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Cited by 12Phagocytosis and Immune RegulationImmune cells in cancerEsophageal Cancer Research and Treatment

Macrophage efferocytosis mediated by the TP63-RAC2 pathway promotes immunosuppressive remodeling in esophageal cancer

Open access
Xi, Yong, Zhao, Zhijie, Zhou, Yundong, Yin, Chengliang, Li, Yuxin, Xu, Xiao, Jin, Wenyi, Lin, Chengbin +7 more·2026·Cell Reports Medicine
DOI 10.1016/j.xcrm.2025.102529PMC PMC12866140
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Abstract

This study explores the role of efferocytosis in esophageal squamous cell carcinoma (ESCC) using single-cell RNA sequencing and in vitro/in vivo assays. Analyzing 27 samples from 9 patients with ESCC, we identify diverse cell types and significant heterogeneity in the tumor microenvironment, with a focus on efferocytosis. Our findings highlight that macrophages engulf apoptotic tumor cells, thereby impairing immune responses and promoting tumor progression. Notably, TP63 and RAC2 emerge as key regulators of this process, influencing efferocytosis and immune modulation. Functional assays demonstrate that disrupting these pathways alters macrophage efferocytosis and impacts tumor growth in vivo. These results suggest that targeting efferocytosis pathways offers potential therapeutic strategies for ESCC, enhancing antitumor immunity and improving patient outcomes. The study underscores the complex interactions between tumor cells and the immune system, with efferocytosis representing a promising therapeutic target.

AI snapshot

Written from the paper’s full text.

TL;DR

Single-cell RNA sequencing of esophageal squamous cell carcinoma (ESCC) identifies a tumor-associated macrophage subtype, C1 FN1+ macrophages, with high efferocytosis activity and immunosuppressive features. The transcription factor TP63 promotes macrophage efferocytosis and M2 polarization by directly regulating RAC2, and disrupting this axis alters tumor growth in vivo.

Key findings

  • In 27 samples from 9 patients with ESCC, scRNA-seq retained 121,038 high-quality cells, identified 36 clusters and 9 cell types, and resolved 7 TAM subtypes; C1 FN1+ macrophages showed the highest efferocytosis activity, the lowest differentiation/stemness by CytoTRACE, and a transitional M1/M2 phenotype.
  • TP63 and KLF5 were among the top regulators of C1 FN1+ macrophages; TP63 knockdown reduced macrophage engulfment of CMFDA-labeled apoptotic EC9706 and HYSE180 cells in vitro, and TP63 was shown to bind the RAC2 promoter at binding sites 2/3 by luciferase and ChIP assays.
  • TP63 depletion reduced M2 macrophages (F4/80+CD163+), increased M1 macrophages (F4/80+CD86+), decreased IL-10 and TGF-β, and increased TNF-α after co-culture with apoptotic cells; RAC2 overexpression reversed these changes.
  • In vivo, shTP63 impaired peritoneal and thymic efferocytosis and shifted macrophages toward M1, while RAC2 overexpression rescued efferocytosis and M2 polarization; in a humanized mouse model, TP63 knockdown suppressed tumor growth, and RAC2 overexpression reversed this effect.

Methods

  • scRNA-seq was performed on 27 surgically resected specimens from 9 untreated ESCC patients (9 tumor tissues and 18 paired lymph nodes); public data from GEO accession GSE196756 were also analyzed. Processing used CellRanger, Seurat, DoubletFinder, Harmony, inferCNV, CytoTRACE, Slingshot, and SCENIC.
  • In vitro assays used human primary macrophages, THP-1-derived macrophages, mouse BMDMs, and RAW264.7 cells, co-cultured with apoptotic ESCC cells (EC9706, KYSE180, AKR) labeled with CMFDA; readouts included confocal microscopy, flow cytometry, western blotting, qPCR, luciferase reporter assays, ChIP, and ELISA.
  • In vivo work used BALB/c mice for peritoneal and dexamethasone-induced thymic efferocytosis assays and NOG mice engrafted with human PBMCs and EC9706 cells; mIHC was performed on clinical samples. Statistical significance was assessed with Student’s t test or one-way ANOVA, with p < 0.05.

