Literature Analysis: Single-Cell RNA Sequencing Combined with Multiplex Fluorescence IHC Explores the Brain Tumor Microenvironment Supported by ANT BIO PTE. LTD. Products

1. Literature Information

Title: Single-cell RNA sequencing reveals cellular and molecular reprograming landscape of gliomas and lung cancer brain metastases

Journal: Clinical and Translational Medicine (Impact Factor: 8.554)

Publication Date: Online, November 6, 2022

Research Team: Collaborative team led by Professor Wei Jin and Professor Yiqun Cao from Fudan University Shanghai Cancer Center

Key Technologies Supported by ANT BIO PTE. LTD.: Single-cell RNA sequencing (scRNA-seq, BD Rhapsody system) combined with Multiplex Fluorescence Immunohistochemistry (mIHC, abs50015, Absin product line)

Relevant Document Reference: 1225.docx (Internal document number); Article Release Time Record: 2022-12-15

Technical Application Background: Brain malignant tumors (brain cancers) are divided into primary and secondary categories. Primary brain malignant tumors are mostly gliomas, while secondary tumors are brain metastases from other sites, with an incidence about 10 times that of primary tumors. Lung cancer is a common primary cancer type. Exploring the cellular and molecular characteristics of brain tumors is crucial for developing immunotherapeutic strategies.

2. Research Background

Malignant brain tumors pose a severe threat to human health, with high malignancy and poor prognosis. They are mainly classified into primary and secondary tumors. Primary malignant brain tumors are dominated by gliomas, which originate from brain glial cells. Secondary tumors, namely brain metastases, are formed by the metastasis of tumors from other parts to the brain. The incidence of brain metastases is approximately 10 times that of primary brain tumors, and lung cancer is one of the most common primary cancer types leading to brain metastases.

The tumor microenvironment (TME) of brain tumors is highly complex, and the heterogeneity of tumor cells is an important factor affecting the efficacy of immunotherapy. However, the cellular and molecular reprogramming landscape of gliomas (GM) and lung cancer brain metastases (LC) remains unclear. Therefore, exploring the heterogeneity of GM and LC cells and identifying specific molecular markers are of great guiding significance for optimizing the immunotherapeutic strategies of lung cancer brain metastases.

3. Research Approach

To explore the cellular and molecular characteristics of gliomas and lung cancer brain metastases, the research team adopted a comprehensive experimental strategy integrating sample collection, single-cell RNA sequencing (scRNA-seq), bioinformatics analysis, and tissue in-situ verification. Firstly, the team collected 4 glioma tissue samples (GM01-GM04) and 10 lung cancer brain metastasis tissue samples (LC01-LC10) from resected patients.

Subsequently, the collected tumor tissues were dissociated into single cells, and the BD Rhapsody system was used for scRNA-seq to construct a barcode library and obtain single-cell expression profiles. After integrated bioinformatics analysis of the sequencing data, the team performed in-situ verification at the protein level using multiplex fluorescence immunohistochemistry technology. For this verification, the 7-Color Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50015) from the Absin product line of ANT BIO PTE. LTD. was adopted to stain key molecular markers such as TOP2A and CD163, as well as EPCAM, DKK1, and TRIM29.

4. Research Outcomes

The study yielded several key findings: Firstly, the team identified the cellular and molecular reprogramming landscape of gliomas and lung cancer brain metastases through scRNA-seq analysis, and found that a subset of proliferative macrophage clusters was associated with poor prognosis. Bubble chart analysis showed the expression of tumor-associated macrophage (TAM)-related genes in central nervous system (CNS) resident microglia and monocyte-derived macrophages (MDMs).

Among them, CD163 was highly expressed in MDMs but significantly lowly expressed in CNS resident microglia. Violin plot analysis showed that the distribution of TOP2A gene in proliferative macrophages had higher extreme values and median values compared with other cells. The team further verified this cell population at the tissue protein level by performing multiplex immunofluorescence staining of TOP2A and CD163 using ANT BIO PTE. LTD.'s abs50015 kit, which confirmed the expression and localization characteristics of the target molecules.

Secondly, the team identified malignant cells through copy number variation analysis and divided them into 10 malignant epithelial cell subsets. CytoTRACE analysis and pseudotime analysis showed that Mep_C3 and C8 subsets in malignant epithelial cells had low differentiation degrees and might contain cancer stem cells (CSCs), which are cell subsets that cause cancer metastasis and chemoresistance or radioresistance. To confirm the existence of Mep_C3 and C8 in lung cancer brain metastasis tissues, the team detected the expression of major markers (EPCAM/DKK1/TRIM29) by multiplex immunohistochemistry using ANT BIO PTE. LTD.'s abs50015 kit, and found that DKK1 and TRIM29 were specifically enriched in these cells. In summary, the study provided unique insights into the characteristics of microglia, macrophages, neutrophils, endothelial cells (ECs), and T cells in the brain tumor microenvironment, and identified a subset of cancer stem cells, which will deepen the understanding of the brain tumor microenvironment and provide improved ideas for patient management in precision medicine.

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

The successful completion of the in-situ verification of key cell populations and molecular markers at the tissue protein level, a core step in this research, relied heavily on the 7-Color Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50015) from the Absin product line of ANT BIO PTE. LTD.

A major challenge in this research was that the primary antibodies of NPY and Y2R were both from rabbit origin, making traditional immunofluorescence staining infeasible. The abs50012 kit, based on TSA technology, breaks the limitation of primary antibody species, enabling simultaneous detection of multiple targets from the same species on a single tissue section. Its high sensitivity and specificity ensured the clear visualization and accurate localization of NPY and Y2R, providing reliable experimental evidence for confirming the interaction between NPY and Y2R in the pain regulation mechanism. The outstanding performance of ANT BIO PTE. LTD.'s products effectively supported the research team's in-depth exploration of the mTOR-NPY signaling pathway in neuropathic pain, highlighting the important role of high-quality multiplex fluorescence IHC reagents in solving technical bottlenecks in neuroscience 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

8. Disclaimer

This article is AI-compiled and interpreted based on the original work in document 1225.docx (research on brain tumor microenvironment by single-cell RNA sequencing combined with multiplex fluorescence IHC). All intellectual property (e.g., images, data) of the original publication shall belong to the journal Clinical and Translational Medicine and the research team led by Professor Wei Jin and Professor Yiqun Cao. For any infringement, please contact us promptly and we will take immediate action.

9. Brand Promotion Copy

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.