Reagents and solutions
The peptides mUNO (sequence: CSPGAK-COOH) and FAM-mUNO (FAM-Ahx-CSPGAK-COOH) were purchased from TAG Copenhagen and doxorubicin from Sigma-Aldrich. St-PGA was kindly provided by Polypeptide Therapeutic Solution S.L. For the rest, see Supplementary Information.
Mayer’s haematoxylin solution was prepared by dissolving 5 g of aluminium potassium sulphate dodecahydrate (Merck Millipore, cat. 1010421000) in 100 mL of water, then 1 g of haematoxylin (Merck, cat. H9627) was added. When it was completely dissolved, 0.02 g of sodium iodide (Merck, cat. 1065230100) was added and waited until completely dissolved. Then 2 mL of acetic acid (Sigma-Aldrich, cat. 33209) was added, and then the solution was brought to boil and cooled. Once ready to use, the solution was filtered using 0.45 µm filter (company).
Eosin (5%) solution was prepared by dissolving 0.5 g of Eosin Y (Sigma-Aldrich, cat. 230251) in 99 mL of water and 1 mL of acetic acid.
Cell culture and experimental animals
4T1 cells were purchased from ATCC, and 4T1-GFP cells were a gift from Ruoslahti laboratory, SBP La Jolla, USA. 4T1 and 4T1-GFP cells were cultured in a Roswell Park Memorial Institute (RPMI-1640) medium (Gibco by Life Technologies, cat. 72400-021) supplemented with 10% v/v foetal bovine serum (FBS, Capricorn Scientific, cat. FBS-11A) and 100 IU/mL penicillin/streptomycin (Capricorn Scientific, cat. PS-B) at + 37 °C in the presence of 5% CO2. For all animal experiments, 8-12-week-old female Balb/c mice were used. Animal experiment protocols were approved by the Estonian Ministry of Agriculture (Project #159). All methods were performed in accordance with existing guidelines and regulations.
Two tumour models were used for homing studies: the orthotopic TNBC model, where 1x106 4T1 cells in 50 µL of phosphate-buffered saline (PBS, Lonza, cat. 17-512F) were injected subcutaneously (s.c.) into fourth mammary fat pad, and the experimental metastases of TNBC model where 5x105 4T1 cells in 100 µL of PBS were injected i.v. into Balb/c mice. Two tumour models were used for treatment studies: the orthotopic TNBC model where 5x104 4T1 cells in 50 µL of PBS were injected s.c. into fourth mammary fat pad; and the experimental metastases of TNBC model where 2x105 4T1 cells in 100 µL of PBS were injected i.v.
Synthesis and characterisation of nanoconjugates
Four different nanoconjugates were used. For in vivo homing studies St-PGA-OG and St-PGA-OG-mUNO were used, and for in vitro cytotoxicity and in vivo treatment studies St-PGA-DOX and St-PGA-DOX-mUNO (“OximUNO”) were used. DOX here denotes doxorubicin. Detailed synthetic procedure for single nanoconjugates can be found in Supplementary Information.
Physico-chemical characterisation methods
Nuclear magnetic resonance (NMR) spectroscopy: NMR spectra were recorded at 27°C (300 K) on a 300 UltrashieldTM from Bruker. Data were processed with the software Mestrenova. Sample solutions were prepared at the desired concentration in D2O or D2O supplemented with NaHCO3 (0.5 M).
UV-Visible (UV-Vis) analysis: The UV-Vis measurements were performed using JASCO V-630 spectrophotometer at 25°C with 1 cm quartz cells and a spectral bandwidth of 0.5 nm. Spectra analysis was recorded three times in the range of 200–700 nm.
Fluorescence analysis: The fluorescence analysis was performed using a JASCO FP-6500 spectrofluorimeter at 25 ºC with 1 cm quartz cells.
Dynamic Light Scattering (DLS): Size measurements were performed using a Malvern ZetasizerNano ZS instrument, supported by a 532 nm laser at a fixed scattering angle of 173°. Nanoconjugate solutions (0.1 mg/mL) were freshly prepared in PBS (10 mM phosphate, 150 mM NaCl), filtered through a 0.45 µm cellulose membrane filter, and measured. Size distribution was measured (diameter, nm) for each polymer in triplicate. Automatic optimisation of beam focusing and attenuation was applied for each sample.