Limitations

  • The paper states that the small sample size (nine patients) may limit generalizability; that the exact mechanistic interactions between TP63, RAC2, and other immune regulators remain unclear; that the focus on macrophage-mediated efferocytosis should be expanded to other immune cells such as T cells and fibroblasts; and that clinical translation requires deeper exploration of molecular mechanisms, delivery strategies, and long-term effects on tumor immunity.
  • The discussion also notes that RAC2 overexpression did not completely restore efferocytosis activity to control levels, suggesting additional factors may regulate this pathway.
  • The future-directions section mentions addressing potential gender bias in the study, which the text makes evident as a stated concern.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.xcrm.2025.102529

Cites

68 of 68 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

24 of 25 cited sources checked · 11 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    In the tumor microenvironment, efferocytosis promotes an immunosuppressive environment by polarizing tumor-associated macrophages toward a tumor-promoting M2 phenotype.

    “While it helps clear apoptotic tumor cells, it also promotes an immunosuppressive environment by polarizing tumor-associated macrophages (TAMs) toward a tumor-promoting M2 phenotype.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [7] Manoharan, 2018, A Computational Approach Identifies Immunogenic Features of Prognosis in Human Cancers, Front. Immunol. source

      The Results section, “CD8+ T Cell-Dependent Long-Term Survival Benefit in Human Cancers” (page unavailable), describes a tumor microenvironment infiltrated by M2-type macrophages as immunosuppressive. But our copy does not say that efferocytosis clears apoptotic tumor cells or causes macrophages to polarize toward M2; searches for efferocytosis, apoptotic-cell clearance, and M2 polarization did not establish that mechanism. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [8] Sun, 2022, An effective therapeutic regime for treatment of glioma using oncolytic vaccinia virus expressing IL-21 in combination with immune checkpoint inhibition, Mol. Ther. Oncolytics source

      The Discussion (page unavailable) describes glioma’s suppressive immune microenvironment and tumor-promoting macrophages, and notes the tumor-promoting properties of M2 macrophages. It does not say that efferocytosis or clearance of apoptotic tumor cells polarizes TAMs toward M2; searches for those processes found no supporting passage. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [9] Ambade, 2016, Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease, World J. Gastroenterol. source

      The discussion of TAMs (section labeled “ROS Steatosis Proliferation Inflammation”; page unavailable) describes them as M2-skewed and says they can suppress adaptive immunity and promote tumors. The paper does not say that efferocytosis clears apoptotic tumor cells or causes TAMs to polarize toward M2; its experimental results instead report mixed M1/M2 markers in the liver. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    After engulfing apoptotic cells, macrophages secrete anti-inflammatory cytokines such as IL-10 and TGF-β to suppress immune responses.

    “After engulfing ACs, macrophages secrete a range of anti-inflammatory cytokines, such as IL-10 and TGF-β, to suppress immune responses.24,25”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [24] Gronski, 2009, An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response, Cell Death Differ. source

      The Results (page unavailable) report that macrophages exposed to apoptotic cells secrete TGF-β, and the Introduction links this response to immunological tolerance. The paper does not identify IL-10 as one of the secreted cytokines. It also finds that recognition of apoptotic cells can induce TGF-β without physical engulfment, so engulfment is not required for the response described. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [25] Kim, 2015, Taurine Chloramine Stimulates Efferocytosis Through Upregulation of Nrf2-Mediated Heme Oxygenase-1 Expression in Murine Macrophages: Possible Involvement of Carbon Monoxide, Antioxid. Redox Signal. source
  • Findingown data

    Genes associated with efferocytosis in the C1 FN1+ subtype, including ABCG1, AXL, and TGM2, regulate apoptosis and lipid metabolism.

    “Genes associated with efferocytosis in the C1 FN1+ subtype, including ABCG1, AXL, and TGM2, were found to regulate apoptosis and lipid metabolism”
    Quote in the paperNot a substantial claim
  • Findingown data

    TAMs were divided into seven distinct subtypes: C0 TREM2+, C1 FN1+, C2 CD14+, C3 LGALS2+, C4 TNFAIP3+, C5 CHCHD6+, and C6 VCAM1+ macrophages.

    “TAMs were divided into seven distinct subtypes: C0 TREM2+ macrophages, C1 FN1+ macrophages, C2 CD14+ macrophages, C3 LGALS2+ macrophages, C4 TNFAIP3+ macrophages, C5 CHCHD6+ macrophages, and C6 VCAM1+ macrophages”
    Quote in the paperSubstantial claim
  • Findingown data

    Different TAM subtypes had different characteristic gene expression patterns.

    “Different TAM subtypes had different characteristic gene expression patterns”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 28
  • May combine two claims. 40
  • The quote only partly states this claim. 2

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In breast and lung cancers, efferocytosis is associated with tumor immune evasion and progression.

