Ablation of Red Stable Transfected Prostate Cancer Cell Lines by C-CPE Gold-Nanoparticle Mediated Laser Intervention

Background: Claudin (CLDN) proteins have been described to be found and accordingly targeted to evaluate novel therapeutic approaches. C-terminus of Clostridium perfringens enterotoxin (C-CPE) binds eciently several claudins and thus recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for cancer cell targeting using gold nanoparticle- mediated laser perforation (GNOME-LP). Cancer cells inoculation is routinely used to generate in vivo models to evaluate novel therapeutic approaches in prostate cancer. However, detailed characterization of cancer spreading and early tumor development and therapeutic response is often limited as conventional cell lines do not allow advanced deep tissue imaging. Methods: two canine prostate cancer cell lines were stably transfected with red uorescent protein (RFP), followed by G418 selection. RFP marker as well as CLDN3, -4 and -7 expression was comparatively conrmed by ow cytometry, qPCR and immunouorescences. For cancer cells targeting, GNOME-LP at a laser uence of 72 mJ/cm² and a scanning speed of 0.5 cm/s was used. Statistical analysis was performed using SAS software 7.1, Dunnett´s Multiple Comparison Test and Student´s two-sided t-test. Differences were considered statistically signicant for p<0.05. Results: we established two canine prostate carcinoma cell lines, stably expressing RFP allowing perspective deep tissue imaging. Directed C-CPE-AuNP binding to native and RFP transfected cells veried the capability to specically target CLDN receptors. Cancer cell ablation was demonstrated in vitro setting using a combination of gold nanoparticle mediated laser perforation and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10 % depending on cell line. Conclusion: the results conrm that this therapeutic approach can be used eciently to target prostate carcinoma cells carrying a marker protein allowing deep tissue imaging. The established cell lines and the veried proof of concept in vitro study provide the basis for perspective Xenograft model in vivo studies. characterization of tumor growth and subsequently possible tumor ablation through C-CPE-AuNPs treatment.

veri ed the capability to speci cally target CLDN receptors. Cancer cell ablation was demonstrated in vitro setting using a combination of gold nanoparticle mediated laser perforation and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10 % depending on cell line.
Conclusion: the results con rm that this therapeutic approach can be used e ciently to target prostate carcinoma cells carrying a marker protein allowing deep tissue imaging. The established cell lines and the veri ed proof of concept in vitro study provide the basis for perspective Xenograft model in vivo studies.
The introduce red uorescence enables deep tissue imaging in living animals and therefore detailed characterization of tumor growth and subsequently possible tumor ablation through C-CPE-AuNPs treatment.

Background
Advanced experimental approaches in cancer research require the establishment of tumor speci c in vivo animal models. Therefore, cell lines represent a powerful tool allowing the generation of neoplasia mimicking closely the initial tumors through xenografts [1,2]. Within these models, the characterization of tumor development and the possibility to monitor tumor cell migration is of major interest for the evaluation of novel therapeutics. Fluorescent proteins and bioluminescent systems provide unique opportunities for non-invasive labeling and tracking of speci c cell types in living organisms in real time.
Herein we established and characterized stable red uorescent canine prostate tumor cell lines, which provide a novel tool for in vitro and in vivo tumor imaging. Further we characterized if the uorescent emission interferes with conventional laser ablation and optimized the therefore required parameters in vitro. Finally, selective C-CPE AuNPs ablation using GNOME-LP in combination was used to eliminate prostate CLDN3, -4 and/or -7 expressing tumor cell lines.

Materials And Methods
Cell lines and culture Canine tumor cell lines TihoDProCarc0840 (DT0840) and TihoDProAdCarc0846 (DT0846) were previously derived by our group from canine prostate carcinomas. DT0840 was generated from a 10-yearold Pit Bull Terrier, castrated [47] and DT0846 was generated from a 6.3-year old intact German Rough Haired Pointer [48]. Both cell lines have been demonstrated to express CLDN3, -4 and − 7 [41].
The cells were cultivated separately in 25 cm 2 cell culture asks in medium 199 (Gibco by Life Technologies, Darmstadt, Germany) supplemented with 10% Fetal Bovine Serum (FBS Superior, Biochrom GmbH, Berlin, Germany) and 2% penicillin/streptomycin (Biochrom GmbH, Berlin, Germany), and incubated in a humidi ed incubator maintained at 37 ˚C with 5% CO 2 . Cultivation medium was replaced twice per week.

