Protein expression of anti-MSLN-LPS BsAbs

Both anti-MSLN amatuximab [IMGT/mAb-DB ID 64] and HN1 [40] were reformatted as BsAbs each with an anti-LPS scFv to allow binding to MSLN and to LPS. The sequences were codon-optimized for expression in Chinese hamster ovary (CHO) cells. The sequence of each BsAb included a mammalian leader sequence from immunoglobulin kappa chain, followed by a 6x Histidine purification tag, then the anti-MSLN scFv sequence, a G₄S linker, the anti-LPS scFv sequence and a C-terminal myc-tag for detection (Fig 2A) as described in Taylor et al. [21]. Both Amatux-LPS and HN1-LPS BsAbs were cloned into the mammalian expression vector pcDNA3.1(+) (Life Technologies), using HindIII and NotI restriction sites. CHO-S cells (Life Technologies) were cultured in CD-CHO with 8 mM GlutaMAX (Gibco) at 37°C, 7.5% CO₂ with shaking at 130 rpm. On the day of transfection, cells were prepared at ~ 3.0 × 10⁶ cells/mL at 98% viability as measured using Cedex HiRes cell counter. The transient transfection for 200 mL culture was prepared as following: 600 μg of plasmid DNA was mixed with 15 mL OptiPRO serum free media (SFM) (Gibco) (15% total volume) and incubated at room temperature (RT) for 30–60 sec. Concurrently, 2400 μL of PEIpro (Polyplus-transfection) was mixed with 30 mL OptiPRO SFM (15% total volume) and incubated at RT for 30–60 sec. The two solutions were then mixed together by gently pipetting the PEIpro: OptiPRO complex into the plasmid DNA: OptiPRO complex, and incubated static at RT for 15 mins. The transfection complex was then added to the culture in a shake-flask and mixed by gentle swirling. The culture was incubated at 37°C, 7.5% CO₂ with shaking at 130 rpm for 4 to 6 hrs, after which the culture was diluted 1:2 (v:v) with CD-CHO supplemented with final concentrations of EfficientFeed A and B (Gibco) 7.5% (v/v), 0.4% Anti-Clumping Agent (Gibco), 8 mM GlutaMAX and then the flasks were kept shaking at 130 rpm at 32°C, 7.5% CO₂ for 12 days. The supernatant was then harvested and 0.22 μm filtered.

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Fig 2. Anti-MSLN-LPS BsAb gene design, synthesis and binding to human mesothelioma cell lines.

(A) A BsAb comprising anti-MSLN scFv (red) and anti-LPS scFv (blue), connected by a glycine-serine linker (G4S). 6-HIS and C-MYC was included into the design for purification and detection purposes. Secretion peptide (SP) was included to enable the BsAbs to be secreted from CHO cells into medium. (B) SDS- PAGE gel showing, starting from left, SeeBlue pre-stained standard MW marker (Invitrogen) (Lane 1), Amatux-BsAb (Lane 2) and HN1-BsAb (Lane 3) purified products (Both MWs are 56 kDa). Flow cytometry was used to test the anti-MSLN designed BsAbs and an anti-EGFR control BsAb (ABX-EGF-BsAb) binding to human mesothelioma cell lines, (C) H226 and (D) MSTO-211H. Binding of anti-CD3-BsAb (black line), ABX-EGF-BsAb (red line), Amatux-BsAb (green line) and HN1-BsAb (orange line) to cells was detected using FITC-conjugated anti-c-myc antibody.

https://doi.org/10.1371/journal.pone.0186137.g002

Protein purification

Both His-tagged BsAbs were purified using immobilized metal affinity chromatography with a 5 mL HisTrap excel column (GE Healthcare). The column was equilibrated in 20 mM sodium phosphate pH 7.4, 500 mM NaCl. The harvested supernatant was loaded onto the column and then washed with equilibration buffer containing 20 mM imidazole to remove non-specifically bound contaminants. The protein of interest was then eluted with 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 500 mM imidazole. The final product was desalted into PBS using HiPrep 26/10 column (GE Healthcare) and then filtered through a 0.22 μm syringe filter. The concentration of protein was determined by measuring the UV absorbance at 280 nm using a Nanodrop 1000 spectrophotometer. Protein purity was analysed by SDS-PAGE using 4–12% Bis-Tris gels (Invitrogen).