    “For instance, in breast33 and lung34 cancers, efferocytosis is associated with tumor immune evasion and progression.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [33] Liang, 2022, Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis, Cancer Commun. source

      Citation 34 is attached to lung cancer, while citation 33 is attached to breast cancer. The Results report that apoptotic cells correlate with tumor-associated macrophages in human lung tumors, and the Discussion links M2-like macrophages generally to immune escape and tumor progression. The paper does not establish that efferocytosis itself is associated with immune evasion or progression specifically in lung cancer. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [34] Kim, 2022, Reprogramming of cancer-associated fibroblasts by apoptotic cancer cells inhibits lung metastasis via Notch1-WISP-1 signaling, Cell. Mol. Immunol. source

      Citation 34 is attached to the lung-cancer portion of the sentence, not the breast-cancer portion. Although the Introduction says phagocyte-mediated clearance can be immunosuppressive in the tumor microenvironment, it also says tumor cells may evade surveillance by avoiding efferocytosis. The Discussion reports the opposite of the claimed progression association in this lung-cancer study: interaction with apoptotic cancer cells reprogrammed phagocytic fibroblasts and inhibited cancer-cell migration and invasion. The supplied copy gives section labels but no page numbers. (LLM, conf=0.85)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The M2-like TAM phenotype supports tumor growth, angiogenesis, and immune evasion.

    “This phenotype supports tumor growth, angiogenesis, and immune evasion, creating a formidable barrier to effective antitumor immunity.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [7] Manoharan, 2018, A Computational Approach Identifies Immunogenic Features of Prognosis in Human Cancers, Front. Immunol. source

      The Results section, “CD8+ T Cell-Dependent Long-Term Survival Benefit in Human Cancers” (page unavailable), describes a microenvironment infiltrated by M2-type macrophages as immune suppressive and predictive of poor survival. This supports the immune-evasion aspect, but the paper does not specifically say that M2-like macrophages support tumor growth or angiogenesis. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [8] Sun, 2022, An effective therapeutic regime for treatment of glioma using oncolytic vaccinia virus expressing IL-21 in combination with immune checkpoint inhibition, Mol. Ther. Oncolytics source
    • cites [9] Ambade, 2016, Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease, World J. Gastroenterol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Proteases have been shown to affect the efferocytosis effect of TAMs.

    “These proteases have been shown to affect the efferocytosis effect of TAMs.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Block, 1986, Identification of pancreas necrosis in severe acute pancreatitis: imaging procedures versus clinical staging, Gut source

      The contrast-enhanced computed tomography results and Discussion assess imaging and clinical staging of pancreatic necrosis. Searches for proteases, efferocytosis, TAMs, and macrophage clearance found no passage in our copy addressing whether proteases affect TAM efferocytosis. The cited paper therefore does not verify the attributed claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Impaired efferocytosis reduces TAM recognition and engulfment of tumor cells, compromising immune surveillance and contributing to resistance to antitumor therapies.

    “Impaired efferocytosis reduces TAM recognition and engulfment of tumor cells, compromising immune surveillance and contributing to resistance to antitumor therapies.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Wei, 2022, Thymosin α-1 Reverses M2 Polarization of Tumor-Associated Macrophages during Efferocytosis, Cancer Res. source

      The Introduction identifies efferocytosis as the removal of apoptotic tumor cells by phagocytes, including TAMs, and says directly impairing it can cause secondary necrosis and drive tumor progression. It does not say impaired efferocytosis compromises immune surveillance or causes resistance to antitumor therapies; instead, it describes chemotherapy-induced efferocytosis as promoting an immunosuppressive TAM phenotype. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous research suggests a role for lipid metabolism in TAM-mediated efferocytosis.

    “This aligns with previous research suggesting a role for lipid metabolism in TAM-mediated efferocytosis.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [16] Gerlach, 2021, Efferocytosis induces macrophage proliferation to help resolve tissue injury, Cell Metab. source

      The Introduction and Discussion describe lipids from apoptotic cells as contributors to pathways in efferocytosing macrophages. The copy does not establish that lipid metabolism has this role specifically in TAM-mediated efferocytosis; its MerTK results address macrophage proliferation and efferocytosis, not a lipid-metabolism link. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Efferocytosis plays a crucial role in maintaining tissue homeostasis and in modulating immune responses.