Zeta potential measurements: The Zeta potential measurements were performed at 25°C using a Malvern ZetasizerNano ZS instrument, equipped with a 532 nm laser using Disposable folded capillary cells, provided by Malvern Instruments Ltd. Nanoconjugate solutions (0.1 mg/mL) were freshly prepared in 1 mM KCl. The solutions were filtered through a 0.45 µm cellulose membrane filter. Zeta potential was measured for each sample per triplicate.
Molecular dynamics simulations
Molecular dynamics (MD) simulations of the nanoconjugate, polyglutamate chains and mUNO peptide were carried out using the ff19SB force field91 in the Amber20 MD engine92. The nanoconjugate system was neutralised using Na + ions and hydrated to account for a total of ~ 920 000 atoms (~ 300 000 TIP3P water molecules) in a truncated octahedral box. A hydrogen mass repartitioning strategy was applied on the resulting topology, allowing us to use a 4 fs integration time step93. We used standard minimisation and equilibration protocols to reach 300 K and 1 atm., followed by 50 ns of production MD run. The simulations were run under the NVT ensemble (constant number of particles, volume, and temperature through Berendsen thermostat94), considering periodic boundary conditions. To fix hydrogen atoms, the SHAKE algorithm was used95. The non-bound cut-off value was set to Angstrom. We parametrised the central moiety of the nanoconjugate using the recommended protocol for the Amber force field. It was necessary to introduce amide bond, angle and dihedral terms using ParmEd module to establish the bond of the central molecule to the polyglutamate chains.
Tumour homing studies
Tumours were induced as described in tumour models’ section. Tumour homing studies were performed on mice bearing orthotopic TNBC or experimental metastases of TNBC. Ten days p.i. of the orthotopic TNBC or the experimental metastases of TNBC, mice were injected i.p. with St-PGA-OG-mUNO (0.41 mg/0.5mL of PBS) or St-PGA-OG (0.35 mg/0.5mL of PBS) (corresponding to 15nmoles of OG, fluorescence measured by UV-Vis). We also analysed the homing of a higher dose of St-PGA-PGA-mUNO (0.82 mg/0.5mL of PBS) or St-PGA-OG (0.7 mg/0.5mL of PBS) (corresponding to 30nmoles of OG) and compared it with the homing of FAM-mUNO (30nmoles/0.5mL of PBS). In every case, the nanoconjugates or free peptide were circulated for 6h, after which mice were sacrificed by anaesthetic overdose followed by cervical dislocation. Organs and tumours were collected and fixed in cold 4% w/v paraformaldehyde (PFA) in PBS at + 4°C for 24h, washed in PBS at RT for 1 h and cryoprotected in 15% w/v sucrose (Sigma Life Science, cat. S9378) followed by 30% w/v sucrose at + 4 °C overnight. Cryoprotected and fixed tissues were frozen in OCT (Optimal Cutting Temperature, Leica, cat. 14020108926), cryosectioned at 10 µm on Superfrost + slides (Thermo Fisher, cat. J1800AMNZ) and stored at -20°C. Immunofluorescent staining was performed as described earlier42. OG was detected using rabbit anti-FITC/Oregon Green (dilution 1/100, Invitrogen by Thermo Fisher Scientific, cat. A889) and Alexa Fluor® 647 goat anti-rabbit antibody (dilution 1/250, Invitrogen by Thermo Fisher Scientific, cat. A21245). CD206 was detected using rat anti-mouse CD206 (dilution 1/150, Bio-Rad, cat. MCA2235GA) and Alexa Fluor® 546 goat anti-rat antibody (dilution 1/250, life technologies, cat. A11081). CD86 was detected using rat anti-mouse CD86 (dilution 100, BioLegend, cat. 105001) and Alexa Fluor® 546 goat anti-rat secondary antibody (dilution 1/250). CD11c was detected using hamster anti-mouse CD11c antibody (dilution 1/75, BioLegend, cat. 117301) and Alexa Fluor® 546 goat anti-hamster secondary antibody (dilution 1/200, life technologies, cat. A21111) Slides were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL, Sigma-Aldrich, cat. D9542-5MG). The coverslips were mounted using mounting medium (Fluoromount-G™ Electron Microscopy Sciences, cat. 17984-25), and sections were imaged using Zeiss confocal microscope (Zeiss LSM-710) and 20x objective. Colocalisation analysis was carried out using the Fiji programme and Pearson’s coefficient, and average values were obtained using three images per mouse per group. OG/FAM mean signal per CD206+ cell analysis was carried our using the ImageJ programme, taking mean OG/FAM signal, and dividing it with the number of CD206+ cells. Average values were obtained from four images per mouse. N = 3 for orthotopic TNBC and N = 2 for the homing in experimental metastases of TNBC.
