Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model

Figures

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  Figure 1. In vivo efficacy of the combined treatment with TZ and HFn-DOX. (A) Tumor progression in D2F2/E2 tumor bearing BALB/c mice (n = 15/group) treated with placebo, HFn-DOX or DOX (1 mg/Kg of DOX, i.v.), TZ (5 mg/Kg, i. p.) and with the combination of them (HFn-DOX + TZ or DOX + TZ). (B) Tumor volume measured at day 24. Plot reported individual values ± s.e. Statistical significance vs placebo *P < 0.05; ***P < 0.005. (C) Immunofluorescence with cleaved caspase 3 antibody (red) on histological slides of OCT embedded tumors excised at day 24. Magnification 40 ×. Nuclei have been labelled with DAPI (blue) and cellular membranes with Alexa Fluor488-Wheat Germ Agglutinin (green). Scale bar = 30 µm. (D) Quantification of apoptosis in tumor tissue upon treatment (n = 8/group). Reported values are the mean of apoptotic cells number/field/sample ± s.e. The count was performed on 10 fields/sample. Statistical significance vs. placebo ***P < 0.0005; ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.005. (E) Quantification of western blot analysis performed on tumors excised at the end of treatment to quantify granzyme release. Granzyme was normalized on tubulin using ImageJ Software. Reported…

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  Figure 2. Analysis of HFn-DOX and TZ mitochondrial cardiotoxicity. Hearts from treated mice were fixed with glutaraldehyde and embedded in epoxy-resin. At least 10 TEM images of ultrathin heart sections have been acquired at 4,200 and 11,500 magnifications for each experimental group. Quantification of mitochondria area and area occupied by cristae were performed on at least 10 images/group, measuring at least 100 mitochondria/sample, while count of mitochondria number were performed on at least 10 images/group. Values represent the mean mitochondria number ± s.e. (A), the mean mitochondrial area ± s.e. (B) and the percentage of mitochondrial area occupied by cristae ± s.e. (C). Statistical significance vs. Placebo ****P < 0.0001; vs TZ ####P < 0.0001 (Kruskal-Wallis test). (D) Representative images of mitochondria from hearts excised at day 24 (n = 3/group) from treated mice acquired at 11,500 magnifications evidenced mitochondria morphological alterations.

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  Figure 3. TZ quantification in heart and tumor tissue. Western blot analysis of hearts (panels A and B) and tumor (panels C and D) excised at the end of treatment to quantify TZ. (A,C) Representative results of western blots performed to quantify TZ, constituted by cropped western blots revealed with anti-human IgG HRP antibody and anti-GAPDH, respectively. (B,D) TZ quantification in heart and tumor, respectively. TZ was normalized on GAPDH using ImageJ Software. Reported values are the mean of 3 samples ± s.e. Statistical significance vs. TZ, *P < 0.05, **P < 0.01, ***p < 0.005. (E) Representative images of confocal microscopy analysis of TZ penetration in tumor from mice treated with placebo,TZ alone or in combination with HFn-DOX or DOX. DAPI signal is white, while TZ is labelled in red. Scale bar = 500 μm. (F) Analysis of spatial distribution of TZ fluorescence signal. Quantification of mean fluorescence intensity (MFI) of anti-Trastuzumab staining along the tumor axis from the edge to the geometric center (mean ± s.e [s.e. bars in grey color]; n = 3 tumor mosaics/staining; statistical comparisons between measurement points are shown). The black line represents the calculated average…