    “Efferocytosis, the process by which macrophages engulf and remove apoptotic cells (ACs), plays a crucial role in maintaining tissue homeostasis and in modulating immune responses.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [5] Zhang, 2023, Nanoliposomal Bcl-xL proteolysis-targeting chimera enhances anti-cancer effects on cervical and breast cancer without on-target toxicities, Adv. Compos. Hybrid Mater. source
    • cites [6] Wu, 2022, Recent advances of tanshinone in regulating autophagy for medicinal research, Front. Pharmacol. source

      The cardiovascular-disease table mentions “repairing efferocytosis and M2 phenotype” in a tanshinone-treated atherosclerosis model, but does not explain efferocytosis’s role in tissue homeostasis or immune responses. Searches for macrophage clearance of apoptotic cells and immune regulation found no passage in our copy addressing the attributed claim. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Within the tumor microenvironment, efferocytosis aids in clearing dead cells and modulates immune responses by secreting anti-inflammatory cytokines.

    “Within the TME, efferocytosis aids in clearing dead cells and modulates immune responses by secreting anti-inflammatory cytokines.21”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [21] Zhu, 2016, Loss of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 Confers Resistance to the Antiatherogenic Effects of Tumor Necrosis Factor-α Inhibition, Arterioscler. Thromb. Vasc. Biol. source

      The Introduction describes efferocytosis as the clearance of apoptotic cells, and the Results state that efficient efferocytosis promotes anti-inflammatory cytokine production. These passages concern atherosclerotic lesions, not the tumor microenvironment; our copy does not establish the claim in that setting. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Efferocytosis involves not only the physical engulfment of cells but also the regulation of the immune system.

    “Efferocytosis involves not only the physical engulfment of cells22 but also the regulation of the immune system.23”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [22] Jiang, 2017, Inactivation of Rab11a GTPase in Macrophages Facilitates Phagocytosis of Apoptotic Neutrophils, J. Immunol. source
    • cites [23] Yang, 2014, High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis, J. Immunol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Current treatment strategies for ESCC primarily include surgery, radiotherapy, and chemotherapy.

    “Current treatment strategies for ESCC primarily include surgery, radiotherapy, and chemotherapy.27,28”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [27] Peng, 2017, Inhibition of glutathione metabolism attenuates esophageal cancer progression, Exp. Mol. Med. source
    • cites [28] Mohlin, 2019, Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma, EMBO Mol. Med. source

      The Introduction lists surgery, radiotherapy, and chemotherapy among current treatments for neuroblastoma. It does not address treatment strategies for ESCC, so the claim’s disease-specific attribution is unsupported. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Efferocytosis is a critical physiological process through which macrophages recognize and engulf apoptotic cells to maintain tissue homeostasis.

    “Efferocytosis is a critical physiological process through which macrophages recognize and engulf ACs to maintain tissue homeostasis.18,19,20”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [18] Roy, 2018, Macrophage-Derived Neuropilin-2 Exhibits Novel Tumor-Promoting Functions, Cancer Res. source
    • cites [19] Anandan, 2021, Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin, Cells source
    • cites [20] Zheng, 2021, bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster, PLoS Genet. source

  • May combine two claims.

    Premisecitation

    This immunosuppressive environment helps tumor cells evade immune surveillance and promotes tumor growth.

    “This immunosuppressive environment helps tumor cells evade immune surveillance and promotes tumor growth.26”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [26] Ma, 2022, Visual Analysis of Colorectal Cancer Immunotherapy: A Bibliometric Analysis From 2012 to 2021, Front. Immunol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Efferocytosis plays a significant role in other cancer types as well.

    “Although this study focuses on ESCC, efferocytosis plays a significant role in other cancer types as well.32”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [32] Tajbakhsh, 2021, The complex roles of efferocytosis in cancer development, metastasis, and treatment, Biomed. Pharmacother. source

  • May combine two claims.

    Findingown data

    Disrupting the TP63/RAC2 pathways alters macrophage efferocytosis and impacts tumor growth in vivo.

    “Functional assays demonstrate that disrupting these pathways alters macrophage efferocytosis and impacts tumor growth in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In GO-BP, C1 FN1+, C2 CD14+, and C4 TNFAIP3+ macrophages had higher leukocyte and immune scores.

    “in GO-BP, C1 FN1+ macrophages, C2 CD14+ macrophages, and C4 TNFAIP3+ macrophages had higher “leukocyte” and “immune” scores”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    T cells predominantly originated from LN samples.

    “T cells predominantly originated from LN samples”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C1 FN1+, C2 CD14+, and C4 TNFAIP3+ macrophages exhibit shared biological effects and enrichment in immune-related pathways such as defense response, immune response, response to cytokine, and cellular response to cytokine stimulus.

