60 mM HEPES pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na3VO4, 1.2 mM DTT) in the presence of 10 μM ATP for 60 minutes at 30°C. Kinase activity was determined by monitoring STAT1 phosphorylation or kinase autophosphorylation. The WB signals were quantified by densitometry using the ImageJ software (National Institutes of Health, Bethesda, US).

For cell transfections, FuGENE6 reagent (Roche) was used according to the manufacturer’s protocol. Vectors (pcDNA3.1) expressing V5-tagged human FGFR1-4 enzymes and their activating mutants were generated as previously described (48).

For growth plate histology, tibias were fixed in paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness for morphological analysis. Haematoxylin-eosin staining was performed on these sections. For in situ hybridization, the Col10a1 plasmid (IMAGp998B1114092Q, Biovalley, France) was linearized using PCR with M13 primers. Sense and antisense DIG-labeled riboprobes were synthesized using SP6 and T7 RNA polymerases, respectively. Hybridization was performed at 60°C overnight, and the sense DIG-labeled probe was used as a negative control. In situ hybridization on tibia sections was conducted as described previously (49).

Total RNA was isolated from micromass cultures and tibias (cleared of bone and soft tissues) using the Mini RNeasy Kit (Qiagen). Reverse transcription was performed with 200 ng of isolated RNA using the SuperScript Vilo cDNA synthesis Kit (Life Technologies). Quantitative PCR (qPCR) was performed with Taqman Universal PCR Master Mix, and probes for FGFR1 (Mm00438930_m1), FGFR2 (Mm01269930_m1), FGFR3 (Mm00433294_m1), FGFR4 (Mm01341852_m1), and β-actin (ACTB, Mm00607939_s1) were obtained from Life Technologies. ΔCT values were calculated and normalized as previously described (50, 51).

AZD4547 injection into chicken limb buds and postnatal mice

Fertilized chicken eggs (ISA brown) were obtained from Integra (Zabcice, Czech Republic) and incubated in a humidified forced air incubator at 37.8°C. Embryos were staged according to the criteria of Hamburger and Hamilton (52). For the experimental treatments, AZD4547 was injected into the right wing bud using a micromanipulator (Leica, Germany) and a microinjector (Eppendorf, Germany) to precisely target the selected area. Three distinct injection sites were used (anterior, posterior, and distal), while the left wings were treated with the TKI vehicle (DMSO) as a control.

Embryos were collected after 10-12 days of incubation, fixed in ethanol, and stained with alizarin red/alcian blue solution. They were then cleared in a KOH/glycerol solution for skeletal analysis.

For the mouse experiments, animals were euthanized by cervical dislocation in compliance with the AVMA Guidelines on Euthanasia. Skeletal preparations were made following a similar procedure as used for the chicken embryos.

Blood tests for the animals were performed by an accredited clinical laboratory (CSN EN ISO 15189:2013) at the University Hospital, Palacky University, Olomouc, following established protocols. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Institute of Animal Physiology and Genetics ASCR, Brno, Czech Republic (No.144/2013).