Spatial Multi-Omics Unveils Tumor Ecosystem Heterogeneity in Advanced NSCLC: The Empowering Role of GeoMx DSP and ANT BIO PTE. LTD.’s Multiplex IHC

Lung cancer remains one of the most prevalent malignant tumors globally and ranks first among the causes of death from malignant tumors in urban populations. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) being the most common subtypes. In recent years, immunotherapy has achieved remarkable progress in the treatment of malignant tumors. However, malignant tumors often exhibit complex intratumoral heterogeneity (ITH), which cannot be fully explored by traditional sequencing techniques. This limitation hinders the in-depth research of immunotherapy, resulting in many patients failing to benefit from it even in the current era where immunotherapy is highly recognized.

In February 2023, Professor Caicun Zhou's team from Tongji University School of Medicine published an online research paper entitled "Spatial multi-omics revealed the impact of tumor ecosystem heterogeneity on immunotherapy efficacy in patients with advanced non-small cell lung cancer treated with bispecific antibody" in Journal for ImmunoTherapy of Cancer. The research team employed Digital Spatial Profiling (DSP) technology for spatial segmentation to identify ITH in advanced NSCLC and distinguish efficacy-related biomarkers derived from tumor or stromal regions. Post-segmentation analysis revealed that stromal regions contained more genetic information, which is conducive to better predicting the efficacy of immunotherapy. Notably, multiplex fluorescence immunohistochemistry (mIHC) kits from the Absin product line of ANT BIO PTE. LTD. played a crucial role in validating the expression of key immune markers in different regions, providing direct evidence for the correlation between spatial molecular characteristics and treatment response.

1. Literature Information

•         Title: Spatial multi-omics revealed the impact of tumor ecosystem heterogeneity on immunotherapy efficacy in patients with advanced non-small cell lung cancer treated with bispecific antibody

•         Journal: Journal for ImmunoTherapy of Cancer

•         Publication Date: Online in February 2023

•         DOI: 10.1136/jitc-2022-006234

•         Research Team: Caicun Zhou et al., Tongji University School of Medicine

•         PMID: 36854570

•         PMCID: PMC9980352

•         Core Reagents from ANT BIO PTE. LTD.: Multiplex Fluorescence IHC Staining Kits (Absin product line), including 4-color to 7-color plus kits (e.g., Catalog No.: abs50012, abs50015) and supporting reagent Antibody Elution Buffer (mIHC-Specific, Catalog No.: abs994)

•         Related Product Link: AntBio - Fueling Research, Feeding Discovery

2. Research Background

Intratumoral heterogeneity (ITH) is a major challenge in the clinical application of immunotherapy for NSCLC. Traditional sequencing technologies, such as bulk sequencing, cannot capture the spatial distribution and interaction of molecules within tumors, making it difficult to comprehensively understand the complexity of the tumor ecosystem and accurately predict treatment efficacy. Bispecific antibodies targeting PD-L1 and CTLA-4 (e.g., KN046) have shown therapeutic potential in advanced NSCLC, but the response rate varies among patients. Therefore, there is an urgent need to develop spatial multi-omics technologies to dissect ITH, identify region-specific efficacy-related biomarkers, and provide a theoretical basis for personalized immunotherapy.

3. Research Strategy

The research team adopted an integrated approach combining spatial multi-omics (DSP-based transcriptomics and proteomics) and multiplex fluorescence IHC to systematically explore the impact of tumor ecosystem heterogeneity on immunotherapy efficacy:

1.       Study Design and Patient Cohort: Enrolled advanced NSCLC patients who had failed or were intolerant to platinum-based chemotherapy and received intravenous KN046 (a bispecific antibody targeting PD-L1 and CTLA-4) every 14 days. Tumor biopsy samples were collected, and treatment response was evaluated.

2.       Spatial Segmentation by DSP Technology: Combined morphological marker staining (Syto 13, PanCK, CD45) with digital optical barcode technology to separate tumor or stromal cells from surrounding cells. Regions of Interest (ROIs) were selected and further segmented into small tumor or stromal Areas of Interest (AOIs) for subsequent DSP detection of proteins and RNA.

3.       Spatial Transcriptomics and Proteomics Analysis: Performed protein DSP assays using UV-photocleavable oligo antibodies and RNA DSP assays using UV-photocleavable oligo probes. Protein counting was conducted by nCounter, and RNA counting by NGS. Correlation analysis of RNA and protein expression was performed between tumor and stromal AOIs.

4.       ITH Evaluation: Analyzed the expression patterns of proteins and RNA in spatially distinct AOIs of each sample to assess ITH. Characterized the molecular profiles of different regions in each sample, and explored the relationship between spatial distance and molecular clustering patterns of AOIs. Pairwise analysis of tumor and stromal AOIs from the same ROI was conducted to compare their molecular correlations.

5.       Validation by Multiplex IHC: Selected 3 samples from the Partial Response (PR) group and Progressive Disease (PD) group for 4-marker 5-color mIHC validation (detection of CD11c, Tim-3, CD45, CD4) using ANT BIO PTE. LTD.’s Absin kits. Compared the expression of these proteins in the stromal regions between the PR and PD groups.

6.       Clinical Correlation Analysis: Calculated scores of spatially resolved markers based on efficacy-related DSP protein markers. Performed multivariate Cox regression analysis to evaluate the impact of stromal and tumor signals on clinical outcomes. Validated the predictive value of stromal region-related molecular signatures in an independent cohort of 65 NSCLC patients receiving immune checkpoint inhibitor (ICI) treatment from public datasets.