Red uorescence expression plasmid
For transfection, the pFusionRed-C (Evrogen, Moscow, Russia) plasmid was used. The vector encodes for red uorescent protein FusionRed as well as a neomycin resistance gene allowing selection of stably transfected cells using Geneticin® Selective Antibiotic (G418) (Life Technologies, Darmstadt, Germany). The vector was inserted into E. coli DH5α competent cells according to standard heat shock transformation procedures for further multiplication. Expanded Plasmid DNA was extracted from bacteria culture using Nucleo Bond® PC 500 plasmid DNA puri cation Kit (MACHEREY-NAGEL GmbH, Düren, Germany).

Transfection of canine tumor cell lines
Both cell lines were seeded with a density of 5*10 5 cells in 6-well plates 24 hours before transfection. The transfection was performed according to the manufacturer's protocol using 6 µl X-treme GENE HP DNA Transfection Reagent (Roche GmbH, Mannheim, Germany) in 200 µl serum-reduced Opti-MEM I media (Life Technologies, Darmstadt, Germany) containing 2.5 µg of the isolated and puri ed pFusionRed-C plasmid. The transfection complex remained for 20 min at room temperature. After adding the transfection complex to the respective cells, the plates were incubated in a humidi ed 5% CO 2 incubator for 48 hours at 37 °C. The expression of the uorescent protein was veri ed using a Leica DMI 6000B uorescence microscope (Leica Microsystem GmbH, Wetzlar Germany).
Geneticin® selective antibiotic kill curve assay The titration of a suitable amount of antibiotics required for the selection of DT0846 and DT0840 cell lines was performed using a kill curve assay. Different G418 concentrations (0, 100, 200, 400, 600, 800, 1000 µg/ml) were applied on 100,000 native DT0846 and DT0840 cells, seeded in 12-well plates. For the selection of positive cells after transfection, the lowest concentration was chosen in which no nontransfected cell survived after seven days of G418 exposure determined by microscopy and ow cytometry analysis.

Selection of positively transfected cells
One day after transfection, the cultivation medium 199 was replaced with medium 199 containing antibiotic G418. A concentration of 200 µg/ml G418 was used for both transfected cell lines.
Subsequently, the selection medium was changed every 48 hours for the rst two weeks, which led to the selection of cells that stably integrated the FusionRed plasmid, with the encoded antibiotic resistance gene, into their genomic DNA. Accordingly, cells not expressing the construct were killed by G418. In vitro imaging using the NightOWL LB 983 in vivo imaging system Viable cells were harvested, counted and plated at a density of 2.5 × 10 6 per well in a 96-well plate and followed by a 1:2 serial dilution until 0.156 × 10 6 cells per well in duplicates. Each well was set to a volume of 250 µl. Unlabeled pre-B-ALL NALM-6 cells were used as controls in equivalent concentrations.
The plate was incubated overnight and images were taken with the NightOWL LB 983 imaging system (Berthold Technologies, Bad Wildbad, Germany). As settings, an excitation lter of 525 nM and emission lter of 655 nM with variable exposure times (4 s/10 s) were used. Images were analyzed with the indiGO software (Berthold Technologies, Bad Wildbad, Germany).

RNA isolation and cDNA synthesis
Total RNA was isolated from transfected and native prostate tumor cell lines using the RNeasy®Mini Kit RNA Puri cation (Qiagen, Hilden, Germany) according to the manufacturer´s instruction. RNA isolation was performed three times per cell line. DNase digestion was carried out with RNase-Free DNase Set (Qiagen) to avoid genomic DNA contamination. Subsequently, cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany), 1000 ng of total RNA, and anchored-oligo (dt) 18 primer according to the manufacturer´s instructions.
Quantitative real-time RT-PCR To verify expression of CLDN genes after transfection process, transfected and native prostate tumor cell lines were comparatively analyzed by quantitative PCR. Primer pairs for CLDN3, CLDN4, and CLDN7 were designed according to the mRNA sequences given by the National Center for Biotechnology Information (NCBI) ( Table 1). Real-time PCR was performed using Fast SYBR™ Green Master mix Kit (Life Technologies, Darmstadt, Germany) according to the manufacturer´s instructions. Quantitative PCR reactions were carried out in real-time PCR cycler peqSTAR 96q (PEQLAB Biotechnologies GmbH, Erlangen, Germany). The qPCR results were analyzed using the delta-delta CT (ΔΔCT) method relative to non-transfected cells. Mean values of three wells were used per measured gene. Normalization was done against housekeeping genes beta-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The experiment was performed three times. iFlour™ 555 Anti-rabbit (AAT Bioquest, CA, USA) were diluted 1:500 in PBS containing 1% BSA and added to the respective cells as secondary antibodies for 1 h at 37 °C. For nuclei staining, DAPI (2 µM) (Sigma-Aldrich) was used. Cells were stored in PBS at 4 °C for further analysis. As a control for unspeci c binding sites, cells were also incubated with only the secondary antibodies. Fluorescent images of cells were taken with a Nikon Eclipse TE2000-E confocal laser scanning microscope (400 nm for DAPI, 555 nm for CLDN3 and − 7 proteins and 488 nm for CLDN4), with a 60x water immersion objective and software EZ-C1 3.80 (Nikon, Düsseldorf, Germany).

