In this study, we evaluated mitochondrial status in ESCC patients who underwent NAC followed by surgery using a recently proposed, novel mitochondrial evaluation system. Our observations, which suggest that pre-NAC mitochondrial status was associated with NAC response, highlight the independent survival impact of pre-NAC mitochondrial status in the tumor entity.

Previous studies have focused on the possible clinical significance of mitochondria-associated genes.17,18,26 A recent study demonstrated that a post-chemoradiotherapy increase in mitochondrial content was associated with treatment resistance in esophageal adenocarcinoma patients.19 These studies highlighted the importance of mitochondrial genes in EC patients; however, to our knowledge, no previous studies have examined mitochondrial status, by employing IHC in ESCC patients receiving surgery after NAC, to elucidate the survival impact of mitochondrial status in patients with these tumors.

Visualizing mitochondria in cancer cells from human pathological specimens might enhance our understanding of cancer biology; however, due to the limited methodologies available, relatively few studies have examined mitochondria in human cancer histological specimens using IHC. Sakashita et al. recently established an objective mitochondrial evaluation system using machine-based processing,11 which sheds light on the metabolic status of malignant tumors.

We revealed that patients with cT3–4 tumors had higher Mito-scores than those with cT1–2 tumors; however, the mechanisms underlying the association between Mito-score and tumor depth remain to be clarified. Mitochondria are multi-functional; bioenergetics and biosynthetic, while also contributing to cellular stress responses such as autophagy and apoptosis. Both glucose and nitrogen metabolisms are altered during the malignant progression of carcinoma,27,28 and they are controlled by nucleotide biosynthesis in the mitochondria.29,30 Therefore, Mito-score is expected to be deeply associated with tumor metabolism and tumor progression;11 however, further analysis is required to confirm this hypothesis and the biological significance of Mito-score, which quantitatively evaluates mitochondria.

Adenosine triphosphate is mainly synthesized in mitochondria by oxidative phosphorylation, which is regulated by COX, the final and rate-limiting step of the respiratory chain.31 We selected an antibody specific for COX4, a marker of the mitochondrial inner membrane, for IHC analysis to evaluate the mitochondria because the granular staining pattern in the cytoplasm clearly indicates the presence of mitochondria. Furthermore, the granular staining pattern of the cytoplasm was better distinguished using COX4 as a marker than voltage-dependent anion-selective channel protein 1, a mitochondrial outer membrane marker.11 We defined the Mito-score as the number of COX4 IHC-positive pixels divided by the number of nuclei. This system was validated using various methods in our prior study.11 Therefore, this system is applicable to evaluating various carcinomas, including ESCC.

Considering that mitochondria are dynamic organelles and their morphology and other factors change in response to external stimuli and metabolic cues,32 we hypothesized that the number of mitochondria recapitulates their function; however, whether number of mitochondria reflects functional status requires further confirmation. Furthermore, we did not analyze the mutation status of mitochondrial DNA in carcinomas or the correlation between the number of mitochondria and the metabolic state. More comprehensive cellular profiling investigations, e.g., RNA-seq or transcriptome profiling, are anticipated to provide more convincing results.

The high-Mito group had significantly higher proportions of patients with cT3–4 tumors and cStage III–IV disease. Furthermore, the high-Mito group showed significantly higher Ki-67 values than the low-Mito group. These results suggest that high-Mito tumors have biological aggressiveness. It is noteworthy that the good responder (therapeutic effect, Grade 2–3) rate was higher in the high-Mito group than in the low-Mito group. Furthermore, the good responder group had a significantly higher pre Mito-score than the poor responder group. Previous studies have revealed that high Ki-67 is associated with good responses to chemotherapy,33,34 suggesting that highly proliferative tumors are sensitive to platinum-based chemotherapeutic drugs.34 In fact, high Ki-67 levels before chemotherapy were reportedly associated with good response to chemotherapy in breast cancer patients35,36 and patients with ESCC.37

Furthermore, recent studies have raised the possibility that mitochondrial functions are associated with chemotherapy resistance in gastrointestinal malignancies.38,39 Our observations, together with those obtained in these earlier investigations, appear to highlight the potential clinical relevance of mitochondrial status to chemotherapy responsiveness. Overall, our observation that tumors with high pre Mito-score had good responsiveness to NAC is reasonable.

It is noteworthy that in our present study, a low Mito-score was independently associated with poor survival outcomes after adjusting for covariates that might be prognostic factors, suggesting the robust survival impact of a low Mito-score. Mitochondria-associated gene changes are reportedly associated with metabolic pathways, immune activity, and survival in gastrointestinal malignancies.15,17 Although glucose and nitrogen metabolism change markedly during the malignant progression of carcinomas, inducing the expressions of various enzymes and leading to mitochondrial dysfunctions,27,29 the precise mechanisms underlying the independent survival impact of a low Mito-score have yet to be elucidated.

Limitations must be taken into account when interpreting the results of this study. First, as mentioned above, a precise evaluation methodology for mitochondria remains to be established and validated for various specific malignancies, including ESCC. Unlike methods based on deep learning, our approach allows for parameter adjustments to tailor the conditions to each facility, suggesting the potential for tuning to suit various settings;11 however, the adaptability of our method has yet to be fully explored. Second, our cohort comprised only patients who underwent NAC followed by surgery, and we evaluated only specimens collected before NAC. Mitochondrial dynamics change markedly after chemotherapy, which reportedly contributes to resistance to chemotherapy in tumors of the gastrointestinal tract.16,19 Third, intratumoral heterogeneity might have affected our results. Although we calculated Mito-scores of two areas in each tumor, in an effort to minimize the influence of intratumoral heterogeneity, evaluation of the tumor microenvironment in small tumors, such as early-stage resections or biopsy samples, are readily affected by intratumoral heterogeneity.40,41 While a recent investigation showed substantial spatial heterogeneity between primary and metastatic tumors,42 we did not evaluate the spatial heterogeneity of the tumors in this study. Fourth, we employed the median value of Mito-score in our cohort as the threshold, although the rationale for the Mito-score threshold is not fully addressed in this study, possibly limiting the application of Mito-score in the clinical setting.