Spatial Transcriptome Analysis Research Articles

Integrated analysis of MIOX gene in prognosis of clear-cell renal cell carcinoma

Clear-cell renal cell carcinoma (ccRCC) is a highly aggressive malignancy that originates in the kidney. It often exhibits a limited response or can be refractory to a wide range of anti-cancer therapies, including tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors. Ferroptosis is a form of oxidative, iron-dependent cell death characterized by lipid peroxidation. Targeting ferroptosis may offer a promising alternative therapeutic strategy for cancer cells that are resistant to existing treatments. The impact of ferroptosis-related genes on the prognosis of ccRCC patients is still not fully understood. In this study, we identified 30 differentially expressed ferroptosis-related genes in ccRCC samples compared to normal tissues using data from The Cancer Genome Atlas (TCGA). Lasso regression analyses, along with Kaplan-Meier analysis, were conducted to identify genes associated with prognosis. Based on scRNA-seq and spatial transcriptome analysis, we identified specificity of MIOX in ccRCC. Furthermore, MIOX demonstrated the highest significance, highlighting its independent prognostic value as a single gene in ccRCC. Our findings suggest that MIOX could serve as potential targets for therapeutic interventions in ccRCC.

Identification of Prognostic Biomarkers of Ovarian High-Grade Serous Carcinoma: A Preliminary Study Using Spatial Transcriptome Analysis and Multispectral Imaging

Ovarian cancer is a lethal malignancy, with most patients initially responding to chemotherapy but frequently experiencing recurrence. Previous studies primarily examined tumor characteristics using limited genetic markers or bulk RNA sequencing. Here, we used spatial transcriptomics via the GeoMx® platform, alongside multispectral immune cell immunofluorescence (IF), to identify biomarkers associated with disease progression following first-line treatment of high-grade serous carcinoma (HGSC). We identified several spatial biomarkers linked to non-recurrence, including elevated NKG7 expression in CD45+ immune cell regions (p = 0.0011) and higher TFPI2 and PIGR expression in tumor areas (p = 2.09 × 10−6), both associated with improved progression-free survival. Multispectral IF revealed significantly higher regulatory T cell (Treg) to CD8+ T cell ratios in the tumor nests and stroma of recurrent patients (p = 0.016, 0.048). Tregs were also found closer to cancer cells or macrophages than CD8+ T cells in recurrent tumors (p = 0.048), correlating with poor survival. Integrated analysis showed that immune cell density and immune pathway scores in the recurrent group positively correlated with cancer pathway scores, except for NF-κB. This comprehensive analysis revealed clues to interactions between different immune cells and identified biomarkers that may be useful for predicting recurrence of HGSC.

DDX24 spatiotemporally orchestrates VEGF and Wnt signaling during developmental angiogenesis

Vascular development is a precisely controlled process, yet how it is spatiotemporally orchestrated remains enigmatic. We previously identified DEAD-box RNA helicase 24 (DDX24) as a pathogenic gene for multiorgan vascular anomalies. Here, we show that DDX24 is expressed in the endothelium during embryonic angiogenesis in zebrafish. DDX24 deficiency causes intersegmental vessel hyperbranching in the trunk, but inhibits central artery angiogenesis in the brain. Mechanistically, DDX24 deficiency enhances VEGFR2 expression by direct binding to its mRNA in nonbrain endothelial cells (ECs), while suppressing GPR124/RECK-mediated Wnt signaling in brain ECs. Additionally, spatial transcriptome analysis profiles DDX24-mediated crosstalk between ECs and neighboring cells. Finally, pharmacological targeting of these two pathways in a temporal manner can rescue the phenotypes induced by DDX24 deficiency. Overall, our findings highlight an essential role for DDX24 in the spatiotemporal regulation of developmental angiogenesis.

Exploration of Mechanism of Action of Nerandomilast in Pulmonary Fibrosis Through Single Cell Rna Sequence and Spatial Transcriptomic Analysis

Exploration of Mechanism of Action of Nerandomilast in Pulmonary Fibrosis Through Single Cell Rna Sequence and Spatial Transcriptomic Analysis

Single-cell and spatial transcriptomics analysis reveals that Pros1+ oligodendrocytes are involved in endogenous neuroprotection after brainstem stroke.

