Methodological Considerations on Neutrophil–Macrophage Crosstalk in Gastric Cancer Under Chemo Immunotherapy

Dear Editor,

We read with great interest the recent study by Nagaoka et al. [1]. The work provides valuable insight into myeloid‐centered remodeling of the gastric tumor microenvironment. We would like to raise several methodological considerations that may merit attention in future studies.

First, the cohort size was modest (total n = 18 across four groups, with some arms as small as n = 3), and clinical response subgroups within the CN arm (PR vs. SD) were too limited to draw firm conclusions regarding predictive transcriptional signatures.

Second, biopsies were obtained cross‐sectionally from different patients rather than in a paired pre−/post‐treatment design; consequently, inter‐patient tumor heterogeneity cannot be fully disentangled from treatment‐driven effects, a concern inherent to scRNA‐seq studies of drug‐exposed human specimens.

Third, although in silico cell–cell communication (CellChat/CrossTalkR) implicated neutrophil‐derived CCL3/4/5 and MIF ligands in driving macrophage polarization, spatial transcriptomics or multiplexed immunohistofluorescence confirming physical proximity and ligand–receptor co‐localization in situ would strengthen the inferred crosstalk.

Fourth, given the known technical challenge of capturing low‐RNA‐content neutrophils, it would be reassuring to see more stringent exclusion of partial monocyte/macrophage contamination in the HLA‐DRA⁺ Neut4 cluster—for example, by demonstrating absence of high‐level monocyte‐specific genes (e.g., FCN1, CD14) on a per‐cell basis or by orthogonal protein validation (HLA‐DR⁺/CD14⁻/CD16⁺).

Finally, the in vivo validation employed the CT26 syngeneic model with Ly6G‐mediated neutrophil depletion; while this supports a functional role for neutrophils in combo‐therapy efficacy, direct pharmacological or genetic blockade of the proposed CCL/CCR or MIF‐CD74 axes would more specifically substantiate the mechanistic pathway identified by scRNA‐seq.

Notwithstanding these points, Nagaoka et al. provide a compelling framework for understanding how chemo‐immunotherapy reshapes innate immune interactions in gastric cancer, and their findings warrant validation in larger, prospectively collected paired‐sample cohorts with integrated spatial profiling.

Author Contributions

Haitao You: writing – original draft. Shunxiong Tang: writing – original draft, writing – review and editing.

Funding

The authors have nothing to report.

Ethics Statement

The authors have nothing to report.

Consent

We reviewed this manuscript and agreed to submit this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Linked Articles

This article is linked to Nagaoka et al. papers. To view this article, visit https://doi.org/10.1111/cas.70384.

Data Availability Statement

The authors have nothing to report.