Plasma half-life evaluation for St-PGA-OG-mUNO
Plasma half-life studies were performed as previously described42. Shortly, healthy female Balb/c mice (N = 3) were injected i.p. with St-PGA-OG-mUNO (0.41 mg/0.5mL of PBS, corresponding to 15nmoles of OG). Ten µL of blood was taken at different timepoints (0, 5, 10, 15, 30, 60, 180, 360, 1440 min) and mixed with 50 µL of PBS-Heparin solution. Blood samples were centrifuged to obtain plasma (300g for 5 min at RT) and OG fluorescence was read with a plate reader (FlexStation II Molecular Devices) at 480nm excitation, 520nm emission.
DOX release studies
The LC-MS was implemented in the determination of free drug, stability, and drug release studies of OximUNO conjugate. The LC-MS consisted of ExionLC LC system and AB Sciex QTRAP 4500, a triple quadrupole ion trap hybrid equipped with Turbo VTM electrospray ionisation source. DOX was determined with an internal standard method: 1 µg/mL of daunorubicin (DAU) was used as internal standard where three calibration curves (in a range from 0.5 to 50 µg/mL DOX) were prepared and used for accurate analysis of DOX in the samples. Both DOX and DAU were detected with positive electrospray ionisation mode by following two mass transitions (544.2 m/z → 397 m/z and 544.2 m/z → 379 m/z for DOX, and 528 m/z → 363.1 m/z and 528 m/z → 321.3 m/z for DAU). The obtained LC-MS optimal conditions were as follows: flow rate 0.5 mL/min; mobile phase – 0.05 % trifluoroacetic acid with 70 % of acetonitrile; LiChrospher 100 C18 column (125x4.0 mm) (Merck); column temperature 40 ºC, 10 µL injection volume.
Stability study of OximUNO conjugate in PBS, pH 7.4
OximUNO conjugate was incubated in 10 mM dPBS at 37°C at the concentration of 3 mg/mL and with 3 µg/mL of DAU. 100 µL of aliquot was collected at defined time points (0, 1, 2, 5, 24, 48, 72 h) extracted with 3x250 µL chloroform and mixed with vortex for 5 min. Organic phases from all three chloroform extracts were collected in one tube, evaporated using speed vacuum until dry, and stored at -20°C. On the day of analysis, dried samples were reconstituted in 300 µL of methanol (LC-MS grade), vortexed for 5 min and centrifuged for 5 min at 30 437g. Supernatants were filtered through 0.45 µm filter and subjected to the LC-MS analysis.
Stability study of OximUNO in the i.p. fluid
The i.p fluid was collected from healthy 8-12-week old Balb/c female mice as performed by Ray and Dittel96 by collecting the supernatant and discarding the pellet after the centrifugation step. A working solution containing 3 mg/mL of OximUNO and 3 µg/mL of DAU in i.p. fluid was incubated at 37°C. 50 µL aliquots were collected at scheduled time points (0, 2, 5, 7, 24 h). The samples were then diluted with 100 µL of methanol and sonicated to aid the dissolution of DOX. The samples were injected into LC-MS after filtration through 0.45 µm filter.
Cathepsin B release kinetic studies.
Cathepsin B (5 IU) was activated in 2 mM EDTA, 5 mM DTT, and 20 mM CH3COONa buffer and incubated at 37°C for 15 min. In a separate tube, a solution containing 3 mg/mL OximUNO and 3 µg/mL of DAU was prepared with 20 mM CH3COONa and incubated at 37°C for 15 min. The two solutions were then combined producing reaction solution that was incubated at 37°C. 100 µL aliquots were collected at scheduled time points (0, 1, 2, 5, 8, 24, 48, 72 h), and after the addition of 900 µL of dPBS (to adjust the pH level to 7.4), the free DOX and DAU were extracted with three portions of 2.5 mL of CHCl3. Samples were proceeded as described under “Stability study of OximUNO conjugate in PBS, pH 7.4”. After CHCl3 evaporation, the samples were reconstituted with 300 µL of methanol, filtered through a 0.45 µm filter and subjected to LC-MS analysis. A blank solution was prepared with the same components as sample solution but without cathepsin B and used as a control sample.
