Literature Analysis: A Novel Inflammatory Checkpoint in KRAS-Mutant Intrahepatic Cholangiocarcinoma Unveiled with Support from ANT BIO PTE. LTD. Products

1. Literature Information

Title: An inflammatory checkpoint generated by IL1RN splicing offers therapeutic opportunity for KRAS mutant intrahepatic cholangiocarcinoma

Journal: Cancer Discovery (Impact Factor: 28.1)

Research Team: Collaborative team led by Professor Qiang Gao, Dr. Jian Lin from Zhongshan Hospital Affiliated to Fudan University, and Researcher Wu Wei from Lingang Laboratory

Key Technology Supported by ANT BIO PTE. LTD.: Multiplex Fluorescence Immunohistochemistry (mIHC)

2. Research Background

Intrahepatic cholangiocarcinoma (iCCA), an adenocarcinoma arising from the epithelium of intrahepatic secondary bile ducts and their branches, constitutes 10% to 15% of primary liver malignancies. Over the past few decades, its incidence and mortality rates have exhibited a steady upward trend. Currently, surgical resection remains the primary therapeutic modality, complemented by immune checkpoint inhibitors (ICIs) and tumor microenvironment (TME)-targeted therapies. Nevertheless, effective treatment options for unresectable iCCA are still lacking, underscoring the imperative to decipher the molecular mechanisms governing iCCA progression and identify novel therapeutic targets.

Prior to this study, a collaborative team led by Academician Jia Fan, Professor Qiang Gao, Researcher Hu Zhou, and Researcher Daming Gao published a seminal work in Cancer Cell at the end of 2021. This proteogenomic characterization study identified TP53, KRAS, FGFR2, IDH1/2, and BAP1 mutations (or fusions) as the major driver genetic alterations in iCCA, laying a pivotal foundation for understanding the molecular subtypes and advancing precision medicine for this disease.

3. Research Approach

Building upon the aforementioned findings, the research team aimed to explore the association between KRAS mutations (a key driver in iCCA) and tumor-related inflammation, as well as to identify potential therapeutic checkpoints. To achieve this, the team integrated a multi-dimensional experimental strategy encompassing multi-omics analyses (RNA sequencing, whole-exome sequencing, BD single-cell RNA sequencing), multiplex fluorescence immunohistochemistry (mIHC) with products from ANT BIO PTE. LTD., fluorescence in situ hybridization (FISH), and flow cytometry.

These techniques were systematically applied to a diverse range of samples, including non-tumor tissues, clinical iCCA specimens, patient-derived cell lines, patient-derived organoids, and KRAS-mutant iCCA mouse models. This comprehensive approach ensured the robustness, reproducibility, and clinical translatability of the research findings.

4. Research Outcomes

The study established a causal link between KRAS mutations and pre-tumor inflammatory responses, confirming that KRAS mutations serve as critical drivers of iCCA tumorigenesis. Through in-depth analysis of multi-omics data from a large cohort of patients, the team discovered that KRAS mutations are correlated with specific alternative mRNA splicing events, which are closely intertwined with myeloid inflammation in iCCA.

A key breakthrough was the identification of a negative feedback regulatory mechanism: interleukin 1 receptor antagonist (IL1RN)-201/203 isoforms are upregulated via alternative splicing, exerting a potent anti-inflammatory effect in KRAS-mutant iCCA. In vivo experiments using KRAS-mutant iCCA mouse models demonstrated that both overexpression of IL1RN-201/203 and Anakinra (a recombinant IL1RN analog) administration elicited robust anti-tumor immune responses by modulating neutrophil recruitment and phenotypic polarization.

Notably, Anakinra treatment synergistically enhanced the efficacy of anti-PD-1 immunotherapy by activating GZMB+CD8+ T cells within tumors. Clinically, high levels of IL1RN-201/203 in KRAS-mutant iCCA patients were found to be strongly associated with favorable responses to anti-PD-1 immunotherapy.

The study further delineated the underlying molecular mechanism of the IL1RN-201/203-mediated negative feedback loop: KRAS mutations activate the ERK signaling pathway, which in turn regulates the expression of TFIP11. TFIP11 then promotes the alternative splicing of IL1RN to generate IL1RN-201/203. Subsequently, IL1RN-201/203 inhibits the ERK-CXCL3-CXCR2 signaling axis, thereby suppressing neutrophil recruitment and polarization. Ultimately, this pathway inhibits KRAS mutation-driven inflammatory cascades, tumor progression, and ICI resistance, highlighting IL1RN splicing-derived inflammatory checkpoint as a promising therapeutic target for KRAS-mutant cancers.

5. Product Empowerment: The Role of ANT BIO PTE. LTD. Products in the Research

Multiplex fluorescence immunohistochemistry (mIHC) technology played an indispensable role in validating key immunological phenotypes at the tissue level, and this critical technical support was provided by ANT BIO PTE. LTD.'s Absin product line.

The research team employed ANT BIO PTE. LTD.'s Absin multiplex fluorescence IHC technology to perform protein-level validation on mouse models. This enabled the successful detection of CD8+ T cell and NK cell (labeled by NK1.1) infiltration, as well as the expression of related molecules, across different treatment groups (Vec, 201, 203, Vec+Ana). The results confirmed that IL1RN-201/203 overexpression or Anakinra treatment significantly increased CD8+ T cell infiltration in Yak and KP mouse models of iCCA.

Furthermore, ANT BIO PTE. LTD.'s mIHC technology was utilized to compare the density of distinct cell subsets between IL1RN_MX PSIhigh and IL1RN_MX PSIlow KRAS-mutant iCCA samples. The analysis revealed that IL1RN_MX PSIlow samples exhibited moderately reduced densities of CXCR4+ and LSP1+ neutrophils, alongside increased densities of CXCL2+ neutrophils and GZMB+CD8+ T cells, compared to IL1RN_MX PSIhigh samples. These findings directly validated the regulatory role of IL1RN alternative splicing in shaping the tumor immune microenvironment, leveraging the high sensitivity, specificity, and multi-target co-detection capabilities of ANT BIO PTE. LTD.'s Absin multiplex fluorescence IHC products.

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.

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work in DOI: 10.1002/advs.202413562. 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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