Single-cell and spatial transcriptomics analysis reveals that Pros1+ oligodendrocytes are involved in endogenous neuroprotection after brainstem stroke.

Diffuse Cyclin D1 and SPINK1 Expression in Gastric Oxyntic Gland Neoplasms: Promising Diagnostic Markers Identified Using Spatial Transcriptome Analysis.

Diffuse Cyclin D1 and SPINK1 Expression in Gastric Oxyntic Gland Neoplasms: Promising Diagnostic Markers Identified Using Spatial Transcriptome Analysis.

Spatial Transcriptomic Analysis of Lung Tissue From Infants With Neuroendocrine Cell Hyperplasia of Infancy

Spatial Transcriptomic Analysis of Lung Tissue From Infants With Neuroendocrine Cell Hyperplasia of Infancy

Spatial Transcriptomic Analysis of Systemic Sclerosis-Associated Interstitial Lung Disease (SSc-ILD) Identifies Fibroblast LIF Receptor as an Amplifier of Lung and Skin Fibrosis

Spatial Transcriptomic Analysis of Systemic Sclerosis-Associated Interstitial Lung Disease (SSc-ILD) Identifies Fibroblast LIF Receptor as an Amplifier of Lung and Skin Fibrosis

FRI-326 Spatial transcriptomic analysis of archived primary sclerosing cholangitis livers reveals distinct molecular signatures

FRI-326 Spatial transcriptomic analysis of archived primary sclerosing cholangitis livers reveals distinct molecular signatures

Spatial Analysis of Hereditary Diffuse Gastric Cancer Reveals Indolent Phenotype of Signet Ring Cell Precursors

Abstract Germline CDH1 loss-of-function mutations are causally linked to an increased lifetime risk of diffuse gastric cancer (DGC). Early, multifocal signet ring cell (SRC) lesions are ubiquitous among CDH1 variant carriers, yet only a subset of patients will develop advanced DGC. A multiomic analysis was performed to establish the molecular phenotype of early SRC lesions and how they differ from advanced DGC using 20 samples from human total gastrectomy specimens of germline CDH1 variant carriers. Spatial transcriptomic analysis demonstrated reduced CDH1 gene expression and increased expression of extracellular matrix remodeling in SRC lesions compared with unaffected adjacent gastric epithelium. Single-cell RNA sequencing revealed an SRC-enriched signature with markers REG1A, VIM, AQP5, PRR4, MUC6, and AGR2. Importantly, SRC lesions lacked alterations in known drivers of gastric cancer (TP53, ARID1A, and KRAS) and activation of associated signal transduction pathways. Advanced DGC demonstrated E-cadherin reexpression, somatic TP53 and ERBB3 mutations, and upregulated CTNNA1, MYC, and MET expression when compared with SRC lesions. Implications: The marked differences in the genomic and transcriptomic profiles of SRC lesions and advanced DGC support the consideration of SRC lesions as precancers in patients with germline CDH1 mutations.

L-DOPA Induces Spatially Discrete Changes in Gene Expression in the Forebrain of Mice with a Progressive Loss of Dopaminergic Neurons.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is effective at alleviating motor impairments in Parkinson's disease (PD) patients but has mixed effects on nonmotor symptoms and causes adverse effects after prolonged treatment. Here, we analyzed the spatial profile of L-DOPA-induced gene expression in the forebrain of mice with an inducible progressive loss of dopaminergic neurons (the TIF-IADATCreERT2 strain), with a focus on the similarities and differences in areas relevant to different PD symptoms. The animals received a 14-day L-DOPA treatment, and 1h after the final drug injection, a spatial transcriptome analysis was performed on coronal forebrain sections. A total of 121 genes were identified as being regulated by L-DOPA. We found that the treatment had widespread effects extending beyond the primary areas involved in dopamine-dependent movement control. An unsupervised clustering analysis of the transcripts recapitulated the forebrain anatomy and indicated both ubiquitous and region-specific effects on transcription. The changes were most pronounced in layers 2/3 and 5 of the dorsal cortex and the dorsal striatum, where a robust increase in the abundance of activity-regulated transcripts, including Fos, Egr1, and Junb, was observed. Conversely, transcripts with a decreased abundance, e.g., Plekhm2 or Pgs1, were identified primarily in the piriform cortex, the adjacent endopiriform nucleus, and the claustrum. Taken together, our spatial analysis of L-DOPA-induced alterations in gene expression reveals the anatomical complexity of treatment effects, identifying novel genes affected by the drug, as well as molecular activation in brain areas relevant to the nonmotor symptoms of PD.