In vitro cytotoxicity assay
Human peripheral blood mononuclear cells (PBMC) were purified from human blood buffy coat using Ficoll Paque Plus (GE Healthcare, cat. 17-1440-02) reagent and CD14+ microbeads (MACS Miltenyi Biotec, cat. 130-050-201) as described before42. 1.2x105 cells in 50 µL of RPMI-1640 medium were seeded on FBS-coated 96-well plate. To obtain optimal macrophage attachment and polarisation towards M2 phenotype, 50 µL of IL-4 (50 ng/mL, BioLegend, cat. 574002) and M-CSF (100 ng/mL, BioLegend, cat. 574802) mixture in the medium was added to the wells. The medium was replenished by substituting the half of the medium with fresh medium containing IL-4 and M-CSF every other day for six days. To obtain M1 macrophages, the monocytes were incubated with M-CSF (100 ng/mL) for six days, replenishing every other day with fresh medium containing M-CSF and on day six, 50 µL of M-CSF, LPS (100 ng/mL, Sigma Aldrich, cat. L4391) and IFN-γ (20 ng/mL, BioLegend, cat. 570202) mixture was added and incubated overnight in the incubator. On day seven, M2 and M1 macrophages were incubated for 15 min at 37 °C with OximUNO, St-PGA-DOX, DOX, or free medium as a control (N = 3 wells per group). Concentrations used were calculated based on DOX: 33µM and 100µM. After incubation, wells were washed, new medium added and incubated for 48 h at 37 °C. After 48 h, 10 µL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT, concentration 5 mg/mL, Invitrogen, cat. M6494) in PBS was added to each well containing culture medium and incubated for 2.5 h at 37 °C after which medium containing MTT was removed without removing formed crystals and 100 µL of isopropanol was added to each well to dissolve crystals. Then, absorbance was read at 580nm using plate reader (Tecan Sunrise) and corresponding Magellan™ 7 programme.
In vivo liver and kidney toxicology studies with OximUNO
Three healthy 12-week-old female Balb/c mice were injected i.p. with OximUNO (704 µg/0.5mL PBS) and circulated for 48 h. Then, mice were anesthetised, and blood was collected through retro-orbital bleeding into Lithium Heparin tubes (BD Vacutainer, cat. 368494). Blood samples were centrifuged at 1800g for 15 min at + 4 °C and 400 µL of plasma was collected for analysis. Samples were analysed in Tartu University Hospital using a Cobas 6000 IT-MW (Roche Diagnostics Gmbh) machine and reagents for creatinine (CREP2, cat. 03263991) and alanine aminotransferase (ALTLP, cat. 04467388).
Monotherapy with OximUNO in the orthotopic TNBC
5x104 4T1 cells in 50 µL of PBS were injected s.c. into the fourth mammary fat pad of 8-12-week-old female Balb/c mice. On day seven, mice were sorted into four groups by their tumour volume measured using digital calliper (Mitutoyo). Tumour volume was calculated based on the formula (W2 x L)/2, where W is the tumour’s width and L is the tumour’s length. The starting volume for each group was ~ 25 mm3, and the number of mice in each group was five. The first i.p. injection of compounds was carried out on day seven, followed by i.p injection every other day; nine injections were performed in total. The dose of the nanoconjugates was calculated based on DOX, 2mg/kg per injection (DOX: 39.5 µg/0.5mL PBS; St-PGA-DOX: 476 µg/0.5mL PBS; OximUNO: 341 µg/0.5mL PBS) giving a cumulative dose of DOX of 18mg/kg. Mice bodyweight and tumour volumes were monitored every other day. The final injection was on day 25 and all mice were sacrificed on day 28. Tumour tissues were processed as described under “In vivo biodistribution studies”, and the lungs and hearts were embedded in paraffin and processed for haematoxylin and eosin (H&E) staining (described below). Tumours were immunostained as described above. CD206 was detected using rat anti-mouse CD206 (dilution 1/200), CD8 using rat anti-mouse CD8 (dilution 1/75 Biolegend, cat. number 100701), and FOXP3 using rat anti-mouse FOXP3 (dilution 1/75, Biolegend, cat number 126401) as primary antibodies. Alexa Fluor® goat anti-rat 647 (dilution 1/300 for CD206 and 1/200 for CD8 and FOXP3) was used as secondary antibody for all markers. Slides were counterstained with DAPI (1 µg/mL in PBS). All 5 tumours from each group were included into the IF analysis. Fluorescent signal intensity was calculated using ImageJ programme, wherein the mean values from at least three images per tumour were included.