Visualization of C-CPE-CLDN binging
The C. perfringens enterotoxin C-terminal fragment (C-CPE) with an N-terminal Strep-tag II was prepared as described previously. For C-CPE-CLDN binding visualization, the C-CPE was conjugated to green uorescent Strep-Tactin® Chromeo 488 dye (IBA, Goettingen, Germany). The complex was generated freshly before usage, by mixing 2.5 µl Strep-Tactin® Chromeo 488 (0.5 mg/mL) as recommended by the manufacturer with C-CPE dissolved in elution buffer. The mix was incubated overnight at 4 °C to allow binding of C-CPE with Strep-Tactin® Chromeo 488. To reach a nal concentration of 20 µg/ml C-CPE, the mixture was diluted with 250 µl culture medium. Cells were cultured in a monolayer and stained for 3 h at 37 °C with 20 µg/ml C-CPE-Chromeo 488 complex. For nuclei staining, 1 µM Hoechst 33258 (Sigma-Aldrich) was used. Thereafter, cells were xed with 4% formaldehyde for 10 min at room temperature and stored in PBS at 4 °C. The cells were imaged with a Nikon Eclipse TE2000-E confocal laser scanning microscope (346 nm for Hoechst 33258 and 488 nm for Chromeo 488) with a 60x water immersion objective and software EZ-C1 3.80 (Nikon).

Tumor cells ablation by GNOME-LP and C-CPE-AuNPs complex interaction
For tumor cell killing using a laser beam, con uent cells in 96-wells were treated with the C-CPE-AuNPs complex for 3 h in a cell culture incubator to allow adhesion of the complex onto the cells. The C-CPE-AuNPs complex was generated as followed: The Strep-tagged C-CPE in elution buffer and Strep-Tactin® conjugated AuNPs (diameter 25 nm) (Aurion, Wageningen, Netherlands) were mixed and incubated overnight at 4 °C. The concentration was adjusted to 20 µg/ml C-CPE and 2.5 × 10 10 AuNPs/mL with cell culture medium.
In addition to non-treated cells, cells incubated with either non-functionalized AuNPs or C-CPE alone, were used as controls. Cells were exposed to a pulsed laser with 72 mJ/cm² at a scanning speed of 0.5 cm/s. Laser treated cells were incubated for 30 min with 1 µM Hoechst 33258 (Sigma-Aldrich) and 10

Electron microscopy
To examine the binding of AuNPs and C-CPE-AuNPs on cell surfaces, the DT0846 and transfected DT0846-FusionRed cell lines were analyzed with scanning electron microscopy (SEM). Con uent cells were treated with AuNPs and C-CPE-AuNPs for 3 h in cell culture incubator to allow complex adhesion to the cells. The cells were exposed to a pulsed laser with 60 mJ/cm² at a scanning speed of 0.5 cm/s. Subsequently, the cells were xed with 4% formaldehyde, washed with PBS, and stored for further processing. For SEM preparation, the coverslips were dehydrated with a graded series of ethanol completed with an acetone step prior to critical point drying with CO2 as an intermedium (Emitech K850 critical point dryer, Emitech/Quorum Technologies Ltd., Laughton, UK). The coverslips were at mounted on SEM-stubs with adhesive carbon tape (Plano, Wetzlar, Germany) and coated with a carbon layer (Leica SCD500, Leica Microsystems, Wetzlar Germany). Specimens were analyzed in a eld-emission SEM (Zeiss Merlin VP compact, Carl Zeiss Microscopy, Oberkochen, Germany) equipped with HE-SE and inlens-Duo detectors operated at 5 kV and images with a size of 1024 × 768 pixels were recorded at different steps of magni cation.

