CoChat
Sign Up
Cited by 129Immune cells in cancerCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma

Open access
Yu, Zhuo, Li, Yuyao, Li, Yue, Zhang, Jinghao, Li, Man, Ji, Longshan, Tang, Yifei, Zheng, Yanxi +8 more·2022·Journal for ImmunoTherapy of Cancer
DOI 10.1136/jitc-2021-004297PMC PMC9125767
Open this paper in CoChat — free accountOpen PDF

Abstract

Background: Immunotherapy for hepatocellular carcinoma (HCC) exhibits limited clinical efficacy due to immunosuppressive tumor microenvironment (TME). Tumor-infiltrating macrophages (TIMs) account for the major component in the TME, and the dominance of M2 phenotype over M1 phenotype in the TIMs plays the pivotal role in sustaining the immunosuppressive character. We thus investigate the effect of bufalin on promoting TIMs polarization toward M1 phenotype to improve HCC immunotherapy. Methods: The impact of bufalin on evoking antitumor immune response was evaluated in the immunocompetent mouse HCC model. The expression profiling of macrophage-associated genes, surface markers and cytokines on bufalin treatment in vitro and in vivo were detected using flow cytometry, immunofluorescence, western blot analysis, ELISA and RT-qPCR. Cell signaling involved in M1 macrophage polarization was identified via the analysis of gene sequencing, and bufalin-governed target was explored by immunoprecipitation, western blot analysis and gain-and-loss of antitumor immune response. The combination of bufalin and antiprogrammed cell death protein 1 (anti-PD-1) antibody was also assessed in orthotopic HCC mouse model. Results: In this study, we showed that bufalin can function as an antitumor immune modulator that governs the polarization of TIMs from tumor-promoting M2 toward tumor-inhibitory M1, which induces HCC suppression through the activation of effector T cell immune response. Mechanistically, bufalin inhibits overexpression of p50 nuclear factor kappa B (NF-κB) factor, leading to the predominance of p65-p50 heterodimers over p50 homodimers in the nuclei. The accumulation of p65-p50 heterodimers activates NF-κB signaling, which is responsible for the production of immunostimulatory cytokines, thus resulting in the activation of antitumor T cell immune response. Moreover, bufalin enhances the antitumor activity of anti-PD-1 antibody, and the combination exerts synergistic effect on HCC suppression. Conclusions: These data expound a novel antitumor mechanism of bufalin, and facilitate exploitation of a new potential macrophage-based HCC immunotherapeutic modality.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1136/jitc-2021-004297

Cites

4 of 4 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Bufalin inhibits tumor growth, regulates metastasis, and enhances antitumor immune responses by polarizing tumor-infiltrating macrophages toward the M1 phenotype.

    “Recent studies have demonstrated that Bufalin inhibits tumor growth, regulates metastasis, and enhances antitumor immune responses by polarizing tumor-infiltrating macrophages toward the M1 phenotype2.”

    From Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha (2025) · cites this paper as [2]

    The Results section reports that bufalin shifts tumor-infiltrating macrophages toward M1, and the Conclusions link that shift to antitumor T-cell responses and an antitumor effect. The Background mentions earlier studies associating suppressed HCC metastasis with immune-cell modulation, but this paper does not establish that M1 polarization regulates metastasis. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Bufalin is a potent anticancer agent used clinically in China to treat various cancers.

    “Bufalin, an active monomer extracted from the Chansu, is a potent anticancer agent used clinically in China to treat various cancers1,2.”

    From Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha (2025) · cites this paper as [2]

  • Recent studies indicated that Bufalin played an important role in the tumor microenvironment by inducing activation of the NF-κB pathway.

    “Recent studies indicated that Bufalin played an important role in the tumor microenvironment by inducing the activation of the NF-κB pathway2.”

    From Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha (2025) · cites this paper as [2]