Binding analysis of BsAbs to human mesothelioma cells by flow cytometry

To evaluate the binding affinity of anti-MSLN BsAbs to MSLN receptors on the human lung mesothelioma cell lines H226 (ATCC, CRL-5826) and MSTO-211H (ATCC, CRL-2081), cells were grown in RPMI-1640 (Sigma) medium supplemented with 10% fetal calf serum (FCS; Bovogen) and Penicillin/Streptomycin (Sigma). Before flow cytometric analysis, the cells were washed twice with PBS and then scraped from T75 flask and centrifuged at 700 x g for 5 mins. The viable cells were counted by trypan blue exclusion method and 8x10⁶ cells/mL were washed twice with cold PBS. A centrifugation step at 700 x g was conducted in between the washes. The sample was divided into five vials (2x10⁶ cells/sample) prior to the final centrifugation step. The pellets were resuspended in 500 μμL Binding Buffer (PBS containing 0.1% sodium azide and 5% bovine serum albumin (BSA)) and incubated for 10 mins to block nonspecific binding sites. Various primary antibodies (4 μg) were added to each tube as follows: (1) Amatux-BsAb, (2) HN1-BsAb, (3) ABX-EGF-BsAb (anti-EGFR BsAb described in [21]), (4) Anti-CD3-BsAb (as a negative control). The tubes were incubated for 45 mins on ice on a gentle shaker. Then the cells were washed twice with Binding Buffer. The cells were incubated with the secondary antibody, anti-cMyc-FITC (Miltenyi Biotec), diluted 1 in 250 in Binding Buffer for 45 mins on ice on a shaker (moderate movement). Afterwards, the cells were washed three times with Binding Buffer, and re-suspended in 400 μL PBS for flow cytometric analysis on a Beckman Coulter, Cytomics CXP500 flow cytometer using the 488 nm laser and the 530/30 nm filter. The data were analysed using FCS Express Flow version 4 (De Novo Software).

Preparation of targeted nanocells

EDVs were manufactured and purified using a cross-flow filtration and lyophilized as previously described [20]. Each lyophilized vial contained 1.1x10¹⁰ EDVs and the lyophilized particles were resuspended in 600 μL of solvent resulting into 1.8x10¹⁰ EDVs/mL. Doxorubicin loading into EDV nanocells to create EDV_Dox was carried out as previously described [20]. Additionally, for some experiments EDV nanocells were labelled with Alexa-Fluor 488 (AF-488) (ThermoFisher) as previously described [21]. Drug-loaded or AF-488 labelled EDVs were then incubated with Amatux-BsAb at RT for 30 mins while shaking at 300 x g, to create ^AmatuxEDV. Samples then underwent 3x PBS spin wash cycles at 9,000 x g for 8 mins each to remove any excess Amatux-BsAb. ^AmatuxEDV nanocells were resuspended to concentrations as required for the experiment.

Internalization analysis of drug-loaded ^BsAbEDV nanocells into H226 cells by confocal microscopy

H226 cells were incubated on coverslips with AF-488 labelled ^AmtauxEDV_Dox, at a ratio of 10,000 EDV nanocells per cell. Cells without any treatment were used as a control. Cells were incubated at 37°C for 3 hrs and 24 hrs which was followed by 3 washes in sterile PBS. Cells were fixed with 4% paraformaldehyde for 10 mins, followed by 3 times PBS washes. After the final wash, 500 μL PBS was added to each well to cover the coverslips completely. An anti-EGFR mAb (528 mAb) was labelled with x-site fluor 670/755 (Kodak) and used as a cell membrane stain; 4 μg was added directly to each sample. Wells were mixed gently and left for 10 mins to allow cells to stain sufficiently. All coverslips were again washed 3 times with PBS followed by a final wash in Milli-Q water and then mounted on clean slides using Fluka Eukitt (Sigma) quick-hardening mounting medium. Cells were imaged using a Carl Zeiss 710 Inverted Laser Scanning Confocal Microscope housed at the Australian National Fabrication Facility, Queensland Node (ANFF) using a plan-apochromat 63x 1.40 Oil DIC M27 objective and ZEN 2008 software for image formatting. The AF-488 labelled EDV nanocells were detected using ex488/em525 nm. Doxorubicin is auto-fluorescent and detected using ex490/em560 nm. Cell surface EGFR was detected with ex633/em670 nm.

