Tumor budding has been found to be of prognostic significance for several cancers, including colorectal cancer (CRC). Additionally, the molecular classification of CRC has led to the identification of different immune microenvironments linked to distinct prognosis and therapeutic response. However, the association between tumor budding and the different molecular subtypes of CRC and distinct immune profiles have not been fully elucidated. This study focused, firstly, on the validation of derived xenograft models (PDXs) for the evaluation of tumor budding and their human counterparts and, secondly, on the association between tumor budding and the immune tumor microenvironment by the analysis of gene expression signatures of immune checkpoints, Toll-like receptors (TLRs), and chemokine families. Clinical CRC samples with different grades of tumor budding and their corresponding PDXs were included in this study. Tumor budding grade was reliably reproduced in early passages of PDXs, and high-grade tumor budding was intimately related with a poor-prognosis CMS4 mesenchymal subtype. In addition, an upregulation of negative regulatory immune checkpoints (PDL1, TIM-3, NOX2, and IDO1), TLRs (TLR1, TLR3, TLR4, and TLR6), and chemokine receptors and ligands (CXCR2, CXCR4, CXCL1, CXCL2, CXCL6, and CXCL9) was detected in high-grade tumor budding in both human samples and their corresponding xenografts. Our data support a close link between high-grade tumor budding in CRC and a distinctive immune-suppressive microenvironment promoting tumor invasion, which may have a determinant role in the poor prognosis of the CMS4 mesenchymal subtype. In addition, our study demonstrates that PDX models may constitute a robust preclinical platform for the development of novel therapies directed against tumor budding in CRC.

S‐nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR‐low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR‐low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR‐Cas9‐mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR‐deficiency conferred higher tumorigenic and tumor‐initiating capacities. Moreover, GSNOR‐KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR‐KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2‐deoxyglucose (2DG), and a fragmented mitochondrial network. Real‐time metabolic analysis revealed that GSNOR‐KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient‐derived xenografts and organoids from clinical GSNOR‐low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.