Self-check

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

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 38

  1. [1]Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin 2020; 70: 7– 30. 10.3322/caac.21590 31912902 · DOI · Paper page
  2. [2]Huang A, Yang X-R, Chung W-Y, et al. Targeted therapy for hepatocellular carcinoma. Signal Transduct Target Ther 2020; 5: 146. 10.1038/s41392-020-00264-x 32782275 PMC7419547 · DOI
  3. [3]Yoh T, Seo S, Taura K, et al. Surgery for recurrent hepatocellular carcinoma: achieving long-term survival. Ann Surg 2021; 273: 792– 9. 10.1097/SLA.0000000000003358 31058698 · DOI
  4. [4]Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet 2018; 391: 1301– 14. 10.1016/S0140-6736(18)30010-2 29307467 · DOI
  5. [5]Kurebayashi Y, Ojima H, Tsujikawa H, et al. Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification. Hepatology 2018; 68: 1025– 41. 10.1002/hep.29904 29603348 · DOI
  6. [6]Lu C, Rong D, Zhang B, et al. Current perspectives on the immunosuppressive tumor microenvironment in hepatocellular carcinoma: challenges and opportunities. Mol Cancer 2019; 18: 130. 10.1186/s12943-019-1047-6 31464625 PMC6714090 · DOI
  7. [7]Binnewies M, Roberts EW, Kersten K, et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med 2018; 24: 541– 50. 10.1038/s41591-018-0014-x 29686425 PMC5998822 · DOI · Paper page
  8. [8]Chan L-K, Tsui Y-M, Ho DW-H, et al. Cellular heterogeneity and plasticity in liver cancer. Semin Cancer Biol 2021; 2. 10.1016/j.semcancer.2021.02.015 33647386 · DOI
  9. [9]Ostuni R, Kratochvill F, Murray PJ, et al. Macrophages and cancer: from mechanisms to therapeutic implications. Trends Immunol 2015; 36: 229– 39. 10.1016/j.it.2015.02.004 25770924 · DOI
  10. [10]Sica A, Invernizzi P, Mantovani A. Macrophage plasticity and polarization in liver homeostasis and pathology. Hepatology 2014; 59: 2034– 42. 10.1002/hep.26754 24115204 · DOI · Paper page
  11. [12]Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol 2010; 11: 889– 96. 10.1038/ni.1937 20856220 · DOI · Paper page
  12. [13]Li X, Yao W, Yuan Y, et al. Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy against hepatocellular carcinoma. Gut 2017; 66: 157– 67. 10.1136/gutjnl-2015-310514 26452628 · DOI
  13. [14]Mantovani A, Marchesi F, Malesci A, et al. Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol 2017; 14: 399– 416. 10.1038/nrclinonc.2016.217 28117416 PMC5480600 · DOI
  14. [15]Li H, Hu S, Pang Y, et al. Bufalin inhibits glycolysis-induced cell growth and proliferation through the suppression of integrin β2/FAK signaling pathway in ovarian cancer. Am J Cancer Res 2018; 8: 1288– 96. 30094101 PMC6079152
  15. [16]Yu Z, Feng H, Sun X, et al. Bufalin suppresses hepatocarcinogenesis by targeting β-catenin/TCF signaling via cell cycle-related kinase. Sci Rep 2018; 8: 3891. 10.1038/s41598-018-22113-2 29497076 PMC5832857 · DOI
  16. [17]Zhang Y, Dong Y, Melkus MW, et al. Role of P53-senescence induction in suppression of LNCaP prostate cancer growth by cardiotonic compound bufalin. Mol Cancer Ther 2018; 17: 2341– 52. 10.1158/1535-7163.MCT-17-1296 30166403 PMC6668018 · DOI · Paper page
  17. [19]Qi F, Inagaki Y, Gao B, et al. Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas- and mitochondria-mediated pathways. Cancer Sci 2011; 102: 951– 8. 10.1111/j.1349-7006.2011.01900.x 21288283 PMC11158054 · DOI
  18. [20]Hu F, Han J, Zhai B, et al. Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation. Apoptosis 2014; 19: 210– 23. 10.1007/s10495-013-0914-7 24114361 · DOI
  19. [21]Yang Z, Tao Y, Xu X, et al. Bufalin inhibits cell proliferation and migration of hepatocellular carcinoma cells via APOBEC3F induced intestinal immune network for IgA production signaling pathway. Biochem Biophys Res Commun 2018; 503: 2124– 31. 10.1016/j.bbrc.2018.07.169 30100060 · DOI
  20. [22]Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity 2014; 41: 14– 20. 10.1016/j.immuni.2014.06.008 25035950 PMC4123412 · DOI · Paper page
  21. [23]Yang R, Mele F, Worley L, et al. Human T-bet governs innate and innate-like adaptive IFN-γ immunity against mycobacteria. Cell 2020; 183: 1826– 47. 10.1016/j.cell.2020.10.046 33296702 PMC7770098 · DOI
  22. [24]Hagemann T, Lawrence T, McNeish I, et al. "Re-educating" tumor-associated macrophages by targeting NF-kappaB. J Exp Med 2008; 205: 1261– 8. 10.1084/jem.20080108 18490490 PMC2413024 · DOI · Paper page
  23. [26]Sly LM, Rauh MJ, Kalesnikoff J, et al. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity 2004; 21: 227– 39. 10.1016/j.immuni.2004.07.010 15308103 · DOI
  24. [27]O'Neill LAJ, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007; 7: 353– 64. 10.1038/nri2079 17457343 · DOI · Paper page
  25. [29]Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004; 25: 280– 8. 10.1016/j.it.2004.03.008 15145317 · DOI · Paper page
  26. [30]Carmody RJ, Ruan Q, Palmer S, et al. Negative regulation of toll-like receptor signaling by NF-kappaB p50 ubiquitination blockade. Science 2007; 317: 675– 8. 10.1126/science.1142953 17673665 · DOI
  27. [31]Yarchoan M, Xing D, Luan L, et al. Characterization of the immune microenvironment in hepatocellular carcinoma. Clin Cancer Res 2017; 23: 7333– 9. 10.1158/1078-0432.CCR-17-0950 28928158 PMC5881396 · DOI
  28. [32]Meng Z, Yang P, Shen Y, et al. Pilot study of huachansu in patients with hepatocellular carcinoma, nonsmall-cell lung cancer, or pancreatic cancer. Cancer 2009; 115: 5309– 18. 10.1002/cncr.24602 19701908 PMC2856335 · DOI
  29. [33]Miao Q, Bi L-L, Li X, et al. Anticancer effects of bufalin on human hepatocellular carcinoma HepG2 cells: roles of apoptosis and autophagy. Int J Mol Sci 2013; 14: 1370– 82. 10.3390/ijms14011370 23344047 PMC3565325 · DOI
  30. [34]Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest 2012; 122: 787– 95. 10.1172/JCI59643 22378047 PMC3287223 · DOI · Paper page
  31. [35]Ma Y, Mattarollo SR, Adjemian S, et al. CCL2/CCR2-dependent recruitment of functional antigen-presenting cells into tumors upon chemotherapy. Cancer Res 2014; 74: 436– 45. 10.1158/0008-5472.CAN-13-1265 24302580 · DOI
  32. [36]Pikarsky E, Porat RM, Stein I, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 2004; 431: 461– 6. 10.1038/nature02924 15329734 · DOI
  33. [37]Maeda S, Kamata H, Luo J-L, et al. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 2005; 121: 977– 90. 10.1016/j.cell.2005.04.014 15989949 · DOI
  34. [38]Wei Y, Zhao Q, Gao Z, et al. The local immune landscape determines tumor PD-L1 heterogeneity and sensitivity to therapy. J Clin Invest 2019; 129: 3347– 60. 10.1172/JCI127726 31112529 PMC6668685 · DOI
  35. [39]Mantovani A, Allavena P, Sica A, et al. Cancer-related inflammation. Nature 2008; 454: 436– 44. 10.1038/nature07205 18650914 · DOI · Paper page
  36. [40]Lawrence T. Macrophages and NF-κB in cancer. Curr Top Microbiol Immunol 2011; 349: 171– 84. 10.1007/82_2010_100 21080151 · DOI
  37. [41]Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12: 252– 64. 10.1038/nrc3239 22437870 PMC4856023 · DOI · Paper page
  38. [42]Qin S, Xu L, Yi M, et al. Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4. Mol Cancer 2019; 18: 155. 10.1186/s12943-019-1091-2 31690319 PMC6833286 · DOI · Paper page