Binding analysis of ^AmatuxEDV_Dox on human mesothelioma cell by flow cytometry

To evaluate binding of ^AmatuxEDV_Dox to MSLN receptors on H226 and MSTO-211H cells, lyophilised EDV_Dox nanocells were reconstituted, loaded with doxorubicin, and then labelled with Amatux-BsAb or with anti-Murine Epithelial Cell Adhesion Molecule (mEpCAM)-BsAb as a negative control. The ^BsAbEDv_Dox complexes were incubated rotating for 1 hr at RT, before washing three times and resuspending in 500 μL of PBS. H226 and MSTO cells were grown as previously described. Cells were washed twice with PBS and then detached using Accutase (Sigma) for 10 mins at 37°C and centrifuged at 300 x g for 5 mins. Viable cells were counted by trypan blue exclusion method on a hemocytometer and 1x10⁷ cells/mL were resuspended in serum free media. The cell suspension was divided into three vials to be treated with: (1) ^Non-targetedEDV_Dox, (2) ^mEpCAMEDV_Dox, and (3) ^AmtauxEDV_Dox. EDVs (5x10⁹ EDV/sample) were added to 5x10⁵ cells (1:10,000 cell:EDV ratio) and incubated rotating at 4°C. After 2 hrs, unbound EDVs were removed by washing the cells with PBS+1%BSA at 300 x g for 5 mins. Cells were fixed with 4% paraformaldehyde for 10 mins at RT, followed by a PBS+1%BSA wash. Bound EDVs were stained with 1 μg of anti-LPS mAb (Biodesign International) labelled with AF-488 (Life Technologies) for 45 mins at RT in the dark. The cells were washed three times with PBS+1%BSA at 300 x g for 5 mins, before analysis on a Beckman Coulter, Gallios flow cytometer using the 488 nm laser and the 530/30 nm filter. The data were analysed using FCS Express Flow version 4 (De Novo Software).

A flow cytometry dose-response of ^AmatuxEDV binding to the aforementioned mesothelioma cell lines, and the murine colon carcinoma CT26 cells (ATCC, CRL-2638), was performed in similar manner, but without loading the EDVs with doxorubicin. The cell suspensions were treated with different amounts of ^AmatuxEDV (1:1000, 1:2500, 1:5000 and 1:10,000 cell: ^AmatuxEDV ratios). EDVs charged with anti-CD3 BsAb were also included as negative controls when we incubated the cell suspensions with constant concentration of ^CD3EDV (1:10,000 cell:^CD3EDV). The data were analysed using FCS Express Flow version 4 (De Novo Software).

Cytotoxicity of ^AmatuxEDV_Dox nanocells

Induction of apoptosis was demonstrated by staining cells with Annexin V-FITC after incubating MSLN-positive H226 cells with ^AmatuxEDV, ^Non-targetedEDV_Dox and ^AmatuxEDV_Dox. H226 cells were grown in DMEM (Sigma) with 10% FCS (Bovogen) and 1% Penicillin/Streptomycin at a seeding density of 3x10⁵ cells per well in 6 well plates. The cells were incubated with ^AmatuxEDV, ^Non-targetedEDV_Dox and ^AmatuxEDV_Dox at a ratio equivalent to 10,000 EDVs per cell, for 2 h at 37°C to allow EDVs to bind to cells. Then the cells were washed 3 times with PBS to remove unbound EDVs, before adding fresh media and incubating at 37°C for 72 hrs to allow EDV internalisation and doxorubicin release. Washing the unbound particles is necessary, because if left in a well more than 24 hrs, they will start to leak out the doxorubicin into media [20, 22]. After 72 hrs, the media were collected and the cells were detached with trypsin-EDTA and collected in media. The cells were washed twice in PBS, then resuspend in 1x Binding Buffer at 1x10⁶ cells in 1 mL. Each sample was divided equally (500 μL) into different tubes, with one treated with 5 μL of Annexin V-FITC while the other was treated with 5 μL of Binding buffer as a control for doxorubicin interference by autofluorescence. The tubes were incubated at RT for 10 min before analysing the fluorescence of the cells on a Beckman Coulter, Cytomics CXP500 flow cytometer, using the 488 nm laser and the 530/30 nm filter. Data were analysed using FCS Express 4 Flow cytometry (De Novo Software).