Statistical analysis
The results are given as mean of at least three independent experiments for quantitative real-time RT-PCR and cell killing using GNOM-LP. Statistical analysis was performed using SAS software 7.1 (SAS Institute Inc., Cary, NC, USA). Signi cant differences in gene expression of CLDN3, -4 and − 7 were calculated using Student's two-sided t-test. Statistical analysis of cell killing using GNOME-LP was performed using Dunnett´s Multiple Comparison Test and Student´s two-sided t-test. Differences were considered statistically signi cant for p < 0.05.

Generation of red uorescent tumor cell lines
Transfected cell lines DT0840-FusionRed and DT0846-FusionRed showed a distinct red uorescence 24-72 h post-transfection all over the cytoplasm whereas native DT0840 and DT0846 cells showed no FusionRed uorescence (Fig. 1A and 1C).
In order to quantify the amount of FusionRed positive cells, both uorescent cell lines DT0840 and DT0846 were compared to native DT0840 and DT0846 cells by ow cytometry. After 50 passages of geneticin selection, 95.6% and 70.5% of cells were found FusionRed positive in DT0840-FusionRed and DT0846-FusionRed respectively ( Fig. 1B and 1D).
In vitro imaging using NightOWL LB 983 in vivo imaging system Transfected DT0840-FusionRed and DT0846-FusionRed cells were further imaged in a NightOWL LB 983 in vivo imaging system to illustrate and quantify uorescent intensity in a whole body imager. Both cell lines showed comparable cell concentration dependent uorescent intensity. The lower limit of detection was 0.3125 × 10 6 cells per well with 2.64 average cps ± 3.734 and 4.485 mm² ± 6.343 for DT0840-FusionRed cell line. For DT0846 the lower detection limit was 0.625 × 10 6 cells per well with 5.69 average cps ± 0.339 and 19.08 mm² ± 5.629 (Fig. 2).

CLDN gene expression in transfected cell lines
Gene expression level of CLDN3, -4 and − 7 in transfected cell lines were examined by quantitative realtime RT-PCR and compared to the native cell lines. Level of CLDN4 expression in DT0840-FusionRed was signi cantly lower in comparison to the reference cell line (Fig. 3). In contrast, levels of CLDN3 and − 4 in DT0846-FusionRed was higher than those of the reference cell line.

CLDN protein immuno uorescence
The presence of CLDN3, -4 and − 7 proteins in native and transfected cell lines were subsequently examined by immunostaining. In DT0840 cell line, CLDN3, -4 and − 7 proteins showed a strong signal and were localized at the cell membranes and in the cytoplasm (Fig. 4). For the DT0840-FusionRed cell line, CLDN3, -4 and − 7 proteins were found at the cell membrane; CLDN7 was weakly expressed (Fig. 4).
Expression of CLDN3 and − 7 proteins in cell line DT0846 was localized at the cell membranes whereas CLDN4 was punctually localized in the cytoplasm and at the cell membrane. In the generated DT0846-FusionRed cell line, CLDN3, -4 and − 7 proteins were strongly distributed along the cell membranes (Fig. 4).
Binding of C-CPE to cell lines C-CPE's capability to target CLDN was determined through visualization of C-CPE-CLDN binding. The experiment was performed by coupling C-CPE was coupled to Strep-Tactin® Chromeo 488. The complex was detected along cell membranes of native and transfected cell lines at cell-cell junction between adjacent cells (Fig. 5).

Electron microscopy
In order to investigate the binding of AuNPs and C-CPE-AuNPs complexes on the cell surface, DT0846 and transfected DT0846-FusionRed cells were examined by scanning electron microscopy (SEM). AuNPs appearing as white bright spheres in high resolution SEM analysis were detected e.g. on microvilli extending from the cell surfaces of DT0846 and DT0846-FusionRed cells (Fig. 6). Uncoupled AuNPs showed a broad distribution at the cell surface while C-CPE AuNPs were found preferentially located in close distance to cell-cell borders. Whereas the presence of AuNPs and C-CPE-AuNPs on microvilli may indicate non-speci c surface binding, at places internalization of AuNPs was clearly visible (see Additional le 1).

