The impact of NDRG1 overexpression on the immunological and metabolic reprogramming of the pancreatic tumour microenvironment

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive, with no effective treatments for the 80% of patients that are diagnosed at an advanced stage. A major hurdle in treating PDAC is the extensive tumour microenvironment (TME) which facilitates resistance to all current therapies. N-myc downstream regulated 1 (NDRG1) is a metastasis suppressor that was found to inhibit tumour progression and metastasis in PDAC. Recent studies also suggest that NDRG1 reduced PDAC-mediated activation of pancreatic stellate cells (PSCs), although the mechanisms behind this remain to be established. Our studies investigated the effect of NDRG1 on PDAC metabolism and how this influences key TME elements including tumour-associated macrophages (TAMs) and PSCs. We generated PDAC cells (MIAPaCa-2 and PANC-1) that stably overexpress NDRG1 and performed extensive metabolomic, proteomic and secretome analysis under normoxia and hypoxia. Using conditioned media or direct 3D spheroid cocultures, we assessed the effect of PDAC cells on THP-1 and U937 monocytes and primary PSCs using flow cytometry, Seahorse metabolic analysis, western blot and immunofluorescence analysis. The findings indicated that NDRG1 expression profoundly affected the metabolism of cancer cells, which led to significant changes in both the immune and fibroblast components of the TME. In cancer cells, NDRG1 reduced the uptake of branched‐chain amino acids (BCAA) leading to inhibition of the mTOR pathway. The secretome of PDAC cells, including exosomes, cytokines and chemokines was also altered by NDRG1. Specifically, NDRG1 increased secretion of TNF-α, while reducing CCL2 and TGF-β production by PDAC cells. This led to re-programming of TAMs from an anti-inflammatory M2 phenotype to a pro-inflammatory M1 phenotype and altered TAM metabolism. NDRG1 expression in PDAC cells also markedly influenced the metabolic cross-talk with PSCs, leading to increased infiltration of M1 polarized TAMs into PDAC/PSC co-culture spheroids. We demonstrate that NDRG1 is highly involved in regulating PDAC metabolism, significantly altering metabolic cross-talk with PSCs and leading to extensive “re-programming” of TAMs into the M1 phenotype. Hence, NDRG1 has the potential to disrupt the oncogenic interactions between PDAC cells and the TME, and promoting the expression of this protein may enhance PDAC vulnerability to current chemo/immunotherapies.