H&E staining on paraffin-embedded formalin-fixed tissues
For H&E staining, 2 µm sections were cut from paraffin-embedded blocks. Then slides were warmed at 60 °C for 2 min before deparaffinising using xylene (3x2 min, 1x1 min) followed by 100% ethanol washes (3x1 min), 80% ethanol wash (1x1 min) followed by 1 min wash in water. Slides were first incubated with ST-1 HemaLast for 30 s, followed by ST-2 Haematoxylin for 5 min after which slides were washed in water for 2 min. Then, ST-3 Differentiator was added for 45 s, and slides were washed in water for 1 min. Next, ST-4 Bluing Agent was added (1 min), washed for 1 min in water followed by 1 min incubation in 80% ethanol, after which ST-5 Eosin was added and incubated for 1 min. For rehydration, incubations in 100% ethanol (2x30 s, 1x2 min) were carried out and finished with incubations in xylene (2x2 min). All washes were carried out in tap water. H&E stainings were performed in Tartu University Hospital by pathologists using Leica staining automat and ST Infinity H&E Staining System (Leica, cat. 38016998). Stained lung sections were scanned using slide scanner (Leica SCN400) and 20x zoom. Pictures were analysed using QuPath programme (version 0.1.2)97. Five levels ~ 1 mm apart were used for each mouse to obtain comprehensive metastases profile of the lungs. Stained heart sections were also scanned using slide scanner and analysed with QuPath programme. Tartu University Hospital pathologists assessed cardiotoxicity in hearts and pulmonary metastases.
Monotherapy with OximUNO in the experimental metastases of TNBC
2x105 4T1 cells in 100 µL of PBS were injected i.v. in the tail vein of 8-12-week-old female Balb/c mice. Treatment with OximUNO, St-PGA-DOX, or DOX was started on day four p.i.; each group had six mice. Doses of different compounds were calculated based on DOX (2mg/kg): DOX: 39.5 µg/0.5mL PBS; St-PGA-DOX: 774.5 µg/0.5mL PBS; OximUNO: 704 µg/0.5mL PBS. The bodyweight of each mouse was monitored every other day. A total of six injections were carried out every other day. The final injection was on day 12, and all animals were sacrificed on day 18 using anaesthetic overdose and perfusion with PBS. Three right lungs from each group went to the flow cytometry (FC) analysis, and three full lungs and three left ones from each group were frozen into block using OCT. Frozen lung tissues were cryosectioned as described earlier, fixed with cold 4% PFA (CD206) or acetone (for CD8 and FOXP3), and stained as described in following section. Immunofluorescent stainings were performed using the same markers and antibodies as shown in “Monotherapy with OximUNO on orthotopic TNBC model” section.
GFP staining and imaging
Six lungs from each group were frozen in OCT. 10 µm sections were cut, and slides were kept at -20 °C until ready to use. Slides were taken out of the freezer at least 30 min before staining. For staining, slides were fixed with 4% PFA for 10 min at RT, washed with PBS for 10 min at RT, counterstained using DAPI (1 µg/mL) for 5 min at RT, washed 3x4 min with PBS and finally mounted using mounting medium. Permeabilisation was not used in this step to improve GFP visualisation. GFP was visualised using its native fluorescence. Slides were imaged using Olympus confocal microscope (FV1200MPE) using a 10x objective.
Macroscopic analysis of GFP signal
Lungs from each group were imaged using Illumatool Bright Light System LT-9900 (LightTool’s Research) in green channel to visualise the fluorescent signal macroscopically, and a photograph of each lung was taken. The total GFP signal of each lung was quantified using ImageJ programme using “IntDen” value.
