ENO3 Emerges as a Lactate-Linked Biomarker in Colorectal Cancer Metastasis

Colorectal cancer kills hundreds of thousands of people each year, and it is rarely the primary tumor that delivers the fatal blow. It is the spread—into the liver, the lungs, and the peritoneal cavity—that turns a potentially curable disease into a lethal one. Nearly half of patients with localized colorectal cancer eventually develop metastasis, and once that happens, five-year survival collapses from over 90 percent to roughly 12 percent. A new study published in Cancer Reports now offers a fresh clue about what drives this deadly transition, pointing to a gene called ENO3 and its role in one of cancer biology’s most talked-about metabolic molecules: lactate.

The research, led by a team at Soochow University in China, began with a puzzle that has been reshaping how scientists think about tumor metabolism. For nearly a century, the dominant paradigm has been the Warburg effect—the observation that cancer cells persistently favor glycolysis, burning glucose even in the presence of abundant oxygen. But recent work has complicated this picture. A landmark 2023 study in Nature found that primary solid tumors can actually exhibit suppressed metabolic activity compared with normal tissues, while metastatic lesions are metabolically hyperactive—a phenomenon the researchers called metabolic reversal. The Soochow team set out to see whether a similar stage-specific pattern could be detected at the transcriptional level in colorectal cancer, and whether the molecular switches governing this transition could be identified.

Using integrated analysis of transcriptomic data from The Cancer Genome Atlas and the Gene Expression Omnibus dataset GSE41568, which profiles primary versus metastatic colorectal cancer lesions, the researchers performed gene set enrichment analysis on metabolic pathways. Their findings mirrored the reversal concept: primary tumors showed broad suppression of signatures related to the tricarboxylic acid cycle, lipid metabolism, and amino acid metabolism compared with adjacent normal tissues. In contrast, metastatic samples showed relative reactivation of several metabolic programs, with glycolysis and the pentose phosphate pathway among the positively enriched pathways. The authors are careful to note that these are transcriptionally inferred metabolic programs, not direct measurements of metabolic flux—but the pattern is striking and consistent with the idea that metastatic cells undergo a distinct metabolic awakening.

Why does this matter? Because one of the key consequences of metabolic reprogramming is the altered secretion of metabolites that act as signaling molecules and reshape the tumor microenvironment. Lactate, once dismissed as metabolic waste, is now recognized as an oncometabolite that can drive tumor progression and immune evasion. By acidifying the tumor microenvironment, lactate suppresses cytotoxic T cells and natural killer cells—the immune system’s front-line assassins. It has also been linked to changes in the myeloid compartment, including the recruitment and activation of tumor-associated neutrophils, which can contribute to metastatic outgrowth and treatment resistance. Clinical and experimental studies have reported substantial neutrophil infiltration in colorectal cancer liver metastases, and neutrophil extracellular traps—web-like structures neutrophils release—have been implicated in metastatic progression.

To identify candidate genes associated with this lactate-linked landscape, the team screened metabolism-related genes for prognostic value using univariate Cox regression combined with LASSO analysis. This yielded a six-gene lactate metabolism-related signature comprising ENO3, TKTL1, ACADL, ACSL6, UGT2A3, and ALAD. Consensus clustering based on the expression of these six genes stratified TCGA patients into two molecular subtypes, and patients in Cluster 1 exhibited significantly shorter overall survival. The researchers then built an exploratory quantitative lactate metabolism-related score, or TLMS, and found that higher scores were associated with the poor-prognosis Cluster 1. Importantly, the signature has not yet been validated in an independent prognostic cohort, so its clinical applicability remains to be established.

What makes the findings particularly intriguing is the connection to neutrophils. Using single-sample gene set enrichment analysis to estimate the abundance of 28 immune-cell populations from transcriptomic data, the team found that the poor-prognosis Cluster 1 had a higher computationally inferred neutrophil score. A similar pattern emerged when primary tumors were compared with liver metastatic samples, with higher inferred neutrophil scores in the metastatic group. Pathway analysis of differentially expressed genes between the clusters showed enrichment of pro-inflammatory and cytokine-related pathways, together with PD-1/PD-L1 checkpoint-related and neutrophil extracellular trap-associated gene signatures. The authors emphasize that these are transcriptome-based estimates, not direct histological measurements of immune-cell abundance, and that enrichment of a NET-associated gene set does not demonstrate NET formation. Still, the associations suggest a potential link between lactate metabolism-related transcriptional features and neutrophil-related biology in colorectal cancer progression.