Effect of ^AmatuxEDV_Dox nanocells on tumour regression

The experiments associated with the effect of Amatux-targeted nanocells on tumour regression were performed essentially as described by Taylor et al. [21]. H226 mesothelioma cells were grown in RPMI with 10% FCS and penicillin/streptomycin. Six week-old female Balb/c mice were injected subcutaneously on the left flank with 5×10⁶ cells in 200 μL of media together with 200 μL of growth factor reduced matrigel (BD Biosciences). Once the tumours reached ~110 mm³ (Day 11) the mice were randomized into treatment groups which included 5–6 mice per group, as follows: Group 1 –Saline, Group 2 −^AmatuxEDV, Group 3 −^Non-targetedEDV_Dox, and Group 4 −^AmatuxEDV_Dox. Each mouse was intravenously injected with 1×10⁹ of the relevant EDVs administered 3 times per week for 3 weeks, and tumour size was monitored throughout the treatment period up to Day 39 post xenograft. The mice were weighed twice a week for 3 weeks, as per previous methodology [20, 27]. A two-sample t-test with equal variance was performed to directly compare the mean tumour volume from each individual treatment to determine statistical significance between treatments and control. Statistical significance was determined as p ≤ 0.05 [47]. The mice were euthanased after a total of 12 injections, tumours were harvested, paraformaldehyde fixed and embedded in paraffin. Note; Experiments were conducted in compliance with the Australian National Health and Medical Research Council guidelines for the care and use of laboratory animals and with the approval of the EnGeneIC Animal Ethics Committee.

Ki67 cell staining and quantification by immunohistochemistry

Paraffin embedded human mesothelioma tumour tissue section (5 μm), prepared from tumours excised from the four xenograft treatment groups at Day 39 (Saline, ^AmatuxEDV, ^Non-targetedEDV_Dox, ^AmatuxEDV_Dox) were dewaxed in xylene, rehydrated in graded alcohols and deionized in distilled water. Heat-induced antigen retrieval (HIER) was performed by immersing the slides for 20 mins in 0.01 M Citric Acid-Based Buffer, pH 6.0 (Vector), at 100°C in a microwave, followed by cooling for 30 mins at RT. The endogenous peroxidase activity was quenched by incubating the slides with methanol containing 1% hydrogen peroxide for 30 min at RT in the dark. Then the slides were washed using PBS with 1% Tween20 (PBS-T) for 5 min and then blocked with 5% goat serum in PBS-T for 30 min at RT. Rabbit anti-human pKi67 antibody (Sino Biological, US) was added to the slides at 20 μg/μL. PBS substituted the primary antibody for the control slide. The slides were incubated overnight at 4°C in a covered humidity chamber. On the next day, the slides were washed in PBS-T and the HRP-conjugated secondary antibody goat anti-rabbit IgG (Life Technologies) was diluted in 5% goat serum in PBS-T at 1:200 before it was incubated with the slides for 2 hrs at RT. Then the slides were washed 3 times in PBS-T, then incubated with AEC (3-amino-9-ethylcarbazole) substrate solution (Vector) until the desired colour developed (10 min). The slides were counterstained with hematoxylin (Blue) for 30 sec and mounted with VectaMount AQ (Vector). The percentage of Ki67 positive cells was estimated by blind counting of Ki67 positive cells (red nuclei) in two different Ki67 expressing regions consisting of 1000 cells in total using 20x magnification. The percentage was estimated from three different mice representing each group. Images were captured using Aperio digital pathology slide scanner and analysed using Aperio ImageScope software (Leica Biosystems). The mean values for the percentage of Ki67 positive cells for all treatments were analysed for variance using one-way ANOVA with Tukey post-hoc test was performed to directly compare the means from each individual treatment to determine if there is any significant difference between treatments and control. Statistical significance was determined as p ≤ 0.05 [47].

