Characterising purified tumour Autophagosomes for their potential to generate citrullinated epitopes

Abstract

There is increasing evidence that citrullinated peptide widely generated in tumours in an autophagic dependent pathway, this generation can potentially induce an antitumour immune response. Therefore, potentially enhancing the efficacy of therapeutic cancer vaccines. The citrullination process likely to be mediated by peptidylarginine deiminase (PAD) enzymes and autophagosomes are likely to be the source of these citrullinated peptides. To this end, we investigated the enrichment of autophagosome from B16F1 tumour cells using density gradient centrifugation. The results showed that density gradient centrifugation has enriched the autophagosomes in specific fractions. Western blot analysis showed that microtubule- associated protein 1A/1B-light chain 3 (LC3) are highly expressed in cancer cells indicating that autophagy is always taking place in the tumour microenvironment. In addition, PAD2 was expressed in cancer cells, resulting in new citrullinated peptides. Taken together, our findings revealed that autophagy in tumour cells could be further induced by rapamycin. Further, autophagosomes fusion to lysosomes could be blocked by chloroquine, resulting in the formation of more autophagosomes. Based on this, we recommend that autophagosomes of cells treated with both rapamycin and chloroquine could be enriched to investigate the presence of citrullinated peptides. Then, measuring the ability of citrullinated peptides to induce an immune response and if they could be used to design new therapeutic cancer vaccines. Keywords: Autophagy, autophagosome, PAD enzymes, peptides, B16F1, rapamycin, chloroquine, LC3