    “C1 FN1+ macrophages, C2 CD14+ macrophages, and C4 TNFAIP3+ macrophages exhibit shared biological effects and demonstrate enrichment in immune-related pathways such as “defense response,” “immune response,” “response to cytokine,” and “cellular response to cytokine stimulus””
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C2 EZH2+ tumor cells and C3 CENPF+ tumor cells primarily originated from within tumor tissues and exhibited heightened proliferative activity.

    “Notably, C2 EZH2+ tumor cells and C3 CENPF+ tumor cells primarily originated from within the tumor tissues and exhibited heightened proliferative activity”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    After quality control and cell filtration, 121,038 high-quality cells were retained.

    “After quality control and cell filtration, 121,038 high-quality cells were finally retained”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The proportion of epithelial cells was highest in tumor tissues, followed by myeloid cells, and LNs had higher immune cell infiltration including B, plasma, and T cells.

    “Among them, the proportion of epithelial cells was the highest in tumor tissues, followed by myeloid cells, and there was higher immune cell infiltration in LNs, including B, plasma, and T cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Although C2 EZH2+ and C3 CENPF+ cells divide vigorously, they dominate at the end of the differentiation trajectory.

    “although these cells divide vigorously, they dominate at the end of the differentiation trajectory.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Proliferating CD4+ T cells and CD4 TIGIT+ Treg cells were primarily derived from tumor tissues.

    “Proliferating CD4+ T cells and CD4 TIGIT+ Treg cells were primarily derived from tumor tissues”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    CD4 TOP2A+ proliferating Treg cells exhibited the most elevated G2M value, whereas CD4 STMN1+ proliferating Treg cells displayed the highest S value.

    “Among these, CD4 TOP2A+ proliferating Treg cells exhibited the most elevated G2M value, whereas CD4 STMN1+ proliferating Treg cells displayed the highest S value”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The scRNA-seq analysis identified 36 different cell clusters.

    “we identified 36 different cell clusters”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Cell cycle scores indicated vigorous proliferation of CD8 ANXA2+ exhausted T cells.

    “the cell cycle scores indicated vigorous proliferation of CD8 ANXA2+ exhausted T cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C2 CD14+ macrophages and C4 TNFAIP3+ macrophages primarily originated from tumor tissues.

    “C2 CD14+ macrophages and C4 TNFAIP3+ macrophages primarily originated from tumor tissues.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    By integrating gene expression, regulon activity, and differentiation pathways, the study constructed a comprehensive map of TAM heterogeneity and identified TP63 and RAC2 as key molecular drivers regulating efferocytosis.

    “By integrating gene expression, regulon activity, and differentiation pathways, we constructed a comprehensive map of TAM heterogeneity and identified key molecular drivers, including TP63 and RAC2, that regulate efferocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The C1 FN1+ macrophage population demonstrated the highest cellular stemness, indicating the lowest degree of differentiation, attenuated functionality, and heightened potential for differentiation.

    “the C1 FN1+ macrophage population demonstrated the highest cellular stemness, indicating the lowest degree of differentiation, attenuated functionality, and heightened potential for differentiation”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Single-cell RNA sequencing of 27 samples from 9 patients with ESCC identified diverse cell types and significant heterogeneity in the tumor microenvironment, with a focus on efferocytosis.

    “Analyzing 27 samples from 9 patients with ESCC, we identify diverse cell types and significant heterogeneity in the tumor microenvironment, with a focus on efferocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The CD8+ T cell population comprised three subtypes of naive T cells, two subtypes of effector T cells, and three subtypes of depleted T cells.

    “The CD8+ T cell population comprised three subtypes of naive T cells, two subtypes of effector T cells, and three subtypes of depleted T cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C0 TREM2+ macrophages were closely associated with energy metabolism and enriched in pathways including cellular lipid metabolic process, small molecule metabolic process, aerobic respiration, and electron transport chain.

    “These macrophages exhibited significant enrichment in pathways such as “cellular lipid metabolic process,” “small molecule metabolic process,” “ aerobic respiration,” and “electron transport chain.””
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Epithelial cells exhibited the highest level of proliferative activity.

    “Moreover, the epithelial cells exhibited the highest level of proliferative activity”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C3 LGALS2+ macrophages and C6 VCAM1+ macrophages were predominantly derived from metastasis-associated LNs.

    “C3 LGALS2+ macrophages and C6 VCAM1+ macrophages were predominantly derived from metastasis-associated LNs”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    CD4+ T cells were classified into nine discrete subtypes, including three naive, two actively proliferating, one effector, one Treg, and two Tfh types.