Hematoxylin and Eosin (H&E) staining for mesothelioma paraffin embedded tumour samples

Paraffin embedded human mesothelioma tumour tissue sections (5 μm), prepared from tumours excised from the four xenograft treatment groups at Day 39 (Saline, ^AmatuxEDV, ^Non-targetedEDV_Dox, ^AmatuxEDV_Dox) were dewaxed in xylene, rehydrated in graded ethanol solutions. Tissue samples were then stained for 7 min in Mayers Haematoxylin and 20 sec in 1% Eosin. Samples were then dehydrated through graded ethanol solutions and cleared with xylene. Finally, slides were coverslipped using DPX solution (Sigma). Samples were visualised using an upright bright-field slide scanner (Zeiss Axio Imager Z2). The percentage of necrotic areas was estimated by blind measuring of multiple scattered necrotic regions (absence of nuclear staining) using 20x magnification. The percentage was estimated from three different mice representing each treatment group. Images were captured using a Zeiss Axio Imager Z2 slide scanner and analysed using ImageJ software v1.50i (National Institutes of Health, Bethesda, MD). The mean values for the percentage of necrotic regions for all treatments were analysed for variance using one-way ANOVA with Tukey post-hoc test. Statistical significance was determined as p ≤ 0.05 [47].

Production of anti-MSLN BsAbs

The BsAbs derived from amatuximab and HN1 were expressed in CHO cells followed by IMAC purification using HisTrap excel affinity chromatography column. SDS-PAGE analysis showed molecular weights of 56 kDa for both BsAbs (Fig 2B). Binding specificity between purified MSLN and purified LPS and the BsAbs was confirmed by conducting indirect ELISA on immobilised MSLN and LPS and detection with HRP-conjugated anti-myc antibody (data not shown).

Binding activity of anti-MSLN BsAbs toward human mesothelioma cells

Flow cytometry was used to assess the binding of Amatux-BsAb and HN1-BsAb to mesothelioma cell lines H226 and MSTO-211H. ABX-EGF-BsAb specific for EGFR [21] was used as a positive control, since malignant mesothelioma frequently shows over-expression of EGFR in either cell lines or the tumour itself [29]. We observed that Amatux-BsAb and ABX-EGFR-BsAb bound strongly to the H226 cells, whereas HN1-BsAb showed no binding (Fig 2C). Both Amatux and HN1-BsAbs showed no binding to MSTO-211H cells (Fig 2D).

^AmatuxEDV_Dox nanocells cellular uptake

^BsAbEDV_Drug nanocells are capable of being internalized by cancer cells and releasing cytotoxic drug intracellularly [22]. Here, we prepared AF-488 labelled ^AmatuxEDV_Dox nanocells to monitor and visualize binding and internalisation of the targeted EDV system to H226 cells at the cellular level by confocal microscopy. Within a 3 hr time-frame, the ^AmatuxEDV_Dox nanocells were co-localized on the cell surface, demonstrated by green fluorescence staining on the H226 cell membrane (Fig 3A). After 24 hrs of incubation, the AF-488 labelled ^AmatuxEDV_Dox nanocells migrated and accumulated within the H226 cells and released doxorubicin intracellularly, demonstrated by purple staining representing doxorubicin autofluorescence (Fig 3B). To confirm that the ^AmatuxEDV nanocells binding to H226 cells was Amatux-BsAb-dependent, we used flow cytometry to test binding of ^Non-targetedEDV_Dox, ^mEpCAMEDV_Dox and ^AmatuxEDV_Dox to H226 and MSTO-211H cell lines_. Only ^AmatuxEDV_Dox nanocells were able to bind to the MSLN on H226 cells, but did not bind to the low MSLN expressing cell line MSTO-211H (Fig 3C and 3D).

