Abstract
Colorectal cancer (CRC) is the second-leading cause of global cancer related death, with New Zealand having one of the leading rates of increased diagnoses in young adults (<50 years old). While public screening programs have improved the prognosis of individuals >60 years old, screening is not available to the general population <58 years old, thus survival outcomes are dictated by the quality of treatment they receive. Immunotherapeutics have largely improved patient outcomes across various solid tumours. They function to enhance a patient’s existing anti-tumour immune response to either induce immune-mediated tumour destruction or increase the efficacy of adjuvant therapy. However, the lack of an active immune response in most CRC cases renders CRC patients largely unresponsive to immunotherapy.
A key driver of the immune suppression in CRC is the tumour microenvironment (TME), a heterogenous composition of non-malignant cells and stroma that buffer the tumour from the surrounding normal tissue. Here, tumour associated macrophages (TAMs) have a critical role, as their highly plastic phenotypes are influenced by the tumour to support tumorigenesis – notably, by promoting immunosuppression in the TME. Long non-coding RNAs (lncRNAs), which are functional RNA molecules >200 nucleotides, contribute to the regulation of dynamic TAM activity. The highly specific expression patterns of lncRNAs render them attractive therapeutic targets in colorectal TAMs. Here, antisense oligonucleotides (ASOs) are a promising therapeutic modality, as they have previously been used to target ncRNAs in tumour models and are employed clinically against mRNA.
Therefore, this research aimed to investigate the impact of ASO-mediated lncRNA knockdown in colorectal TAMs, a novel strategy to improve the response of CRC to immunotherapy. 12 candidate lncRNA targets were identified from sequenced CRC patient TAMs, and prioritised to identify the most promising oncology targets, for which ASOs were designed – KCNQ1OT1, MIR4435-2HG, and MALAT1. These ASOs were tested in the previously optimised transfection system, the HCT116 CRC cell line. In HCT116 cells, the MALAT1-targeting ASO induced a significant knockdown in MALAT1 expression, 50.28% (P < 0.005), but did not affect cell proliferation. No knockdown of KCNQ1OT1 was observed, and MIR4435-2HG could not be detected by RT qPCR. To assess the impact of MALAT1 knockdown on macrophages, an in vitro primary macrophage model was established from peripheral blood mononuclear cells from multiple human donors. Effective gymnotic ASO uptake was observed, as well as a dose-dependent reduction in MALAT1 expression, which was significant in all but one treatment group (P < 0.05). Two markers of macrophage activity, IL-10 and HLA-DRA, were assessed with qPCR in macrophages treated with MALAT1-targeting ASO compared to a non-targeting ASO control, but no change in these markers was observed.
These results highlight the challenges of therapeutically targeting lncRNAs, where improved lncRNA structural predictions are critical to enhance ASO design pipelines, which may improve the efficacy of future lncRNA-targeting ASOs. The novel work in the PBMC-derived macrophage model provides the foundation for MALAT1 ASO knockdown in a 3D multicellular co-culture model of the colorectal TME.