To Investigate The Effects Of Recombinant Interferon-Gamma (IFN-Y) In A Murine Breast Cancer Model

Abstract

Advances in the understanding of tumour biology have allowed targeted therapies such as immunotherapy against specific molecular targets to develop. Interferon gamma (IFN-ƴ), the sole Type II interferon cytokine, is a pleiotropic cytokine, is apleiotropic cytokine endowed with potent immunomodulatory effects on a variety of immune cells in vitro and in vivo. IFN-ƴ has been used to treat several diseases and malignancies in severely immunocompromised patients including chronic granulomatous disease and osteopetrosis and leukemia. However, there are a few key elements of the immune system of a tumour microenvironment such as metastatic tumour biology, cytokine and antigen-specific lymphocyte production, and the role of T-regulatory (Treg) cells, that need further understanding in order to develop an effective therapeutic agent to combat diseases such as breast cancer.

The work described in the study aimed to develop a functional BALB/c murine model of breast cancer and to approximate the time of metastatic onset, to investigate the therapeutic effects of IFN-ƴ in the murine breast cancer model, to elucidate the various immune responses and immunomodulatory effects following metastasis in the breast cancer model and therapeutic administration of IFN-ƴ, to study the roles of various cytokines and phenotypic cell surface markers in the growth and spread of breast cancer of breast of breast cancer in model and to determine the ratio of T-regulatory cells to helper cells (Treg:Th) in a metastatic breast cancer murine model and following therapeutic administration of IFN-ƴ.

For the in vitro work, effect of IFN-ƴ and tumour necrosis factor alpha (TNF-α)treatment on 4T1 cells was assessed using MTT assay to elucidate the viability of the cells in the presence of the cytokines, which may shed light on their anti-tumoural and anti-angiogenic properties. Production of IFN-ƴ, TNF-α and GM-CSF were assessed using ELISA to understand their role in the growth and progression of 4T1 Tumour cells. Cell surface markers such as MHC Class II, CD95 and CD95l were analysed using flow cytometry to evaluate their role of immune response induction in the microenvironment of tumour cells. IFN-ƴ and TNF-α proved to be efficient therapeutic agents when administered to 4T1 cells, with 37.42% and 51.68% of cells inhibited when exposed to highest concentration of IFN- ƴ at 20pg/mL and highest concentration of TNF-α 20pg/mL respectively. 4T1 cells elicited highest production of TNF-α at 176.95pg/mL, IFN-ƴ at 86.98pg/mL and the least with GM-CSF at 64.36pg/mL. No expression of MHC Class II, CD95 and CD95L were detected in 4t1 Cells. Our study has successfully characterized various immune responses elicited in the tumour microenvironment, induced by the tumour cells alone and also with prior stimulation of certain cytokines.

For the metastasis and IFN-ƴ treatment study, six-week-old female BALB/c mice were inoculated with 4T1 murine breast cancer cells. The treatment group were administered with 40pg/mL. IFN-ƴ every other day following the day the tumour was palpable. Mice weight and primary tumour mass volume were regularly recorded to study the physical effects of a vigorously growing and spreading of cancer cell line. Gross and histological studies were carried out to determine the approximate day of metastatic onset. Production of IFN-ƴ and TNF-α was assessed by ELISA to understand its role in tumour growth and metastasis. Production of antigen-specific T Cells were analysed through mixed lymphocyte reactions, and this was to elucidate immunological response following tumour invasion. Lymphocyte markers such as CD4⁺,CD8⁺,CD16⁺and CD19 were analysed to elucidate its role in tumour growth and progression. Role of Treg cells were envisaged to understand the immune escape mechanisms employed by these cells and their immunosuppressive effects in a tumour model. Our study showed that the metastatic onset occurs approximately 14 days after the mice were inoculated with 4T1 murine breast cancer cells. Gross studies showed hepatosplenomegaly and slight congestion in the spleen. The breast cancer cells from primary tumour were found to spread rapidly to the liver on day 14. Following IFN-ƴ treatment, the size of primary tumour was reduced by 76% and there was less invasive metastatic clusters seen in liver sections. IFN-ƴ production and TNF-α was slightly higher in inoculated mice serum and splenocytes compared to the treatment group. Production of antigen-specific T Cells in the treatment group was higher than the untreated breast cancer-laden group. CD4⁺and CD16 were seen to be higher in the breast cancer-laden mice compared to control mice and CD8 and CD19 production were regressed in the experimental group CD4⁺,CD8⁺,CD16⁺and CD19 were slightly elevated in the treatment group than the untreated breast cancer-laden group, but the numbers towered those of control group. Elucidation of the factors underlying breast cancer progression and metastasis has been tremendously fruitful from mouse models in which multiple stages of tumour progression are further confirmed. We have successfully envisaged that the 4T1 murine breast cancer cells can migrate and metastasise rapidly to the liver., eliciting various immune responses, and the therapeutic administration of IFN-ƴ was effective in arresting the growth of primary tumour and a retardation of metastasis, also eliciting potent immune responses.