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Fig 3. BsAb targeting and internalisation of EDV_Dox to MSLN on H226 cells.

Internalisation was detected using confocal imaging of H226 cells incubated with Amatux-BsAb targeted AF-488-EDV nanocells (Green: Ex 488; Em 490–540) loaded with doxorubicin (Purple: Ex 488; Em 540–590) at 3 hr (A) and 24 hr (B) timepoints (Scale bars 5 and 10 μm, respectively). H226 cell membranes were pre-stained with anti-EGFR-AF-647 (Red: Ex 633; Em 640–750). Specificity of ^Non-targetedEDV_Dox (red line), ^mEpCAMEDV_Dox (black line) and ^AmatuxEDV_Dox (green line) binding to human mesothelioma cell lines, H226 (C) and MSTO-211H (D), and dose-response assay of ^AmatuxEDV_Dox on H226 (E), MSTO-211H (F) and CT26 (G) were excuted by flow cytometry using anti-LPS AF-488 and 488 nm laser and 530/30 nm filter.

https://doi.org/10.1371/journal.pone.0186137.g003

A dose-response shift was generated when H226 mesothelioma cells were treated with different concentrations of ^AmatuxEDV (Fig 3E). The intensity of the binding shift was concentration-dependent, wherein the strongest shift was observed when the H226 cells were treated with 10,000 ^AmatuxEDV per cell, while a gradual reduction of the binding intensity was observed each time we reduced the ^AmatuxEDV:cell ratio by half (Fig 3E). Under the same conditions, ^AmatuxEDV showed no binding to both MSTO-211H and murine colon carcinoma CT26 cells (MSLN negative cell line) (Fig 3F and 3G). Also, as a negative control we included 1:10,000 cell:^CD3EDV, which showed no binding to the three cancer cell lines.

Cytotoxicity of ^AmatuxEDV_Dox nanocells

Apoptosis causes externalization of phosphatidylserine from the inner leaflet of the plasma membrane phospholipid bilayer to the cell surface, which can be probed with Annexin V for detection, while living cells remain Annexin V negative [48]. Therefore, we stained ^AmatuxEDV, ^Non-targetedEDV_Dox and ^AmtauxEDV_Dox treated cells with Annexin V-FITC and performed flow cytometry to evaluate the induction of apoptosis. Approximately 37.7% of ^AmatuxEDV_Dox targeted H226 cells showed Annexin V positive staining, which was around 9-fold greater than that of ^AmatuxEDV and ^Non-targetedEDV_Dox samples (Fig 4). The same samples were also analysed in the absence of Annexin V; the ^AmatuxEDV_Dox sample showed a small increase in fluorescence attributed to the autofluorescence of doxorubicin, which has a comparable emission wavelength to FITC, yet the shift remained within the Annexin V negative gate (data not shown). These results suggest that ^AmatuxEDV_Dox nanocells are capable of targeted killing of MSLN-expressing cells. Consequently, tumour regression studies were performed using ^AmatuxEDV_Dox nanocells in a H226 cell line xenograft model.

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Fig 4. internalisation of ^AmatuxEDV_Dox apoptosis induction.

The induction of apoptosis due to release of doxorubicin internally was measured using Annexin V-FITC staining, on untreated cells (1), cells treated with ^AmatuxEDV (2), ^Non-targetedEDV_Dox (3), or ^AmatuxEDV_Dox (4). Fluorescence was measured using 488 nm laser and 530/30 nm filter. The red dot plot represents Annexin V-FITC positive cells and the green dot plot represents Annexin V-FITC negative cells.

https://doi.org/10.1371/journal.pone.0186137.g004

^AmatuxEDV_Dox nanocells mediate anti-tumour effect in human mesothelioma xenograft models

