Lalani, El-Nasir Mussa Ahmed; (1996) Development of syngeneic and transgenic mouse models to evaluate immune responses against the human MUC1 gene product. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A Balb/C syngeneic mouse adenocarcinoma cell line (410.4) and a fibrosarcoma cell line (L-cell) derived from aC3H mouse were both transfected with different constructs : a) a full length human MUC1 cDNA, b) a genomic EcoR1 MUC1 fragment, c) MUC1 cDNA devoid of the transmembrane region. Broadly, the resultant transfectants (epithelial) expressed the MUC1 gene product (Polymorphic Epithelial Mucin - PEM) either as a membrane associated and cyloplasmic form or secreted, membrane associated and cytoplasmic form. In addition the PEM variants expressed by the transfectants (based on core protein monoclonal antibody immunoreactivity) were similar to PEM expressed by either normal glandular epithelial or PEM expressed by neoplastic cells. The genomic epithelial transfectants (E3 and E4) which expressed PEM on the cell surface and cytoplasm were grown in syngeneic Balb/C mice at different inocula. At a low inoculum of 104 cells, the parental cell line 410.4 was found to induce tumours with an efficiency of 66%, and the 410.4 hygro-induced tumours with an efficiency of 100%. At the higher innoculum of 105 cells all the mice developed tumours. The incidence of tumour formation by the E3 transfectants at 104 and 105 was significantly reduced to only 7.5% and 40% respectively and by the E4 transfectants to 17.5% and 60%. A statistical analysis (non-paired t test) comparing the frequency of tumour development between the four cell lines at the 105 cell inoculum demonstrates that there is a significant delay in tumour growth of the transfected clones (p≤0.001) compared to the controls. The full length cDNA and the transmembrane deleted cDNA transfectants did not show statistically significant delay in growth. In addition the genomic transfectants (E3 and E4) when used as immunogens at low inocula of 104 conferred immunity to further challange by a higher inoculum of E3 and E4 cells. Use of allogeneic L-Cell transfectants as immunogens in Balb/C mice did not result in an immune response sufficient to protect mice against a challenge by syngeneic MUC1 transfectants. A 40 kilobase pair (kbp) Sall-Cla1 and a smaller Sac11 10.6 kbp fragment of a cosmid clone GPEM-1, were used to generate transgenic mice. Three transgenic mice containing the 40 kbp fragment were identified one of which did not transmit and one of two transgenic mice carrying the 10.6 kbp fragment transmitted. Detailed immunohistochemical analyis for the expression of PEM in organs from mice which transmitted, demonstrated tissue specific expression identical to that observed in humans. The development of both the syngeneic and transgenic mouse models offer excellent in vivo systems to analyse immune responses against PEM. Studies utilising these models will enhance our understanding of cancer immunology and help develop therapeutic modalities in combating cancer.

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