Community Research and Development Information Service - CORDIS


FIGHT-CANCER Report Summary

Project ID: 617978
Funded under: FP7-IDEAS-ERC
Country: Italy

Mid-Term Report Summary - FIGHT-CANCER (Long non-coding RNAs of tumor infiltrating lymphocytes as novel anti-cancer therapeutic targets)

Although tumor tissues can be infiltrated by T cells specific for tumor antigens, the effector functions of these lymphocytes are generally suppressed by CD4+ regulatory T cells (Tregs). Since tumor infiltrating Tregs can display function heterogeneity, depending on both the tumor type and the inflammatory milieu, only inhibition of the right Tregs activity should result in the unleash of an effective anti-tumor T cell responses. To identify the Tregs that truly inhibit anti-tumor T cells, we will profile by RNA-Seq the transcriptome of Tregs infiltrating both tumor and healthy tissues. In particular, we will focus on long non-coding RNAs (lncRNAs) and the gene networks they modulate, since they have recently emerged as relevant epigenetic regulators of cell differentiation and identity.
We will exploit this new knowledge to create a panel of regulatory transcripts, which will be assessed at single cell level on tumor infiltrating Tregs, so to determine the association of specific transcripts with different Treg populations. Moreover, we will develop new molecules composed by an aptamer, single stranded oligonucleotides that bind to cell surface markers, and a siRNA (Aptamers-siRNA-Chimeras) that specifically target lncRNAs of interest (with either positive or negative effects on antitumor T cell responses) as novel antitumor therapeutic targets. This project will provide new knowledge on tumor infiltrating Tregs possibly allowing definition of molecular signatures of Tregs whose modulation may not only provide new perspectives for anti-tumor therapy but more in general may be relevant to any immunomodulatory therapeutic strategies.

Since the project started we isolated different CD4+ lymphocytes subsets from bot healthy donors and two different tumors, NSCLC and CRC, from the normal tissue adjacent to the tumors, and from peripheral blood samples. From all these tissues, we purified CD4+ Treg (36 samples), Th1 (30 samples) and Th17 (22 samples) cells. We have extracted total RNA from these samples, RNA-seq analysis has been performed on NSCLC and CRC tumor-infiltrating lymphocytes and data have been analysed to define specific gene signature for tumor infiltrating lymphocytes.
We have set-up procedures for single-cell RNA-sequencing protocol using both the Fluidigm C1 microfluidic platform and the TenX Genomics drop-sequencing platform along with the generation of the relevant bioinformatics analysis pipelines.
We also characterized a CD4+Th1 specific lincRNA- lincMAF4 and showed its expression contributes to the definition of CD4 Th1 cells identity. Other Treg cell specific lincRNAs are under investigation and tools to knock out the lincRNA genes in Treg cells are under development (based on the CRISPR-Cas9 System)
Concerning the task aimed at the identification of therapeutic targets for the modulation of immune response we have started the set-up of cell SELEX procedure to identify specific aptamers against CTLA-4. Moreover we are interrogating the RNA-seq data on tumor infiltrating Treg cells to identify membrane proteins that might be expressed with high specificity by these cells and can be used as target for Tregs-specific aptamers generation.

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