Eleven days after tumour subcutaneous-implantation when the volume of the tumours reached ~110 mm³, mice were randomly sorted into four different groups (n = 6 per group): saline control group, ^AmatuxEDV, ^Non-targetedEDV_Dox and ^AmatuxEDV_Dox. By the end of the treatment period (Day 39), all the mice in saline, ^AmatuxEDV and ^Non-targetedEDV_Dox treatment groups had comparable tumour volumes (p > 0.1), while the ^AmatuxEDV_Dox treated group showed significant tumour regression (p < 0.001) with the tumour volume being 3-fold less than all other treatment groups (Fig 5A). Within the ^AmatuxEDV_Dox treated group, one mouse showed a partial response (≥30% tumour regression) during the whole study period. Three mice showed transient partial response followed by slight tumour growth and then tumour stasis (no tumour growth or progressive disease). One mouse demonstrated stable tumour size during the whole treatment course (data not shown). The ^Non-targetedEDV_Dox did not evoke any significant anti-tumour effect through passive targeting. Thus, active targeting through BsAbs is essential for the efficient delivery of EDV nanocells to tumours.

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Fig 5. Effect of ^AmatuxEDV_Dox on xenograft models.

(A) Xenograft implanted mice were treated with either saline as a control, or EDV variants ^AmatuxEDV, ^Non-targetedEDV_Dox or ^AmatuxEDV_Dox all at a dose equal to 1 x 10⁹ EDVs at different time points, indicated with a red triangle. The average tumour size at the start is ~110 mm³. (B) The mice were weighed twice a week throughout the treatment. Mean ± SEM is shown. (*** = p < 0.001).

https://doi.org/10.1371/journal.pone.0186137.g005

All mice survived throughout the treatment period, except one mouse in the ^AmatuxEDV_Dox group that died of unknown cause(s) on the first day of the experiment, thus n = 5 in subsequent analysis of data for this group. Physical observation of all the mice showed no abnormalities in behaviour nor physical condition, and there was no difference in weight throughout the treatment period between the groups of mice (Fig 5B). This suggests the doxorubicin-loaded EDV nanocells are well tolerated despite the high and repeated dose regimen.

^AmatuxEDV_Dox nanocells mediate anti-proliferative effect and reduce necrosis in human mesothelioma xenograft models

The nuclear protein Ki67 is a proliferation marker and an independent prognostic factor in malignant mesothelioma that can be utilised to evaluate the effectiveness of a chemotherapy regimen [49, 50]. The Ki67 biomarker was therefore utilised to detect the proliferation rate differences between the four treatment groups. The Ki67 immunohistochemical staining of tissue sections from excised xenograft tumours from the ^AmatuxEDV_Dox treated group showed a significant decrease in the number of proliferating cells, compared to the saline (p < 0.01), ^AmatuxEDV (p < 0.01) and ^Non-targetedEDV_Dox (p < 0.05) treated groups (Fig 6A and 6B). Using H&E staining, tissue sections from saline treated mice revealed larger areas of necrosis (12.52%) compared to ^AmatuxEDV (10.9%), ^Non-targetedEDV_Dox (9.5%) and ^AmatuxEDV_Dox (5.65%) treatment groups (Fig 6C and 6D). Statistically, the ^AmatuxEDV_Dox treated mice had significantly smaller necrotic areas compared to the saline group, ^AmatuxEDV and ^Non-targetedEDV_Dox (p ≤ 0.0005, p < 0.01 and p < 0.05, respectively).

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Fig 6. Immunohistochemical staining of paraffin embedded mesothelioma xenograft tumour samples.

(A) Malignant mesothelial cells showed strong and diffuse staining by anti-human pKi67 antibody. Tissue stained with secondary antibody only showed no staining. Scale bar 200 μm. (B) The percentage of Ki67-positive cells were calculated blindly from tumour tissue sections obtained from three different mice representing each group (Mean ± SD). (C) Tumour necrotic areas in the tissue sections are shown by reduced H&E staining. Representative tissue sections for the four treatment groups show necrotic areas (N) (absence of nuclear staining). Scale bar 500 μm. (D) The percentage of necrosis was calculated blindly using ImageJ from tumour tissue sections obtained from three different mice representing each group (Mean ± SD). (* = p < 0.05, ** = p < .01 and *** = p < 0.0005).

https://doi.org/10.1371/journal.pone.0186137.g006