microrna-amplification-recognition-through-locked-nucleic-acid-situ

MicroRNA Amplification and Recognition through Locked-nucleic-acid <em>In situ</em> Hybridization as a Novel Detection and Quantification Method

When using traditional probe-based in situ hybridization (ISH) imaging detection methods, microRNAs (miRNAs) are difficult to capture due to their size, ...

rna-situ-hybridization-with-sequential-protein-immunofluorescence

RNA <em>In situ</em> Hybridization with Sequential Protein Immunofluorescence in Tandem Assay

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest ...

dual-modality-molecular-cartography-integrating-multiplex-mrna

Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival and what cues it receives as it ...

The University of Texas MD Anderson Cancer Center

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) AML patients. Cells co-expressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated AML patients, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.

Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney neoplasm; bone metastasis (BM) develops in 35% to 40% of metastatic patients and results in substantial morbidity and mortality, as well as medical costs. A key feature of ccRCC is the loss of function of the von Hippel-Lindau protein, which enhances angiogenesis via vascular endothelial growth factor release. Consequently, antiangiogenic tyrosine kinase inhibitors (TKI) emerged as a treatment for ccRCC. However, limited data about their efficacy in BM is available, and no systematic comparisons have been performed. We developed mouse models of bone and lung ccRCC tumors and compared their anticancer efficacy, impact on mouse survival, and mechanisms of action, including effects on tumor cells and both immune and nonimmune (blood vessels and osteoclasts) bone stromal components. This approach elucidates the efficacy of TKIs in ccRCC bone tumors to support rational interrogation and development of therapies.

Antiangiogenic Tyrosine Kinase Inhibitors have Differential Efficacy in Clear Cell Renal Cell Carcinoma in Bone.

Cancer-associated fibroblasts (CAFs) are a multifaceted cell population essential for shaping the tumor microenvironment (TME) and influencing therapy responses. Characterizing the spatial organization and interactions of CAFs within complex tissue environments provides critical insights into tumor biology and immunobiology. In this study, through integrative analyses of over 14 million cells from 10 cancer types across 7 spatial transcriptomics and proteomics platforms, we discover, validate, and characterize four distinct spatial CAF subtypes. These subtypes are conserved across cancer types and independent of spatial omics platforms. Notably, they exhibit distinct spatial organizational patterns, neighboring cell compositions, interaction networks, and transcriptomic profiles. Their abundance and composition vary across tissues, shaping TME characteristics, such as levels, distribution, and state composition of tumor-infiltrating immune cells, tumor immune phenotypes, and patient survival. This study enriches our understanding of CAF spatial heterogeneity in cancer and paves the way for novel approaches to target and modulate CAFs.

Conserved spatial subtypes and cellular neighborhoods of cancer-associated fibroblasts revealed by single-cell spatial multi-omics.

Acute myeloid leukemia (AML) resides in an immune-rich microenvironment, yet, immune-based therapies have faltered in eliciting durable responses. Bridging this paradox requires a comprehensive understanding of leukemic interactions within the bone marrow microenvironment. We optimized a high-throughput tissue-microarray-based pipeline for high-plex spatial immunofluorescence and mass cytometry imaging on a single slide, capturing immune, tumor, and structural components. Using unbiased clustering on the spatial K function, we unveiled the presence of tertiary lymphoid-like aggregates in bone marrow, which we validated using spatial transcriptomics and an independent proteomics approach. We then found validated TLS signatures predictive of outcomes in AML using an integrated public 480-patient transcriptomic dataset. By harnessing high-plex spatial proteomics, we open the possibility of discovering novel structures and interactions that underpin leukemic immune response. Further, our study's methodologies and resources can be adapted for other bone marrow diseases where decalcification and autofluorescence present challenges.

Multimodal spatial proteomic profiling in acute myeloid leukemia.

Subunits of the SWI/SNF chromatin remodeling complex are altered in ∼20% of human cancers. Exemplifying the alterations is the ARID1A mutation that occurs in ∼50% of ovarian clear-cell carcinoma (OCCC), a disease with limited therapeutic options. In this study, we showed that ARID1A mutations create a dependence on alanine by regulating alanine transporters to increase intracellular alanine levels. ARID1A directly repressed the alanine importer SLC38A2 and simultaneously promoted the alanine exporter SLC7A8. ARID1A inactivation increased alanine utilization predominantly in protein synthesis and passively through the tricarboxylic acid cycle. Indeed, ARID1A-mutant OCCCs were hypersensitive to the inhibition of SLC38A2. In addition, SLC38A2 inhibition enhanced chimeric antigen receptor T-cell assault in vitro and synergized with immune checkpoint blockade using an anti-PD-L1 antibody in a genetically engineered mouse model of OCCC driven by conditional Arid1a inactivation in a CD8+ T-cell-dependent manner. These findings suggest that targeting alanine transport alone or in combination with immunotherapy may represent an effective therapeutic strategy for ARID1A-mutant cancers.

Selective Alanine Transporter Utilization Is a Therapeutic Vulnerability in ARID1A-Mutant Ovarian Cancer.

The co-evolution of different cell subsets in the progression of precursor lesions to lung adenocarcinoma (LUAD) is incompletely understood. We generated spatial transcriptomic maps of 56 human precursor lesions and LUADs from 25 patients and of an independent cohort of 36 lesions from 19 patients, analyzing a total of 486,519 spots and 5.4 million cells. We identify region-specific programs that distinguish precursors from LUADs. Spatially resolved clonal architectures reveal patient-specific heterogeneity in evolution of precursors to LUADs. We find epithelial alveolar progenitors expressing tumor-associated meta-programs and residing in niches enriched with proinflammatory subsets including IL1B high macrophages. Epithelial-proinflammatory niches are prevalent in precursor lesions but become less frequent in LUADs. These niches are conserved in mice and promote alveolar progenitor growth. Targeting inflammation alone or in combination with immune checkpoint blockade in precancerous phase reduces alveolar progenitors. Epithelial-inflammatory niches are stage-specific, shape early LUAD development and represent promising targets for interception.

ArticleTotal : 3	Year
Publication title Cited by 1	2025
Publication title	2025
Publication title	2025

Article	Year
Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion. Cancer immunology research\| PubMed ID: 37163233	2023
Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses. Cancers\| PubMed ID: 38473208	2024
Antiangiogenic Tyrosine Kinase Inhibitors have Differential Efficacy in Clear Cell Renal Cell Carcinoma in Bone. Cancer research communications\| PubMed ID: 39248577	2024
Conserved spatial subtypes and cellular neighborhoods of cancer-associated fibroblasts revealed by single-cell spatial multi-omics. Cancer cell\| PubMed ID: 40154487	2025
Multimodal spatial proteomic profiling in acute myeloid leukemia. NPJ precision oncology\| PubMed ID: 40394148	2025
Selective Alanine Transporter Utilization Is a Therapeutic Vulnerability in ARID1A-Mutant Ovarian Cancer. Cancer research\| PubMed ID: 40658600	2025
Multimodal spatial-omics reveal co-evolution of alveolar progenitors and proinflammatory niches in progression of lung precursor lesions. Cancer cell\| PubMed ID: 41202811	2025

Akshay Basi

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