الفهرس 
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Abstract
Immunotherapy has become a promising method for cancer treatment as it has some benefits in breast cancer treatment over traditional chemotherapies and molecularly targeted agents. Transforming growth factor beta (TGFβ) signaling pathway has diverse roles in each of the following: regulating cell growth, differentiation, apoptosis, motility as well as invasion, extracellular matrix production, angiogenesis, and immune response.
So, the aim of the present work was to study the effect of monoclonal anti- TGFβ antibody on the malignant mammary tumor implanted in Swiss albino mice using histopathological, histochemical and immunohistochemical techniques.
Sixty female mice were randomly divided into 5 groups; control mice (n=10) were administered PBS as vehicle and experimental control mice (n=10) were received 0.1 mg/kg body weight of anti -TGFβ antibody dissolved in PBS in 2 doses weekly for 4 weeks either IM or IP. Mice of protective group (n=10) were injected with anti -TGFβ antibody either IM or IP in the same dose and time schedule as experimental control mice before EAC implantation.
Thirty mice were implanted with EAC in a dose of two million cells in 0.1 ml PBS in right thigh muscle; 10 mice were served as Ehrlich group and twenty mice were divided equally after 10 days of tumor implantation and treated with anti- TGFβ antibody either by IM or IP administration in the same dose and time schedule as experimental control group & protective group.
Ten mice of treated group were sacrificed after 1 week of treatment and the other 10 after 4 weeks. Five mice of Ehrlich group and mice of protective group were sacrificed 10 days post tumor implantation. The other 5 mice of Ehrlich group in addition to mice of control and experimental control groups were sacrificed at the end of experiment.
The results of the current study could be summarized as following:
1-Tumor size:
The collected data revealed statistical significant reduction in mean tumor sizes of mice after IM administration in protective group (0.08±0.03 (as compared with IP administration (0.17 ± 0.03) and Ehrlich group (0.21 ± 0.09) 10 days post tumor implantation. No significant difference in tumor sizes was found between treated groups after 4 weeks of treatment and Ehrlich group at the end of experiment.
2-Histopathological findings:
Hematoxylin and Eosin (H&E):
Histopathological observations showed focal degenerative changes and scattered inflammatory cellular infiltrations in muscle tissue of experimental control mice IM treated with anti-TGFβ antibody. Preserved bundles structures were seen in muscles of control mice as well as experimental control mice IP treated with anti -TGFβ antibody. Tumor sections of Ehrlich group either post 10 days of tumor implantation or at the end of experiment showed discohesive sheets of malignant cells with large pleomorphic nuclei and abundant cytoplasm.
Tumor sections in protective group revealed necrotic areas with separating sheets of malignant cells after IM administration and large area of necrosis in addition to diminution of malignant cells after IP administration.
Tumor sections of treated group revealed increased eosinophilia, single scattered apoptotic cells and nuclear alteration after 1st week of IM administration and no changes were observed in features of malignancy in IP administrations. After the 4th week of treatment tumors sections revealed area of necrosis after IM administration and the dense eosinophilia in addition to fibrosis after IP administration.
3-Histochemical findings
Masson’s trichrome stain
Muscle sections in IM administration in experimental control mice stained with Masson’s trichrome revealed high collagen deposition in striated muscle bundles in comparison with IP administration and control muscle sections. Stroma with excessive collagenous deposition was seen post 10 days of tumor implantation in Ehrlich group. Protective group after IP administration of anti -TGFβ antibody revealed less noticeable deposition of collagen in comparison with IM administration as well as the Ehrlich group post 10 days of tumor implantation. After the 1st week of treatment, tumor sections revealed moderate stained fibrous stroma tissue in IM administration and abundant stromal inflammatory reaction in IP administration. After the 4th week of treatment, tumor sections showed less collagen content in IP administration and no obvious changes in IM administration in comparison with Ehrlich group at the end of experiment.
Image analysis to calculate area percentage of collagen deposition showed that control group revealed lower significant mean area (2.06 ± 0.72) in comparison with IM administration in experimental control group (10.20 ± 3.24) and no significant value as comparing with IP administration (4.31 ± 1.98). Ehrlich group at the end of experiment showed mean area% with higher significant difference (42.18 ± 1.80) in comparison with both IM and IP administration in experimental control mice. Significant increase in area percentage was observed in IM administration in protective group (35.26 ± 3.62) comparing with Ehrlich group 10 days post tumor implantation (21.06 ± 2.36) and no significant difference was seen as compared with IP administration (18.42 ± 1.77). Insignificant increase was observed in IM administration in treated group after the 4th week of treatment (44.07 ± 2.46) comparing with Ehrlich group (42.18 ± 1.80) at the end of experiment. Lower significant difference was encountered comparing IP administration in treated group after the 4th week of treatment (32.22 ± 3.70) with Ehrlich group at the end of experiment.
4- Immunohistochemical findings:
Caspase-3 intensity
Immunohistochemichal assessment of caspase-3 expression in mice tumor sections was detected as intensity of brown staining color in cytoplasm of tumor cells and intense staining color in necrotic area. Muscle thighs of experimental control and control mice indicated negative caspase-3 immune reaction. Ehrlich group at the end of experiment revealed intense immune reaction compared with treated groups at the same time. Treated z
group IM administered with anti-TGFβ showed less caspase-3 intensity comparing with IP administration. Only necrotic area in protective group revealed high caspase-3 intensity after IP administration of anti-TGFβ antibody.
Caspase-3 color intensity was calculated by image analysis. It revealed insignificant decrease difference between mean caspase-3 intensity of Ehrlich group (164.35 ±2.79) post 10 days of tumor implantation and IM administration in protective group (164.13 ± 3.76), as well as a significant decrease (131.66 ±9.34) with IP administration. A significant increase in mean intensity between Ehrlich group (179.07 ± 4.09) at the end of experiment and IM administration in treated group (168.32 ± 3.07) after the 4th week of treatment. Insignificant increase comparing IP administration in treated group (180.69 ± 2.84) after the 4th week of treatment with Ehrlich group (179.07± 4.09) at the end of experiment.
Conclusions:
The role of TGFβ either as a tumor promoter or suppressor at the level of cancer cell is a controversial subject, owing to its differential effects at the early and late stages of carcinogenesis. At microenvironment level, TGFβ adds to generating a fortunate microenvironment for tumor growth and metastasis during all the stages of carcinogenesis, therefore the inhibition of TGFβ signaling could be a developing plan for cancer therapy at different doses and durations of the monoclonal anti-TGFβ antibody other than used one in the present study especially if combined with other therapy type that has less inflammatory effect on the histological level.