ChIP-seq Combined with Multiplex IHC: ANT BIO PTE. LTD. Reagents Unveil the Potential Mechanism of p53 Regulating Tumor Immune Microenvironment

p53 mutation, the most common genetic alteration in human cancers, occurs in over half of all malignancies, leading to transcriptional inactivation. However, the mechanism by which p53 regulates the "immune landscape" to facilitate tumor immune escape remains elusive. A pivotal study published in a high-impact journal (IF=25.5) revealed that cancer stem cells (CSCs) establish an interleukin-34 (IL-34)-orchestrated niche to promote tumorigenesis and progression in p53-deficient hepatocellular carcinoma (HCC). Mechanistically, IL-34 is a gene transcriptionally repressed by p53, and p53 deletion induces CSCs to secrete IL-34. IL-34 upregulates CD36-mediated fatty acid oxidation (FAO) metabolism, thereby driving M2-like polarization of foam-like tumor-associated macrophages (TAMs). These IL-34-orchestrated TAMs inhibit CD8+ T cell-mediated anti-tumor immunity, promoting immune escape. Blocking the IL-34-CD36 axis elicits anti-tumor immunity and synergizes with anti-PD-1 immunotherapy to achieve a complete response. This study uncovers the potential mechanism of p53 regulating the tumor immune microenvironment (TME) and provides a promising target for immunotherapy of p53-inactivated cancers. Notably, multiple core reagents from ANT BIO PTE. LTD., including ChIP kits, multiplex IHC kits, and specific antibodies, played a crucial role in validating the key mechanisms of this research.

1. Literature Information

Title: Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation

Impact Factor: 25.5

Publication Date: Relevant data based on research context: 2024

Core Reagents from ANT BIO PTE. LTD.: Chromatin Immunoprecipitation (ChIP) Kit (Catalog No.: abs50034); DNA Pull Down Kit (Animal, Catalog No.: abs50074); 4-Color Multiplex Fluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody, Catalog No.: abs50012); BODIPY 493/503 (Catalog No.: abs825191); In Vivo anti-Mouse PD-1 Recombinant Monoclonal Antibody (R071, Catalog No.: abs172153); FITC Rat anti-Mouse F4/80 Antibody (BM8, Catalog No.: abs1850175); PE Mouse anti-Human CD206 Antibody (15-2, Catalog No.: abs1840268); FITC Mouse anti-Human IFN-γ Antibody (GAMMA3-11.1, Catalog No.: abs1840791)

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2. Research Background

Hepatocellular carcinoma (HCC) is a highly malignant tumor with high morbidity and mortality. The p53 tumor suppressor gene plays a critical role in regulating cell cycle, apoptosis, and DNA repair. However, p53 mutation or deletion is frequent in HCC, leading to the loss of its tumor suppressor function and promoting tumor progression. Increasing evidence suggests that p53 inactivation is closely associated with the establishment of an immunosuppressive tumor microenvironment, which is a key driver of tumor immune escape. Nevertheless, the specific molecular pathways linking p53 inactivation to immune microenvironment remodeling remain unclear.

IL-34, a cytokine involved in monocyte proliferation, survival, and differentiation into macrophages, has been reported to be associated with tumor progression. However, the regulatory relationship between p53 and IL-34, as well as the role of IL-34 in p53-deficient HCC immune escape, has not been fully elucidated. This study aimed to fill this knowledge gap by exploring the potential mechanism of p53 regulating the tumor immune microenvironment through IL-34, providing a theoretical basis for the development of targeted immunotherapies for p53-inactivated HCC.

3. Research Methodology

The research team adopted a multi-technical approach, integrating transcriptomics, molecular biology, immunology, and imaging techniques to systematically explore the regulatory mechanism of p53 in the HCC immune microenvironment. Key methodologies included:

•         Transcriptomic Analysis: RNA sequencing was performed on Trp53 wild-type (Trp53WT) and Trp53-deficient (Trp53null) tumors to identify differentially expressed genes, with a focus on IL-34.