Selective cancer cells ablation using GNOME-LP and C-CPE-AuNPs complex
Laser exposure of DT0840 native and transfected CLDN expressing cells in combination with C-CPE functionalized AuNPs signi cantly reduced amount of vital cells down to 32.73% and 26.86% respectively in comparison to untreated cells (Fig. 7). In native and transfected DT0846 cell lines in comparison to untreated cells, GNOME-LP in combination with C-CPE functionalized AuNPs signi cantly decreased cell survival to 8.55% and 5.52% respectively.
In cells treated with C-CPE alone, GNOME-LP application did not signi cantly impair cell survival. Application of GNOME-LP in presence of non-functionalized AuNPs reduced cells by 41

Discussion
In vivo models are the key to understand the pathogenesis of prostate cancer and the development of novel therapeutic approaches [2]. Although in vitro systems offer several possibilities for basic drug evaluation, they remain limited for the evaluation of complex interactions. The use of long-wavelength emitting uorescent proteins to label cancer cells enables deep tissue imaging; thereby allow real-time tracing of cancer growth, metastasis and determination of e cacy of candidate antitumor and antimetastatic therapies. An optimal uorescent protein for whole-body imaging should have excitation and emission spectra within 650-950 nm. This "near-infrared optical window" has the lowest absorbance by hemoglobin, melanin and water in mammalian tissue [9].
Herein, two canine prostate cancer cell lines, stably expressing red uorescent protein, were established. These cell lines can be detected in living animals using uorescence imaging systems. The generated cells have shown stable long time expression of red uorescent protein; therefore represent a valuable tool for studying canine prostate cancer in vivo models such as xenograft mice.
It is well documented that CPE receptors CLDN3, -4 and/or -7 are abnormally regulated in many tumor types [10-13, 49, 50], which also was con rmed for the used 0840 and 0846 cell lines [41]. CLDN3, -4 and − 7 expressions in generated uorescent cell line DT0840-FusionRed revealed no signi cant difference in comparison to native DT0840 for CLDN3 and − 7, however CLDN4 was signi cantly decreased. Analysis of CLDN7 in DT0846-FusionRed showed no difference in expression, whereas CLDN3 and − 4 were even higher expressed after transfection.
Immuno uorescence staining showed strong expression of all three CLDN proteins in all cell lines.
Therefore, DT0840-FusionRed still represents a su cient model for further experiments despite signi cant decrease in CLDN4 mRNA level measured by qPCR. Interestingly, immuno uorescence staining revealed that CLDN3, -4 and − 7 in DT0840 cells, as well as CLDN4 in DT0846, were punctually located in the cytoplasm. Such apparent miss-localizations were also described for the CLDN4 protein in human prostate cancer-derived cell lines and may be related to loss of cellular organization due to a defect in tight junction formation or cell polarity; features common in tumor cells [43].
Binding of CPE to CLDN3 and − 4 can trigger cell death [51][52][53][54]. Therefore, it proposed to use CPE for tumor therapy. However, studies in vivo revealed that systemic administration of full-length CPE in mice was toxic and thus limited its use to local therapies [52]. Previously we demonstrated that the noncytotoxic C-terminal domain of CPE, which preserves CPE's binding a nity to CLDN receptors, is capable to functionalize AuNPs [36]. Imaging of C-CPE binding to the canine tumor cell lines proved that the protein can speci cally target CLDN3, -4 and − 7 demonstrating that the functionalization did not alter the binding capacity to CLDN.
Further, electron microscopy images indicated that the C-CPE conjugated AuNPs retain the a nity to its receptors (CLDN3, -4 and − 7).
The GNOME-LP technology has been used for the cellular introduction of dyes as well as siRNA into different cell types via transient cell permeabilization [55][56][57][58]. The present report shows that C-CPE coupled to Strep-Tactin conjugated AuNPs in combination with GNOME-LP technique can be used for speci c targeting of CLDNs expressing tumor cell lines.
In the previous study, we showed that the energy power of the applied laser at 60 mJ/cm 3 and a scanning speed of 0.5 cm/s in combination with C-CPE-AuNPs reduced cell survival to less than 30% of claudin expressing cell lines [36]. In a rst experiment, GNOME-LP with the same settings accordingly reduced cell survival to about 30% in native DT0846 cells, but showed no effect on the transfected uorescence cells (data not shown). At 532 nm (laser wavelength) the red uorescent dye FusionRed has approx. 50% absorption (50% of dye molecules absorb light at 532 nm). Therefore, depending on dye concentration in the cells, a signi cant amount of laser light might be absorbed and thus reduced the overall effect on AuNPs. Therefore, GNOME-LP was applied at the maximal laser uence (72 mJ/cm 3 ) on native and uorescent cell lines. Using the new setting, GNOME-LP in combination with C-CPE functionalized AuNPs reduced cell survival to down to 30% in DT0840 and less than 10% in DT0846 (native and uorescent) cells.