Single-nucleus RNA sequencing and spatial transcriptomics reveal the mechanism by which Xiaozhiling injection treats internal hemorrhoids.

Hemorrhoids, a prevalent chronic condition globally, significantly impact patients' quality of life. While various surgical interventions, such as external stripping and internal ligation, procedure for prolapse and hemorrhoids, and tissue selecting technique, are employed for treatment, they are often associated with postoperative complications, including unsatisfactory defecation, bleeding, and anal stenosis. In contrast, Xiaozhiling injection, a traditional Chinese medicine-based therapy, has emerged as a minimally invasive and effective alternative for internal hemorrhoids. This treatment offers distinct advantages, such as reduced dietary restrictions, broad applicability, and minimal induction of systemic inflammatory responses. Additionally, Xiaozhiling injection effectively eliminates hemorrhoid nuclei, prevents local tissue necrosis, preserves anal cushion integrity, and mitigates postoperative complications, including bleeding and prolapse. Despite its clinical efficacy, the molecular mechanisms underlying its therapeutic effects remain poorly understood, warranting further investigation. To investigate the molecular mechanism underlying the therapeutic effect of Xiaozhiling injection in the treatment of internal hemorrhoids. An internal hemorrhoid model was established in rats, and the rats were randomly divided into a modeling group [control group (CK group)] and a treatment group. One week after injection, Stereo-seq and electron microscopy were used to study the changes in gene expression and subcellular structures in fibroblasts. Single-cell sequencing revealed differences in the expression and transcript levels of the genes collagen 3 alpha 1, decorin, and actin alpha 2 in fibroblasts between the CK group and the treatment group. Spatial transcriptome analysis revealed that genes of the sphingosine kinase 1 (Sphk1)/sphingosine-1-phosphate (S1P) pathway spatially overlapped with key genes of the transforming growth factor beta 1 pathway, namely, Sphk1, S1P receptor, and transforming growth factor beta 1, in the treatment group. The proportion of fibroblasts was lower in the treatment group than in the CK group, and Xiaozhiling treatment had a significant effect on the proportion of fibroblasts in hemorrhoidal tissue. Immunohistochemistry revealed a significant increase in the expression of a fibroblast marker. Electron microscopy showed that the endoplasmic reticulum of fibroblasts contained a large amount of glycogen, indicating cell activation. Fibroblast activation and the expression of key genes of the Sphk1-S1P pathway could be observed at the injection site, suggesting that after Xiaozhiling intervention, the Sphk1-S1P pathway could be activated to promote fibrosis. Xiaozhiling injection exerts its therapeutic effects on internal hemorrhoids by promoting collagen synthesis and secretion in fibroblasts. After Xiaozhiling intervention, the Sphk1-S1P pathway can be activated to promote fibrosis.

Macrophage Lyn Kinase Is a Sex-Specific Regulator of Post-Subarachnoid Hemorrhage Neuroinflammation.