Among the six genes, ENO3 stood out. Also known as beta-enolase, ENO3 has traditionally been associated with muscle energy metabolism, but previous work has also implicated it in colorectal cancer cell glycolysis and progression. In the current study, ENO3 was upregulated in colorectal cancer tumors compared with normal tissues, and higher ENO3 expression was associated with shorter overall survival. To probe its functional contribution, the researchers generated ENO3 knockout MC38 murine colon adenocarcinoma cell lines using CRISPR/Cas9 technology, verifying loss of ENO3 protein by immunoblotting. CCK-8 proliferation assays showed that ENO3 loss reduced MC38 cell proliferation over a 96-hour period. More dramatically, in an experimental lung colonization model using immunodeficient NSG mice, mice injected with ENO3 knockout cells developed a significantly lower pulmonary lesion burden than mice injected with wild-type cells, as quantified by blinded counting of surface pulmonary nodules and confirmed by histological evaluation.

The authors are careful about what these experiments do and do not show. The NSG tail-vein model was selected to provide a preliminary assessment of the tumor-cell-intrinsic effect of ENO3 loss on experimental lung colonization in an immunodeficient background. It was not designed to evaluate an intact tumor immune microenvironment, and it bypasses earlier steps of the metastatic cascade, including local invasion and intravasation. The findings support a potential tumor-cell-intrinsic contribution of ENO3 to proliferation and experimental lung colonization, but they do not establish ENO3 as a central orchestrator of metastasis or demonstrate that its effects are mediated by lactate metabolism. The study also does not directly show that ENO3 regulates lactate production, chemokine secretion, or neutrophil recruitment—questions that will require dedicated mechanistic work using immune-competent models, metabolic measurements, and tumor cell-neutrophil interaction assays.

Several other limitations frame the conclusions. Bulk transcriptomic data do not allow definitive assignment of ENO3 expression to tumor, stromal, or immune-cell populations, and the MC38 experiments confirm ENO3 expression and functional relevance in tumor cells but do not exclude expression in other components of the tumor microenvironment. The TLMS was developed and initially evaluated in the same TCGA cohort, and the independent prognostic value of ENO3 was not assessed using a comprehensive multivariable model incorporating TNM stage and molecular characteristics. The enrichment of PD-1/PD-L1 checkpoint-related gene sets in the high-risk subgroup raises interesting questions about immunoregulatory phenotypes—particularly given that durvalumab, a PD-L1-blocking antibody, showed an exploratory overall-survival signal in the Phase II CO.26 study when combined with tremelimumab in refractory colorectal cancer—but the current results do not demonstrate PD-L1 protein expression or establish TLMS as a predictive biomarker for PD-L1 blockade.

Looking ahead, the researchers propose that future work should combine direct metabolic measurements with immune-competent syngeneic or orthotopic colorectal cancer models, neutrophil profiling, and single-cell or spatial approaches to resolve cell-type-specific ENO3 expression and potential tumor-immune interactions. Artificial intelligence and machine-learning approaches may also facilitate integration of transcriptomic, pathological, spatial, and clinical information for refined risk stratification, though such approaches require large multicenter datasets, external validation, and careful control of algorithmic bias. For now, the study nominates ENO3 as a candidate prognosis-associated factor in colorectal cancer metastasis and provides preliminary evidence of a tumor-cell-intrinsic functional contribution. Whether ENO3 ultimately proves to be a genuine molecular switch linking lactate metabolism to neutrophil-driven metastatic progression remains an open and compelling question—one that could shape how scientists approach the metabolic and immune dimensions of one of the world’s deadliest cancers.

Subject of Research: A transcriptomic study identifies ENO3 as a candidate lactate metabolism-associated biomarker in colorectal cancer metastasis.

Article Title: Integrated Transcriptomic Analysis Identifies ENO3 as a Candidate Lactate Metabolism‐Associated Biomarker in Colorectal Cancer Metastasis

Article References: Qian, J., Wang, M., Liu, J., & Gao, M. (2026). Integrated Transcriptomic Analysis Identifies ENO3 as a Candidate Lactate Metabolism‐Associated Biomarker in Colorectal Cancer Metastasis. Cancer Reports, 9(10), Article e70723. https://doi.org/10.1002/cnr2.70723

Image Credits: AI Generated

DOI: 10.1002/cnr2.70723

Keywords: colorectal cancer, metastasis, ENO3, lactate metabolism, tumor microenvironment, neutrophils, biomarker, transcriptomics, Integrated, Transcriptomic, Analysis, Identifies