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•         Molecular Biology Validation: Quantitative real-time PCR (q-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot were used to verify the expression level of IL-34 in CSCs of Trp53WT and Trp53null tumor samples.

•         Chromatin Immunoprecipitation (ChIP) Assays: The ChIP Kit from ANT BIO PTE. LTD. (Catalog No.: abs50034) was used to perform ChIP-seq, ChIP-qPCR, and DNA pull-down experiments to confirm whether p53 directly represses IL-34 transcription.

•         Immune Cell Composition Analysis: CIBERSORT was used to analyze the composition of liver tumor immune cells and explore the correlation between IL-34 expression and macrophage proportion.

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•         Spatial Transcriptomics and Multiplex IHC Staining: Spatial transcriptome sequencing and ANT BIO PTE. LTD.'s 4-color multiplex fluorescence IHC kit (Catalog No.: abs50012) were used to determine the spatial distribution of TAMs in Trp53null tumors and their colocalization with IL-34-secreting CSCs.

•         Functional Assays: BODIPY fatty acid probes (Catalog No.: abs825191, ANT BIO PTE. LTD.) were used to stain TAMs to detect lipid accumulation. Co-culture experiments of Trp53+/+ or Trp53-/- cells with bone marrow-derived macrophages were conducted to evaluate the effect of IL-34 on TAM lipid accumulation and pro-tumor polarization.

•         In Vivo Animal Experiments: Il34-overexpressing CT cells (Trp53+/+) and Il34-knockout PT cells were inoculated into mice to verify the role of IL-34 in tumor growth. Anti-IL-34, anti-CD36 neutralizing antibodies, and anti-PD-1 antibodies (Catalog No.: abs172153, ANT BIO PTE. LTD.) were used for therapeutic intervention to evaluate the anti-tumor effect of blocking the IL-34-CD36 axis alone or in combination with anti-PD-1 immunotherapy.

4. Key Research Findings

Through systematic investigation, the research team uncovered the key mechanism by which p53 inactivation drives HCC immune escape via the IL-34-CD36 axis. The key findings are summarized as follows:

4.1 p53-Inactivated CSCs Are the Main Source of IL-34 Secretion

RNA sequencing, q-PCR, ELISA, and Western blot results showed that IL-34 expression in CSCs of Trp53null tumor samples was significantly higher than that in Trp53WT tumors. Multiplex IHC staining results (using ANT BIO PTE. LTD.'s 4-color multiplex IHC kit) demonstrated that IL-34 and EpCAM (a CSC marker) were colocalized in Trp53null tumors, and EpCAM+ CD47+ CSCs in Trp53null tumors did not express p21. These results collectively indicated that p53-inactivated CSCs are the main cell subset secreting IL-34.

4.2 p53 Directly Represses IL-34 Transcription

To verify the hypothesis that p53 may repress IL-34 transcription, the research team combined ChIP-seq, ChIP-qPCR, and DNA pull-down experiments using ANT BIO PTE. LTD.'s ChIP Kit (abs50034) and DNA Pull Down Kit (abs50074). The results confirmed that p53 can directly bind to the promoter regions of IL-34 and CDKN1A (p21, positive control), thereby directly repressing IL-34 transcription.

4.3 IL-34 Induces Foam-Like TAM Formation Near CSCs

CIBERSORT analysis showed that IL-34 expression was positively correlated with macrophage proportion. Spatial transcriptome sequencing and multiplex IHC results indicated that most TAMs in Trp53null tumors were monocyte-derived macrophages (MDMs), and these TAMs accumulated near IL-34-secreting CSCs. Gene set enrichment analysis revealed that TAMs from Trp53null tumors were enriched in lipid droplet characteristics. Staining of TAMs with BODIPY fatty acid probes (abs825191, ANT BIO PTE. LTD.) showed that TAMs in Trp53null tumors were similar to foam-like macrophages. Differential gene analysis showed that the proportions of CD206+ and CD163+ macrophages (M2-like TAMs) in Trp53null tumors were increased. Co-culture experiments further confirmed that IL-34 promoted lipid accumulation and pro-tumor polarization of TAMs in Trp53null tumors.