Lyn kinase is a member of the Src family of tyrosine kinases, primarily known for its role in regulating immune cell signaling. It can phosphorylate and modulate the activity of various proteins involved in immune responses, including Toll-like receptor 4 (TLR4). TLR4-mediated inflammatory pathways have been extensively studied; however, the sex-specific interaction of TLR4 and Lyn in neuroinflammation after aneurysmal subarachnoid hemorrhage (SAH) has yet to be investigated. SAH occurs due to a ruptured aneurysm, and the consequences often lead to neuroinflammation and cognitive impairments. In our study, we investigated the sex-specific involvement of Lyn kinase in regulating TLR4 signaling to understand the TLR4-mediated inflammatory response after SAH. Cell-specific Lyn knockout mice of both sexes were used for this study. Wild-type and conditional knockout mouse brains were analyzed by multicolor flow cytometry, immunohistochemistry, and western blotting at postoperative day 7 following SAH surgery. An unbiased spatial transcriptomic analysis was performed with the frozen mouse brain tissues. A 3-dimensional brain stroke model and cerebrospinal fluid samples of patients with SAH were also used for this study. Our overall animal and patient data from flow cytometry, immunohistochemistry, western blot, cognitive function tests, and spatial transcriptomic data revealed that Lyn kinase is a sex-specific regulator in inflammatory cytokine production, red blood cell phagocytosis, neuronal apoptosis, and cognitive function, as well as a negative regulator of TLR4 signaling pathways. Our results highlight sex-specific modulation of Lyn kinase activity in TLR4 signaling after hemorrhagic stroke and indicate that successful treatment of neuroinflammation may require sex-specific treatments.

Pathway Enrichment-Based Unsupervised Learning Identifies Novel Subtypes of Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma.

Existing single-cell clustering methods are based on gene expressions that are susceptible to dropout events in single-cell RNA sequencing (scRNA-seq) data. To overcome this limitation, we proposed a pathway-based clustering method for single cells (scPathClus). scPathClus first transforms the single-cell gene expression matrix into a pathway enrichment matrix and generates its latent feature matrix. Based on the latent feature matrix, scPathClus clusters single cells using the method of community detection. Applying scPathClus to pancreatic ductal adenocarcinoma (PDAC) scRNA-seq datasets, we identified two types of cancer-associated fibroblasts (CAFs), termed csCAFs and gapCAFs, which highly expressed complement system and gap junction-related pathways, respectively. Spatial transcriptome analysis revealed that gapCAFs and csCAFs are located at cancer and non-cancer regions, respectively. Pseudotime analysis suggested a potential differentiation trajectory from csCAFs to gapCAFs. Bulk transcriptome analysis showed that gapCAFs-enriched tumors are more endowed with tumor-promoting characteristics and worse clinical outcomes, while csCAFs-enriched tumors confront stronger antitumor immune responses. Compared to established CAF subtyping methods, this method displays better prognostic relevance.

Single-cell and spatial transcriptomic analysis reveals that an immune cell-related signature could predict clinical outcomes for microsatellite-stable colorectal cancer patients receiving immunotherapy.

Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4+ T, regulatory CD4+ T, CD4+ Th17, exhausted CD8+ T, cytotoxic CD8+ T, proliferated CD8+ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.

Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development: a Sample Preparation Protocol.

Maize is an important crop that contributes to the modern economy in various ways, including use for human consumption, as animal feed, and in industrial products. Research on maize is crucial for understanding plant development, which in turn provides valuable insight into improvement of maize crops to meet the food demands of a growing population. Maize embryogenesis, which is the primordial stage of the corn life cycle, determines the fundamental body plan and developmental programs that organize the tissue patterning and subsequent growth and reproduction of the corn plant. Investigating maize embryogenesis at high cellular resolution can enhance our understanding of the homology, ontogeny, and developmental genetic mechanisms of embryonic organ morphogenesis. However, until recently, no published studies have used methods for analyzing maize embryo development at single-cell resolution. This protocol describes single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic analyses, which are powerful, combinatorial tools that can be used to study maize embryogenesis at the single-cell level within a spatial context. These tools have the power to reveal transcriptomic relationships between tissues/organs, and to provide insight into the gene regulatory networks operating during embryogenesis. In this protocol, we describe a detailed procedure to prepare maize embryo samples for construction of scRNA-seq and Visium spatial transcriptomic libraries that are suitable for massively parallel sequencing. Our protocol borrows from prior published studies and manufacturer's instructions and is optimized for studies of the maize embryo.

Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development.

Plant embryogenesis encompasses the biological processes wherein the zygote (fertilized egg) undergoes cell division, cell expansion, and cell differentiation to develop histological tissue layers, meristems, and various organs comprising the primordial body plan of the organism. Studies of embryogenesis in the agronomically important maize crop advance our understanding of the fundamental mechanism of plant development, which, upon translation, may advance agronomic improvement, optimization of conditions for somatic embryogenesis, and plant synthetic biology. Maize embryo development is coordinated temporally and spatially and is regulated by interactive genetic networks. Single-cell RNA sequencing (RNA-seq) and spatial transcriptomics are powerful tools to examine gene expression patterns and regulatory networks at single-cell resolution and in a spatial context, respectively. Single-cell technology enables profiling of three-dimensional samples with high cellular resolution, but it can be difficult to identify specific cell clusters due to a lack of known markers in most plant species. In contrast, spatial transcriptomics provide transcriptomic profiling of discrete regions within a sectioned, two-dimensional sample, although single-cell resolution is typically not obtained and fewer transcripts per cell are detected than in single-cell RNA-seq. In this review, we describe the combined use of these two transcriptomic strategies to study maize embryogenesis with synergistic results.

Abstract 2177: Spatial transcriptomic and functional analysis of HLA/PD-1/CTLA4 multi-gene edited CD19 CAR-iNK cells targeting pericytes in the glioblastoma tumor microenvironment to inhibit tumor migration and growth

Abstract Targeting glioblastoma (GBM) stem cell-derived pericytes in the perivascular niche offers a promising strategy to overcome intratumoral heterogeneity and inhibit tumor migration. By targeting CD19-positive pericytes, CD19 chimeric antigen receptor (CAR)-expressing natural killer (NK) cells can mitigate off-tumor toxicity; however, their interactions within the GBM tumor microenvironment (TME) require further investigation. In this study, we developed a GBM TME xenograft model by transplanting GBM-blood vessel assembloids (GBVA) into immunodeficient mice to study the effects of CD19 CAR-expressing iPSC-derived NK (CAR-iNK) cells on pericytes and their interactions within the TME. Additionally, we enhanced the efficacy of CD19 CAR-iNK cells by knocking out PD1 and CTLA4, enabling these cells to better target GBM within an immunosuppressive TME. As a result, GBVA xenografts recapitulated the GBM TME including human pericytes and macrophages, providing a robust in vivo platform for validating CD19 CAR-iNK cell efficacy against GBM. Optimization of CAR-iNK cell dosing and efficacy resulted in significant antitumor effects. Spatial transcriptome analysis using SLACS (Spatially Linked Antibody and cDNA Sequencing) revealed that CD19 CAR-iNK cell infiltration altered gene expression in the TME, elucidating mechanisms of GBM lysis, migration inhibition, and evasion of immunosuppressive influences. These findings underscore the therapeutic potential of CD19 CAR-iNK cells for GBM treatment through precise targeting of pericytes and modulation of the TME. Citation Format: Kyung-Sun Kang. Spatial transcriptomic and functional analysis of HLA/PD-1/CTLA4 multi-gene edited CD19 CAR-iNK cells targeting pericytes in the glioblastoma tumor microenvironment to inhibit tumor migration and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2177.