    “CD4+ T cells can be further classified into nine discrete subtypes, encompassing three types of naive T cells, two types of actively proliferating T cells, one effector T cell type, one regulatory T (Treg) cell type, and two types of follicular helper T (Tfh) cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Even when samples were taken from the same site, the main cell subtypes were not exactly the same among different patients.

    “even if the samples were taken from the same site, the main cell subtypes were not exactly the same among different patients.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    shTP63-treated macrophages exhibited a decreased M2 phenotype and increased M1 polarization in vivo.

    “shTP63-treated macrophages exhibited a decreased M2 phenotype and increased M1 polarization”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Thymic macrophages from shTP63-treated mice showed M1 polarization, and RAC2 overexpression restored M2 polarization.

    “Thymic macrophages from shTP63-treated mice showed M1 polarization, and RAC2 overexpression restored M2 polarization (Figures S16C and S16D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Human primary macrophages engulfed CMFDA-labeled EC9706 and HYSE180, whereas the ability of TP63-depleted macrophages to take up apoptotic cells was notably reduced.

    “human primary macrophages engulfed CMFDA-labeled EC9706 and HYSE180, whereas the ability of TP63-depleted macrophages to take up ACs was notably reduced”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RAC2 overexpression restored efferocytosis levels and M2 polarization in vivo.

    “RAC2 overexpression restored efferocytosis levels and M2 polarization (Figures S17D–S17F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ChIP experiment showed that TP63 directly binds to the RAC2 gene, which is notably repressed by siTP63.

    “The results from the chromatin immunoprecipitation (ChIP) experiment showed that TP63 directly binds to the RAC2 gene, which is notably repressed by siTP63”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RAC2 overexpression partially rescues the TP63 knockdown-induced reduction in efferocytosis and restores macrophage polarization toward the M2 phenotype.

    “while RAC2 overexpression partially rescues this effect, restoring efferocytosis and macrophage polarization toward the M2 phenotype.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with shTP63 reduced RAC2 expression in mouse peritoneal macrophages, while RAC2 overexpression restored it.

    “Treatment with shTP63 reduced RAC2 expression, while RAC2 overexpression restored it (Figure 7A).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    siTP63-3 or siKLF5-3 induced the most significant reduction of TP63 or KLF5 in human primary macrophages, THP-1, mouse primary macrophages, and RAW264.7 cells.

    “siTP63-3 or siKLF5-3 induced the most significant reduction of TP63 or KLF5 in human primary macrophages”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A distinctive subtype of TAMs within tumors demonstrates pronounced proliferative characteristics, robust immunosuppressive functions, heightened efferocytosis activity, and significant differentiation potential.

    “the above data indicate the identification of a distinctive subtype of TAMs within tumors. This subtype demonstrates pronounced proliferative characteristics, robust immunosuppressive functions, heightened efferocytosis activity, and a significant differentiation potential.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dimensionality reduction and cluster analysis of TAM subtypes based on gene expression and regulon activity revealed distinct subgroups, with significant differences in regulon activity among TAM subtypes but no variation between different cell phases.

    “Dimensionality reduction and cluster analysis of TAM subtypes based on gene expression and regulon activity revealed distinct subgroups, with significant differences in regulon activity among TAM subtypes but no variation between different cell phases”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    A subtype of TAMs with vigorous division was found in the microenvironment of the primary tumor, which may be closely related to tumor immune imbalance.

    “This suggests that a subtype of TAMs with vigorous division was found in the microenvironment of the primary tumor, which may be closely related to the tumor immune imbalance.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: TAMs are the most abundant immune cells in the tumor microenvironment.

  • May combine two claims.

    Interpretationreasoning

    C2 EZH2+ tumor cells and C3 CENPF+ tumor cells have decreased differentiation ability and a relatively restricted degree of diversification.

    “suggesting a decreased differentiation ability and a relatively restricted degree of diversification”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: When tumor cells actively proliferate, their tissue differentiation ability gradually decreases.

  • May combine two claims.

    Interpretationown data

    The primary tumor site has a stronger immunosuppressive environment, potentially making macrophage efferocytosis a promising therapeutic target for ESCC.

    “This suggests a stronger immunosuppressive environment at the primary tumor site, potentially making macrophage efferocytosis a promising therapeutic target for ESCC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    C1 FN1+ macrophages appeared to be in a transitional state between M1 and M2 phenotypes, influencing their immune function.