4.4 IL-34-CD36 Axis Promotes Tumor Immune Escape by Inhibiting CD8+ T Cell Function

The research team demonstrated that IL-34 promotes fatty acid uptake through CD36 and induces pro-tumor polarization of macrophages through FAO metabolic reprogramming. In vivo experiments showed that the proportion of TAMs in Il34-overexpressing tumors was higher than that in control CT tumors. Systemic depletion of macrophages by injecting clodronate liposomes eliminated the difference in tumor growth between Il34-deficient PT tumors and control PT tumors. Flow cytometry and co-culture experiments indicated that TAMs orchestrated by the IL-34-CD36 axis promote tumor immune escape by inhibiting T cell-mediated anti-tumor immunity in Trp53null HCC.

4.5 Blocking the IL-34 Signal Inhibits Tumor Growth and Synergizes with Anti-PD-1 Immunotherapy

In mouse experiments, treatment of Trp53/PT tumor-bearing mice with anti-IL-34 or anti-CD36 neutralizing antibodies significantly inhibited tumor growth. Analysis of public single-cell RNA-seq data of TP53-mutated HCC showed that tumor cells highly expressed IL-34, and TAMs showed strong CD36 expression, while their expression in all other cell types was weak. Furthermore, combined treatment with anti-IL-34 and anti-PD-1 antibodies (abs172153, ANT BIO PTE. LTD.) achieved a more significant anti-tumor effect than single-agent treatment, indicating that blocking IL-34 signaling may be a potential immunotherapeutic strategy for patients with TP53-mutated cancers.

5. Product Empowerment: The Critical Role of ANT BIO PTE. LTD.'s Reagents

The validation of key molecular mechanisms and cellular interactions in this study relied heavily on high-quality experimental reagents. A series of core products from ANT BIO PTE. LTD. provided reliable technical support for the smooth progress of the research, ensuring the accuracy and credibility of the experimental results.

5.1 Core Products and Their Application Value

5.2 Technical Value in Mechanistic Exploration

The exploration of the molecular mechanism in this study involved multiple technical links, from transcriptional regulation verification to cellular interaction observation and in vivo therapeutic effect evaluation. ANT BIO PTE. LTD.'s products provided comprehensive technical support for these links. The ChIP Kit and DNA Pull Down Kit ensured the accurate verification of the direct transcriptional regulation relationship between p53 and IL-34; the 4-color multiplex IHC kit enabled the clear observation of the spatial colocalization of CSCs and IL-34, as well as TAMs and CSCs, providing direct evidence for cellular interactions; specific antibodies and BODIPY probes facilitated the accurate identification of cell subsets and the detection of key biological characteristics (lipid accumulation, cytokine expression); the in vivo anti-PD-1 antibody provided a reliable tool for evaluating the synergistic therapeutic effect. These high-quality products collectively ensured the smooth progress of the research and the credibility of the conclusions, highlighting the important role of reliable experimental reagents in cutting-edge life science research.

6. Brand Mission

As a professional supplier of life science reagents, ANT BIO PTE. LTD. is dedicated to providing high-quality, reliable products and comprehensive solutions to empower global life science research. The company's three specialized sub-brands cover the full spectrum of research needs in the life science field: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. Our core mission is to bridge the gap between cutting-edge scientific research and practical applications, accelerate the pace of scientific discovery, and contribute to the advancement of human health and regenerative medicine.

7. Related Product List

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work titled "Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation" (IF=25.5). All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

9. Brand Promotion Copy

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