Signi cant killing of DT0846 and DT0840 (native and transfected) cells treated with non-functionalized AuNPs may be related to endocytosis activity allowing them to internalize the AuNPs. However, the results show that the functionalization of AuNPs with C-CPE increases the ablation e ciency of CLDN expressing tumor cell lines in comparison to cells treated only with AuNPs. This interpretation is supported by SEM analysis showing the presence of many uncoupled AuNPs that are bound nonspeci cally on the cell surface microvilli even after three hours of incubation whereas fewer C-CPE functionalized AuNPs are present on the cell surface, mostly restricted along cell-cell borders. This suggests that C-CPE-AuNPs e ciently bind to their protein targets and are rapidly internalized into the cells as can be traced at places with SEM (see Additional le 1).
In the current study, the generated DT0840-FusionRed and DT0846-FusionRed cell lines showed stably strong red uorescence protein expression. This was further analyzed by NightOWL LB 983 in vivo imaging system, where the uorescent detection potential of FusionRed transfected DT0840 and DT0846 cell lines were veri ed. Small cell numbers were su cient to monitor the FusionRed labeled cells in the whole body imager [59]. For in vivo imaging studies a cell number from 1 × 10 6 cells per tumor is detectable with this setting already in early tumor stages [60]. Red uorescent proteins showed high sensitivity in subcutaneous, abdominal as well as deep tissue cell implantation. Thus, in vivo imaging with this stably transfected cell lines provides a tool for long-term imaging and monitoring longitudinal tumor development and drug response per individual mouse [61].
The results con rm for the rst time that the therapy concept of C-CPE functionalized AuNPs can be used e ciently against prostate carcinoma cells. By using GNOME-LP system and C-CPE functionalized AuNPs an irreversible laser ablation of prostate tumor cells was achieved in vitro. Cells, which were irradiated with maximal laser power without C-CPE-AuNPs, maintained viability. Likewise, cells incubated with C-CPE and irradiated with the maximal laser uence maintained viability as well. A combination of laser treatment and C-CPE-AuNPs, however, reduced tumor cell viability down to less than 10% in DT0846.
To further extend the presented in vitro ndings, in vivo studies need to be carried out in a next step. The same cell lines used for the in vitro ndings can also be detected in vivo through deep tissue imaging and therefore enabling to observe tumor growth and subsequently possible tumor ablation through C-CPE treatment in a living animal e.g. mouse xenograft models. In vivo studies could allow the characterization if the C-CPE complex is able to diffuse through the extracellular matrix and bind to tumor tissues. If successful, a combination between GNOME-LP and functionalized AuNPs may establish a treatment option for canine prostate cancer.

Conclusion
Page 13/23 The established cell lines and the veri ed proof of concept in vitro provide the basis for perspective xenograft in vivo studies. The introduced red uorescence enables deep tissue imaging in living animals and therefore detailed characterization of tumor growth and subsequently possible tumor ablation through C-CPE-AuNPs treatment.
Since dogs represent an excellent model for prostate cancer, the development of therapeutic strategies provides an important contribution to translational research directed to treat humans, thus providing bene t for both species. Availability of data and materials All data generated or analyzed during the current study are included in this publication and its supplementary information les without restriction.

Competing interests
The authors declare that they have no competing interests.    Expression of CLDNs protein in native and transfected tumor cell lines. Cells were subjected to immunostaining with corresponding antibodies using uorescein-conjugated secondary antibodies, showing red signals for CLDN3 and -7 and green signals for CLDN4. DAPI was used for blue nuclei staining. Images were observed under confocal microscopy. Arrows indicate CLDN localization on cellcell contact. Scale bars = 20 µm.