4. Key Research Findings

4.1 Moderate Positive Correlation Between RNA and Protein Expression in NSCLC Spatial Multi-Omics

DSP-based spatial transcriptomics and proteomics analysis of advanced NSCLC samples showed a moderate positive correlation between RNA and protein expression. This finding confirms the reliability of integrating multi-omics data to explore the molecular characteristics of tumor ecosystems, providing a solid foundation for subsequent efficacy-related biomarker screening.

4.2 Distinct Expression Patterns of AOIs in Tumors Identified by DSP

Significant differences in protein and RNA levels were observed between tumor and stromal AOIs from the same sample. Immunocyte markers (such as CD3, CD4, CD8, CD45, CD11c) were significantly increased in stromal regions. The characteristic scores of the immune cell profiling core and immune cell typing modules were significantly higher in stromal AOIs. Analysis of 12 tumor samples with clear positional relationships between different AOIs showed that the spatial distance of tumor AOIs was consistent with the molecular clustering pattern in 8 samples. Molecular clustering inferred that the relationships between different regions reflected the tumor development path. In addition, the correlation between tumor and stromal regions from the same ROI was significantly higher than that between different ROIs, and the correlation between tumor AOIs was the strongest at both protein and RNA levels.

4.3 DSP Enables Assignment of Biological Observations to Specific Phenotypic Cell Types in Complex Tumor Ecosystems

Quantification of transcripts marking different immune cell types by DSP revealed that the abundance of lymphocytes in tumor AOIs was significantly lower than that in stromal AOIs. Macrophages, CD4+ T cells, fibroblasts, monocytes, B cells, and CD8+ T cells were the most frequently detected cell types in stromal AOIs. Scores of lymphocyte trafficking and lymphocyte regulation signaling pathways were associated with stromal AOIs. Evaluation of the expression programs of AOIs in each patient revealed cancer-related cellular functions, signaling pathways, and metabolic processes.

4.4 Identification of DSP Markers Reveals the Impact of ITH on Screening Efficacy-Related Biomarkers

DSP protein data and simulated bulk sequencing data consistently showed that the expression of CD45, CD4, GITR, and CD11c was increased in the PR group. The fold changes of most protein markers (PR/PD) in stromal regions, tumor regions, and simulated bulk sequencing data were consistent, with a few proteins showing differences. Multiplex IHC validation results (using ANT BIO PTE. LTD.’s kits) showed that the expression of CD11c, Tim-3, CD45, and CD4 in the stromal regions of the PR group was significantly higher than that of the PD group. In addition, the correlation between tumor and immune DSP markers was distinguishable, indicating different responses of tumor and immune cell populations to treatment efficacy. The abundance of four types of immune cells in the stromal regions of PR patients was significantly higher than that of PD patients. Analyses such as IO drug and stromal pan-tumor signature scores also showed that the proportion of tumor regions in PR patients was significantly higher.

Further analysis of tumor and stromal changes under different treatment methods showed that after KN046 treatment, the levels of Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Keratin 5 were significantly increased, indicating that high ALCAM expression was associated with poor prognosis of NSCLC. Subsequently, the team constructed a panel using related genes to explore the differences in immune exhaustion-related pathways before and after treatment, and found that immune exhaustion signatures were significantly reduced after resistance to bsAb-KN046.

4.5 Spatially Resolved Signatures of Stromal Regions Have Greater Clinical Response Correlation Than Tumor Regions

There were significant differences in the signature scores of tumor and stromal regions between PD and PR patients. Multivariate Cox regression showed that stromal and tumor signals were favorable factors affecting clinical outcomes. Compared with tumor markers and traditional biomarkers such as PD-L1 and Tumor Mutation Burden (TMB), markers from stromal regions showed stronger clinical correlation. Patients with high stromal signature scores had longer median Overall Survival (OS) than those with low scores. Validation in an independent cohort showed that molecules from stromal regions may have greater potential in evaluating the clinical efficacy of immunotherapy, with an AUC of 0.776 for the ROC curve of the validation cohort.

5. Product Empowerment: The Critical Role of ANT BIO PTE. LTD.’s Multiplex IHC Kits

Validating the expression differences of key immune markers in stromal regions between PR and PD groups was a key step in this study, as it provided direct evidence for the clinical significance of stromal region immune infiltration. ANT BIO PTE. LTD.’s Absin multiplex fluorescence IHC kits delivered the high-performance multiplexing capability, sensitivity, and specificity required for this critical validation, enabling the simultaneous detection of multiple immune markers in NSCLC tissue sections.

5.1 Core Products and Their Application Value

5.2 Technical Value in Tumor Immunology Research

In tumor immunology research, the accurate detection of immune cell infiltration and marker expression in specific tumor regions is critical for understanding the tumor immune microenvironment and predicting immunotherapy efficacy. ANT BIO PTE.

LTD.’s multiplex fluorescence IHC kits enable researchers to simultaneously detect multiple key immune markers on a single tissue section, preserving the native spatial context of immune cell distribution—a significant advantage over traditional single-marker IHC. In this NSCLC research, the kits’ high sensitivity and specificity ensured clear detection of low-abundance immune markers in complex tumor tissue samples, while their ability to multiplex multiple markers allowed for the comprehensive analysis of immune cell infiltration patterns in stromal regions. This technical empowerment was instrumental in validating the clinical significance of stromal region immune infiltration, highlighting the value of ANT BIO PTE. LTD.’s products in tumor immunology research and personalized immunotherapy guidance.

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 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.

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