Methodological Challenges in Studying Immune-Suppressive Neutrophils in HCC Models

Immunosuppressive CD10+ALPL+ neutrophils promote resistance to anti-PD-1 therapy in HCC by mediating irreversible exhaustion of T cellsJournal of HepatologyVol. 79Issue 6PreviewRemodeling the tumor microenvironment is a critical strategy for treating advanced hepatocellular carcinoma (HCC). Yet, how distinct cell populations in the microenvironment mediate tumor resistance to immunotherapies, such as anti-PD-1, remains poorly understood. Full-Text PDF Open Access The authors declare no conflicts of interest. The authors received no financial support to produce this manuscript. Delin Ma: manuscript writing, critical revision; Xianzhi Li: critical revision. We have read with great interest the article conducted by Meng et al. published in Journal of Hepatology.1Meng Y, Ye F, Nie P, et al. Immunosuppressive CD10+ALPL+ neutrophils promote resistance to anti-PD-1 therapy in HCC ​by mediating irreversible exhaustion of T cells. Journal of hepatology, S0168-8278(23)05078-X.Google Scholar In this study, the authors identified a CD10+ALPL+NEU cluster closely associated with resistance to PD-1 treatment. They demonstrated that this cluster is generated through NAMPT secretion by tumors, which leads to NTRK1 reprogramming, and that it can induce irrevocable exhaustion of T cells, resulting in immune suppression. The role of neutrophils in tumor immunity has been underappreciated, and the findings by Meng et al. represent a significant breakthrough in this field. Their discoveries contribute to a deeper understanding of neutrophils' role in tumor immunotherapy and provide valuable insights for clinical practitioners in identifying appropriate candidates for immunotherapy, bearing profound clinical implications. While reading the mentioned study, we have some concerns regarding certain details. Firstly, the authors employed intraperitoneal injection of neutrophils for adoptive transfer. However, we have doubts about whether this method effectively enhances the presence of CD10+ALPL+NEU cells at the liver tumor site. Neutrophils themselves are fragile, and intraperitoneal injection may not guarantee their efficient delivery to the tumor site. We are curious to know if the authors validated the increase of neutrophils within liver tumors after intraperitoneal injection and whether they considered other injection methods to establish the model, such as the tumor site injection or tail vein injection as employed in the study by Chang et al.2Chang Y, Cai X, Syahirah R, et al. CAR-neutrophil mediated delivery of tumor-microenvironment responsive nanodrugs for glioblastoma chemo-immunotherapy. Nature communications, 14(1), 2266.Google Scholar Secondly, in Figure 2A, the UMAP plot of neutrophils reveals noticeable batch effects, with cells from each sample not mixing well. However, this study does not mention how these batch effects were addressed. Dealing with batch effects is critical when analyzing single-cell data to ensure result accuracy and reproducibility. Therefore, we suggest that the authors provide additional details on how they processed and corrected batch effects in their study to better explain and address this issue. Furthermore, in Figure 2B, the authors validate the spatial adjacency between CD10+ALPL+NEU cells and PD-1+ T cells using multicolor IHC staining. However, they did not employ T cell-specific markers to confirm that the cells expressing high levels of PD-1 are indeed T cells. Therefore, we find the description in the text concerning this aspect to be insufficiently rigorous. Lastly, in Figure S2C, we noticed that NEU2 was mislabeled as NEU1, and in the same supplementary figure (Figure S2E), there are labeling errors in CD10+ALPL+NEU1 with NEU2, as well as mislabeling between NEU5 and NEU6. We kindly request the authors to rectify these labeling errors to ensure the accuracy and readability of the results. We truly thank authors for the excellent and important work.