Background
The Chromosome 19 MicroRNA Cluster (C19MC) is a group of 46 microRNAs that span approximately 100 Kb of the human genome. C19MC is exclusively expressed in the placenta, regulating trophoblast migration and acting as an antiviral mechanism, but can become overexpressed in human cancers.1 Additionally, C19MC has been hypothesized to suppress maternal immunity to protect the developing fetus.2 We hypothesize that cancer cells that overexpress C19MC exploit this function to evade anti-tumor immunity. Therefore, we are investigating how C19MC-overexpression can facilitate immune evasion to bolster oncologic fitness.
Methods
A tri-lentiviral infection of a CRISPRa (‘Activation’) system was used to develop a C19MC overexpressing melanoma cell line (526-C19), as well as a nontargeted matched control (526-NT). The parent 526 cell line was used specifically due to the availability of an HLA-A-matched (MCC15781) tumor infiltrating lymphocytes (TILs) that are reactive to the melanoma cell line. C19MC overexpression was confirmed using a TaqMan RT-PCR panel. Immune cell killing assays were analyzed on the Incucyte SX5. Antigen presentation rates on cancer cell lines were identified using a flow panel of HLA class I and class II pan-antibodies and transcriptional analysis of these cells was performed using SYBR RT-PCR. No-contact co-cultures were performed using 0.6 um Transwell plates or exosome-only media. TIL proliferation was analyzed using CellTrace Violet, survival was analyzed using Caspase GLO reagent, and activation was read through quantitative ELLA.
Results
The C19MC overexpressing 526-C19 cells exhibited reduced TIL cytotoxic activity compared to 526-NT in direct co-culture experiments (80% vs. 60%, p = 0.0380) but showed no difference when cultured alone. C19MC overexpression produced C19MC miRNAs were found in greater abundance in 526-C19 exosomes than in the control. In no-contact co-culture experiments, we observed increased intracellular levels of C19MC microRNAs in TIL that were co-cultured with 526-C19 cells relative to those co-cultured with 526-NT cells, suggesting transference of C19 miRNAs between cancer cells and TIL via exosomes. TIL co-cultured directly or indirectly with C19MC overexpressing cancer cells demonstrated a less proliferative and less activated phenotype.
Conclusions
C19MC overexpression leads to decreased T cell-mediated cell death and diminished T cell fitness in a melanoma cell line co-cultured with HLA-matched TIL, suggesting that this microRNA cluster may contribute to immune tolerance when overexpressed in cancer.
References
Setty BA, Jinesh GG, Arnold M, et al. The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss. PLoS Genet 2020;16(4):e1008642.
Bullerdiek J, Flor I. Exosome-delivered microRNAs of ‘chromosome 19 microRNA cluster’ as immunomodulators in pregnancy and tumorigenesis. Mol Cytogenet 2012;5(1):27.
Ethics Approval
MCC15781 was approved by USF IRB approval number Ame5_107905.