Cited by

Sorted by citations

Authors
Yu, Z., Li, Y., Li, Y., Zhang, J., Li, M., Ji, L., Tang, Y., Zheng, Y., Sheng, J., Han, Q., Li, F., Guo, J., Wang, L., Sun, X., Gao, Y., & Feng, H.
Affiliations
  • Shanghai University of Traditional Chinese Medicine (CN)
  • Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China
  • Department of Hepatopancreatobiliary Surgery, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China
  • Jilin University (CN)
Corresponding author
  • Sun, Xuehua
  • Gao, Yueqiu
  • Feng, Hai
ORCID
Venue
Journal for ImmunoTherapy of Cancer
Publisher
BMJ
Published
2022-05
Volume
10
Issue
5
Pages
e004297
ISSN
2051-1426 (ISSN-L)
DOI
10.1136/jitc-2021-004297
PMC
PMC9125767
OpenAlex
W4281569644
Cited by
129
References
4
Citations by year
2026: 25 · 2025: 41 · 2024: 29 · 2023: 27 · 2022: 7
FWCI
10.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal for ImmunoTherapy of Cancer (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Natural Science Foundation of China (81774240, 2018–2020, 81874436, 82074154, shslczdzk01201, 81904117)
  • Shanghai University of Traditional Chinese Medicine (81774240, 81874436, 2018-2020)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
bufalin, hepatocellular carcinoma, tumor-infiltrating macrophages, M1 macrophage polarization, M2 macrophage polarization, antitumor immunity, NF-κB signaling, p50/p65 heterodimer, immunosuppressive tumor microenvironment, effector T cell activation
MeSH terms
Animals, Bufanolides, Carcinoma, Hepatocellular, Humans, Immunity, Liver Neoplasms, Macrophages, Phenotype, Cytokines, NF-kappa B, 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.

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.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review