The role of fibroblast ENPP1 expression on anti-tumour immunity
Keywords: cGAS-STING
ENPP1
oesophageal adenocarcinoma
T cell
ECM
2'3'-cGAMP

Background:
Highly chromosomally unstable (CIN-high) tumours such as oesophageal adenocarcinoma (OAC) chronically activate the cGAS-STING pathway to produce immunostimulatory 2’3’- cGAMP. This cyclic dinucleotide is exported from tumour cells and can induce type I interferon secretion by surrounding stromal and immune cells. Ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1), the only known 2’3’-cGAMP regulator, is a transmembrane glycoprotein involved in the extracellular hydrolysis of 2’3’-cGAMP, thus preventing its paracrine signaling and STING-dependent anti-tumorigenic activity. Our lab has previously reported that CIN-high cancers upregulate ENPP1 expression to create a paradoxical immunosuppressive microenvironment through the conversion of 2’3’-cGAMP- derived AMP to adenosine (Li J. et al, 2021). Now, we have identified that fibroblasts, which are found abundantly in desmoplastic solid tumours, express significantly higher ENPP1 when compared to tumour or immune cells. The significance of fibroblast ENPP1 expression on tumorigenesis remains unexplored and may impact immune infiltration. Here, we study the regulation of fibroblast ENPP1 expression and its immunomodulatory functions in the tumour microenvironment.

Method:
Patient-derived cancer-associated fibroblast (CAF) cell line was previously generated in Parkes Lab. First, the expression of CAF markers, cGAS and other 2’3’-cGAMP regulators in this cell line was studied by immunoblot analysis. Novel CAFs were then treated with tumour-derived signals such as transforming growth factor beta (TGF-β) and 2’3’-cGAMP to identify factors that could potentially regulate fibroblast ENPP1 expression, measured by immunoblot analysis, tissue microarray (TMA) analysis and confocal microscopy. Normal human (TIG-1) fibroblasts were used as a control. RNA sequencing was also performed on patient-derived OAC CAFs to identify differentially expressed genes and enriched functional pathways. Subsequently, ENPP1 expression in fibroblasts was modulated using lentivirus. ENPP1KO and ENPP1OE fibroblast lines were then co-cultured with ex-vivo human immune cells isolated from patient blood to identify the role of ENPP1 in immune responses.

Results:
SORB81 fibroblasts highly express established CAF markers (α-SMA, FAP, vimentin, PDGFR-α) and ENPP1. Compared to normal fibroblasts, SORB81 fibroblasts show a TGF-β- responsive, α-SMAhigh, PDGFRlow phenotype consistent with a tumour-proximal, myofibroblast-like CAF (myCAF) profile. SORB81 fibroblasts upregulated ENPP1 in response to extracellular 2’3’-cGAMP in a dose-dependent manner. The mechanism of CAF 2’3’-cGAMP sensing and transport, as well as the importance of intracellular/extracellular 2’3’-cGAMP gradient in its signalling require further investigation. Transcriptomic analysis of patient-derived CAFs show that multiple matrisome and matrisome-associated pathways were enriched in CAFs compared to normal fibroblasts. Of the 26 genes that were differentially expressed in CAFs, 8 were genes encoding for known matrix proteins such as MMP3, ADAMTS8 and CD36 with reported functions in extracellular matrix (ECM) regulation, secretion, proteoglycan and glycoprotein synthesis. These findings suggest that CAFs could additionally play a role in ECM reprogramming through the production of pro-tumorigenic matrix proteins. Co-culture experiments also show that CAFs could alter T cell responses in a contact-independent fashion.

Conclusion:
MyCAFs in OAC could be potential drivers of immune exclusion from the tumour microenvironment through ENPP1-mediated T cell immunomodulation and dysregulated ECM remodeling to create immune-exclusive niches in the tumour stroma.