Flow cytometry (FC) analysis
Three mice were sacrificed using anaesthetic overdose, perfused with PBS and right lung tissues were place in cold RPMI-1640 medium supplemented with 2% v/v FBS. Lungs were cut into small pieces on ice in a solution containing collagenase IV (160 U/mL, Gibco cat.17104019)/dispase (0.6 U/mL, Gibco, cat. 17105-041)/DNase I (15 U/mL; AppliChem, cat. A3778) mixture. To obtain single cell suspension, lung pieces were incubated in 10 mL of the same mixture at 37 °C on a rotating platform for 45–60 min, pipetting every 10 min to improve digestion. The cells were washed with 5 mL of RB (“running buffer”: 4 mL 0.5M EDTA, 100 mL v/v FBS in 1L of PBS), centrifuged (all centrifugations at 350g for 7 min at + 4 °C), and red blood cells were lysed with 3 mL of Ammonium-Chloride-Potassium Lysing Buffer (ACK) at RT. Ten mL of RB was added, cells were centrifuged and filtered using 100 µm cell strainer (Falcon, cat. 352360). Cells were counted using the brightfield mode of LUNA™ Automated Cell counter (Logos Biosystems). The cells were taken up in RB at a concentration of 5x106/100µL, placed on a 96-well plate with conical bottom and incubated for 30 min in FcR-blocking 2.4G2 hybridoma medium at + 4 °C. The cells were then stained for either macrophage or T cell markers for 25–45 min in the dark at + 4 °C, centrifuged and washed twice with RB. Antibodies used are listed in Table 2. For intracellular staining of T cells, cells were fixed using eBioscience™ FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher, cat. 00-5523-00) according to the protocol provided. Following permeabilisation, cells were stained for 25–45 min in the dark at RT and washed twice using RB. All the cells were taken up in 150 µL of RB, filtered through a 70 µm filter (Share Group Limited) and 150 µL of RB was used to wash the filter. BD LSRFortessa Flow Cytometer and FCS Express 7 Flow (De Novo Software) were used for analysis.
Antibodies used in FC analysis: macrophage and T cell markers.
PerCP/Cyanine5.5 anti-mouse CD206 (MMR)
1/200, BioLegend, clone C068C2, cat. 141715
PE anti-mouse CD86
1/400, BioLegend, clone PO3, cat. 105105
PE/Cyanine7 anti-mouse F4/80
1/200, BioLegend, clone BM8, cat. 123114
PE/Dazzle™ 594 anti-mouse/human CD11b
1/ 800, BioLegend, clone M1/70, cat. 101255
eBioscience™ Fixable Viability Due eFluor™ 506
1/800, Thermo Fisher Scientific, cat. 65-0866-18
T cell markers
Brilliant Violet 570™ anti-mouse CD4
1/400, BioLegend, clone RM4-5, cat. 100542
Brilliant Violet 605™ anti-mouse CD8a
1/400, BioLegend, clone 53 − 6.7, cat. 100744
PE/Dazzle™ 594 anti-mouse CD279 (PD-1)
1/200, BioLegend, clone 29F.1A12, cat. 135228
Alexa Fluor® 488 anti-mouse FOXP3
1/100, BioLegend, clone MF-14, cat 126406
PerCP/Cyanine5.5 anti-mouse CD3ε
1/200, BioLegend, clone 145-2C11, cat. 100328
Brilliant Violet 421™ anti-mouse CD152 (CTLA4)
1/200, BioLegend, clone UC10-4B9, cat. 106312
eBioscience™ Fixable Viability Due eFluor™ 506
1/800, Thermo Fisher Scientific, cat. 65-0866-18
H&E staining on PFA fixed cryosections
Ten µm sections were cut from unfixed tissues in a frozen block; sections were stored at -20 °C until ready to use. When ready, slides were taken out of the freezer at least 30 min before staining. Room temperature slides were fixed with cold 4% PFA for 10 min at RT followed by washing in PBS for 10 min at RT. After washing, slides were dipped into Mayer’s haematoxylin solution (see preparation under “Reagents and solution”) for 10 s, followed by washing in running tap water for 5 min. Then, slides were dipped into Eosin (5%) solution (see preparation under “Reagents and solution”) for 20 s, followed by washing in running tap water for 5 min. For rehydration, slides were placed first in 96% ethanol (2x2min) followed by 100% ethanol (2x2min). For clearance slides were placed in RotiClear® solution (Roth, cat. A538.5) for 2x5 min after which slides were mounted using Eukitt® quick-hardening mounting medium (Merck, cat. 03989). Slides were scanned using Leica DM6 B microscope and Leica Aperio Versa 8 slides scanner with 20x zoom and pictures were analysed using ImageScope programme (version 12.3.3).
All statistical analysis was carried out using One-Way ANOVA, Fisher LSD test, using a Statistica programme (release 7).