    “C1 FN1+ macrophages appeared to be in a transitional state between M1 and M2 phenotypes, influencing their immune function”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Macrophages engulf apoptotic tumor cells, thereby impairing immune responses and promoting tumor progression.

    “Our findings highlight that macrophages engulf apoptotic tumor cells, thereby impairing immune responses and promoting tumor progression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    The vigorous proliferation of CD8 ANXA2+ exhausted T cells is potentially linked to their involvement in cellular metabolism.

    “potentially linked to their involvement in cellular metabolism”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    C1 FN1+ macrophages had low differentiation and a high degree of malignancy, which may be related to the rapid progression of ESCC and poor prognosis.

    “CytoTRACE and Slingshot analyses showed that C1 FN1+ macrophages had low differentiation and a high degree of malignancy, which may be related to the rapid progression of ESCC and poor prognosis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    These TAMs play a crucial role in the TME and contribute to immune resistance.

    “These TAMs play a crucial role in the TME and contribute to immune resistance.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: TAMs are the most abundant immune cells in the tumor microenvironment.

  • May combine two claims.

    Interpretationnone given

    Targeting TP63 and RAC2 can alter TAM functions, reduce their immunosuppressive effects, and enhance the ability of the immune system to attack tumors.

    “Specifically, targeting TP63 and RAC2 can alter TAM functions, reduce their immunosuppressive effects, and thereby enhance the ability of the immune system to attack tumors.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: M1 macrophage polarization is pro-inflammatory and antitumor.
    • Rests on an assumption: Current treatment strategies for ESCC have limited efficacy, particularly in advanced-stage patients.

  • May combine two claims.

    Interpretationreasoning

    Intervention in the efferocytosis process can alter the immune status within the tumor microenvironment, thereby inhibiting tumor progression.

    “Our study further suggests that intervention in the efferocytosis process can alter the immune status within the TME, thereby inhibiting tumor progression.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: M1 macrophage polarization is pro-inflammatory and antitumor.

  • May combine two claims.

    Interpretationown data

    TP63 and KLF5 play critical roles in regulating the carcinogenic mechanisms of C1 FN1+ macrophages, with TP63 emerging as a key driver in the regulation of TAM biological functions.

    “These findings suggest that TP63 and KLF5 play critical roles in regulating the carcinogenic mechanisms of C1 FN1+ macrophages, with TP63, in particular, emerging as a key driver in the regulation of TAM biological functions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The RNA sequencing analysis was performed in three independent experiments.

    “Three independent experiments were performed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Written informed consent was obtained from all participants.

    “Written informed consent was obtained from all the participants.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study was approved by the Ethics Committee of Ningbo Medical Center, Lihuili Hospital.

    “This study adhered to all pertinent ethical regulations and was approved by the Ethics Committee of Ningbo Medical Center, Lihuili Hospital.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All human and mouse cell lines used were authenticated using STR profiling.

    “All human and mouse cell lines (EC9706, KYSE180, THP-1, AKR, and RAW264.7) were authenticated using short tandem repeat (STR) profiling.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The study's relatively small sample size (nine patients) may limit the generalizability of its findings.

    “The relatively small sample size (nine patients) may limit the generalizability of our findings”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Animal experiments were approved by the IACUC of Ningbo University.

    “All animal experiments were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) of Ningbo University (Approval No. 13124).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All cell lines were regularly tested and confirmed free of mycoplasma contamination.

    “All cell lines were regularly tested and confirmed to be free of mycoplasma contamination.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the multiplex immunohistochemistry analysis of human samples, two specialist pathologists blinded to patient information evaluated all specimens.

    “Two specialist pathologists (blinded to the patient’s information) evaluated all specimens.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The data in this study are expressed as the mean ± SEM of three independent replicates.

    “Data are expressed as the mean ± SEM of three independent replicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The truncated RAC2 promoter constructs used in the luciferase reporter assay were verified by sequencing.

    “Truncated RAC2 promoter constructs were cloned into pGL3-Basic and verified by sequencing.”
    Quote in the paperNot a substantial claim
65 of 65

  • The paper relies on this without support.

    TAMs are the most abundant immune cells in the tumor microenvironment.

    “TAMs are the most abundant immune cells in the TME, and they can evolve a variety of malignant phenotypes to interfere with the development of ESCC and occupy a dominant position in the immune microenvironment of ESCC.”

  • The paper relies on this without support.

    When tumor cells actively proliferate, their tissue differentiation ability gradually decreases.

    “When tumor cells actively proliferate, their tissue differentiation ability gradually decreases.”

  • The paper relies on this without support.