Abstract 5863: Mapping tumor metastasis and EMT-associated immune changes in HNSCC through spatial transcriptomic analysis

Abstract Head and neck squamous cell carcinoma (HNSCC) is a globally prevalent malignancy. Standard treatments typically include surgical resection, radiation therapy, and chemotherapy. However, for patients with recurrent or distantly metastatic tumors where surgery or radiation is no longer feasible, the primary treatment strategy shifts to combining targeted therapies with chemotherapy or immune checkpoint blockade (ICI) therapy. Despite these approaches, the overall prognosis remains poor. Therefore, identifying key changes in immune cell composition within the tumor microenvironment of malignant or metastatic tumors is crucial for improving the management and outcomes in HNSCC therapy. The aim of this study is to analyze changes in the immune microenvironment composition across different HNSCC specimens. We utilized tools including the GeoMx Digital Spatial Profiler (DSP) for imaging-based digitized gene expression analysis. We identified region of interesting (ROI) from distinct tumor regions, such as the tumor invasive front, tumor core, and paired lymph node metastases. Through the Cancer Transcriptome Atlas (CTA) analysis, we identified two distinct epithelial-mesenchymal transition (EMT) gene signatures, which were highly expressed in particularly aggressive tumor tissues. Additionally, within the myeloid activation signature, we observed that immune cell composition in primary tumor site was predominantly neutrophil-activated, whereas in lymph node metastases, macrophages were the dominant immune cells. To further validate these observations, we applied other spatial transcriptomics tools, including 10x Visium and 10x Xenium, to analyze specimens from six HNSCC patients. Transcriptomic trajectory analysis and ligand-receptor pair analysis revealed significant changes in the immune microenvironment of malignant HNSCC tissues. These results provide crucial insights into head and neck cancer treatment strategies tailored to different tumor microenvironment (TME) characteristics. Citation Format: Chih-Hung Chung, Muh-Hwa Yang. Mapping tumor metastasis and EMT-associated immune changes in HNSCC through spatial transcriptomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5863.

Abstract 5298: Impact of targeted therapy on the tumor immune microenvironment in EGFR-mutated NSCLC

Abstract Background: In EGFR-mutated lung cancer, osimertinib, a third-generation EGFR tyrosine kinase inhibitor, has demonstrated durable efficacy in many cases. However, early resistance arises in some patients during treatment. The tumor immune microenvironment is believed to play a pivotal role in shaping treatment outcomes, though the detailed molecular mechanisms are not fully understood. Methods: This study analyzed four cases with paired tissue samples collected both before and after osimertinib treatment. Using Visium-HD, we performed analyses comparing the pre-treatment samples between the early resistance and long-term efficacy groups, as well as paired samples collected before and after treatment. We converted the Visium-HD spot data into single-cell data using StarDist, a deep learning-based method for nuclei/cell detection and segmentation. Furthermore, by adding the calculated coordinate data derived from StarDist to the single-cell data, we performed spatial transcriptome analysis using single-cell coordinates instead of spot coordinates. This enabled spatial transcriptomic analysis at the single-cell level while preserving the actual spatial relationships between cells. Results: In the post-treatment samples from early resistance cases, the proportion of macrophages, particularly SPP1-positive macrophages, was increased compared to that in long-term efficacy cases. In the comparison of pre-treatment samples between early resistance and long-term efficacy cases, the former exhibited a high degree of intermixing among diverse cell populations (e.g., lymphocytes, macrophages, and fibroblasts), whereas the latter showed minimal intermingling between tumor cells and other cell types. Furthermore, in early resistance cases, the expression levels of CXCR4 and CD74 were higher compared to long-term efficacy cases. Analysis of cell-cell interactions suggested potential effects of tumor cells on tertiary lymphoid structures. Conclusions: EGFR-mutated lung cancer is typically characterized by reduced immune cell infiltration, often referred to as “cold tumor.” However, early resistance cases exhibited increased immune cell infiltration and the presence of tertiary lymphoid structures, indicating a “hot tumor” phenotype. This finding suggests that EGFR-mutated lung cancer cases prone to early resistance might possess a tumor immune microenvironment resembling that of EGFR wild-type tumors. Citation Format: Daisuke Shibahara, Satoshi Nakamura, Yasuyuki kishikawa, Keita Nakatomi, Noriaki Nakagaki, Kohei Otsubo, Yoshimasa Shiraishi, Yasuto Yoneshima, Eiji Iwama, Kentaro Tanaka, Isamu Okamoto. Impact of targeted therapy on the tumor immune microenvironment in EGFR-mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5298.