Modification of the red fluorescent protein TagRFP to reduce its immunogenicity in establishing fluorescent model tumors in immunocompetent Balb / C mice

Background. Long-wave fluorescent proteins (FP) exhibit significant potential for in vivo investigations involving fluorescent tumor models in laboratory animals. If tumors expressing FP exhibit enhanced immunogenicity in a specific strain of immunocompetent mice as compared with the original tumors, it becomes challenging to delineate the immune component contributing to the antitumor effects of cytotoxic therapy.

Aim. To establish mouse tumor cell lines 4T1 that consistently express the original protein TagRFP and the mutant protein TagRFP-L228A, to develop tumor models based on these cell lines, and to assess the humoral immune response of Balb / c mice to such tumors.

Materials and methods. Eukaryotic plasmids pcDNA3 containing genes encoding the expression of the original protein TagRFP (TagRFP-WT) and the L228A mutant protein (TagRFP-L228A) were obtained from prokaryotic plasmids by genetic engineering methods. Individual 4T1 tumor cell clones expressing TagRFP-WT and TagRFP-L228A were obtained by sequential liposomal transfection of cells with eukaryotic plasmids, selection, and cloning. Tumor models were obtained by subcutaneous inoculation of suspensions of 4T1, 4T1-TagRFP-WT, and 4T1-TagRFP-L228A cells into female Balb / c mice. After 4 weeks, the immune response to TagRFP protein was evaluated by enzyme-linked immunosorbent assay (ELISA) by binding of mouse venous blood sera to the parent TagRFP-WT protein.

Results. Plasmids for the expression of TagRFP-WT and TagRFP-L228A proteins in eukaryotic cells and 4T1 cell clones stably expressing these proteins have been obtained. After inoculation of 4T1-TagRFP-WT, 4T1-TagRFP-L228A and 4T1 cells, tumors developed in all mice of the corresponding groups. Using an ELISA on microplates with adsorbed TagRFP-WT protein, the presence of a humoral immune response to the original fluorescent protein was shown in Balb / c mice with fluorescent tumors, in contrast to mice with 4T1 tumors. Compared to the sera of mice with the 4T1-TagRFP-WT tumors, the binding to the original protein TagRFP-WT of the sera of mice with the 4T1-TagRFP-L228A tumors decreases by more than 4 times.

Conclusion. The single amino acid substitution L228A in the TagRFP protein leads to a significant reduction in the humoral immune response to the TagRFP-L228A protein expressed by tumors compared to the original TagRFP-WT. This will allow using TagRFP-L228A expressing cell lines to create optimized tumor models for Balb / c mice.

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