    Immune function in tumors is closely linked to the efferocytosis activity of TAMs.

    “Immune function in tumors is closely linked to the efferocytosis activity of TAMs.”

  • The paper relies on this without support.

    M1 macrophage polarization is pro-inflammatory and antitumor.

    “shifted macrophage polarization toward the M1 phenotype, which is pro-inflammatory and antitumor.”

  • The paper relies on this without support.

    Current treatment strategies for ESCC have limited efficacy, particularly in advanced-stage patients.

    “However, these methods have limited efficacy, particularly in advanced-stage patients.”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different mouse strains reported for the peritoneal efferocytosis model

    Major

    The Figure 7 caption identifies the efferocytosis model as C57BL/6, whereas the method for that experiment says the apoptotic cells were injected into BALB/c mice.

  • Thymus assay mouse sex conflicts with stated protocol

    Major

    The animal-culture section says all in vivo experiments used female mice, but the thymus efferocytosis assay specifies male mice.

  • HYSE180 and KYSE180 are given as the second cell-line name

    Minor

    The functional-assay description calls the second esophageal carcinoma cell line HYSE180, whereas the cell-culture methods identify it as KYSE180.

  • Figure data summaries conflict with the statistical-analysis section

    Minor

    The Figure 5 caption reports mean ± SD from five independent experiments, while the statistical-analysis section says data are expressed as mean ± SEM from three independent replicates.

4 of 4

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1080/21655979.2022.2060720

    Info

    The cited work was retracted on 2026-10-01 (notice 10.1080/21655979.2026.2728890), not before this paper was published (2026-01).

    Cited work 10.1080/21655979.2022.2060720 · retracted 2026-10-01 · notice 10.1080/21655979.2026.2728890 · this paper published 2026-01

1 of 1

Cites

1 retracted

Flagged first, then by citations

Cited by

Sorted by citations

Authors
Xi, Y., Zhao, Z., Zhou, Y., Yin, C., Li, Y., Xu, X., Jin, W., Lin, C., Zhao, K., Kuang, J., Hong, S., Yu, B., Conde, J., Liu, S., & Shen, W.
Affiliations
  • Ningbo University (CN)
  • Shanghai Jiao Tong University (CN)
  • University of Malaya (MY)
  • Peking University (CN)
  • Fudan University (CN)
  • City University of Hong Kong (HK)
  • Yunnan University of Traditional Chinese Medicine (CN)
  • Guangxi Medical University (CN)
  • First Affiliated Hospital of Nanchang University (CN)
  • Comprehensive Health Research Centre (PT)
  • Second Affiliated Hospital of Xi'an Jiaotong University (CN)
Corresponding author
  • Hong, Senlian
  • Yu, Bentong
  • Conde, João
  • Liu, Shiyuan
  • Shen, Weiyu
ORCID
Conde, João
Venue
Cell Reports Medicine
Publisher
Elsevier BV
Published
2026-01
Volume
7
Issue
1
Pages
102529
ISSN
2666-3791 (ISSN-L)
DOI
10.1016/j.xcrm.2025.102529
PMC
PMC12866140
OpenAlex
W4417534926
Semantic Scholar
6a81bd6bb24c34770a6006f22eeac8bfbd1de423
Cited by
12
References
57
Citations by year
2026: 12
FWCI
3.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Medicine (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, other-oa)
  • CityU Scholars (submittedVersion, cc-by-nc-nd)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
Funders
  • Fundação para a Ciência e a Tecnologia (UID/04923, LISBOA2030-FEDER-00862500-14998)
  • Ningbo Municipal Natural Science Foundation (2022J256)
  • Natural Science Foundation of Jiangxi Province (20252BAC240428)
  • National Key Research and Development Program of China (2022YFC3400803)
  • National Natural Science Foundation of China (82560462)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Health Sciences › Medicine › Surgery › Esophageal Cancer Research and Treatment
Keywords
macrophage efferocytosis, p63, Rac2, esophageal squamous cell carcinoma, tumor microenvironment, immunosuppression, antitumor immunity, tumor progression, therapeutic target
MeSH terms
Esophageal Squamous Cell Carcinoma, Efferocytosis, RAC2 GTP-Binding Protein, Animals, Esophageal Neoplasms, Female, Humans, Macrophages, Male, Phagocytosis, Transcription Factors, Signal Transduction, Apoptosis, rac GTP-Binding Proteins, Tumor Suppressor Proteins, Cell Line, Tumor, Mice, Tumor Microenvironment

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-19.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-19.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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