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

Advanced high-grade serous ovarian cancer (HGSC) is an aggressive disease that accounts for 70% of all ovarian cancer deaths. Nevertheless, 15% of patients diagnosed with advanced HGSC survive more than 10 years. The elucidation of predictive markers of these long-term survivors (LTS) could help identify therapeutic targets for the disease, and thus improve patient survival rates. To investigate the stromal heterogeneity of the tumor microenvironment (TME) in ovarian cancer, we used spatial transcriptomics to generate spatially resolved transcript profiles in treatment-naïve advanced HGSC from LTS and short-term survivors (STS) and determined the association between cancer-associated fibroblasts (CAF) heterogeneity and survival in patients with advanced HGSC. Spatial transcriptomics and single-cell RNA-sequencing data were integrated to distinguish tumor and stroma regions, and a computational method was developed to investigate spatially resolved ligand-receptor interactions between various tumor and CAF subtypes in the TME. A specific subtype of CAFs and its spatial location relative to a particular ovarian cancer cell subtype in the TME correlated with long-term survival in patients with advanced HGSC. Also, increased APOE-LRP5 cross-talk occurred at the stroma-tumor interface in tumor tissues from STS compared with LTS. These findings were validated using multiplex IHC. Overall, this spatial transcriptomics analysis revealed spatially resolved CAF-tumor cross-talk signaling networks in the ovarian TME that are associated with long-term survival of patients with HGSC. Further studies to confirm whether such cross-talk plays a role in modulating the malignant phenotype of HGSC and could serve as a predictive biomarker of patient survival are warranted.

Spatial Transcriptomics Depict Ligand-Receptor Cross-talk Heterogeneity at the Tumor-Stroma Interface in Long-Term Ovarian Cancer Survivors.

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.

Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross-species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while reexpression of Nkx6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis.

Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential.

Inactivating TP53 mutations leads to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promotes tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome and find that by targeting minichromosome maintenance complex components (MCMs), GOF mutp53 predisposes cells to replication stress and chromosomal instability (CIN), leading to a tumor cell-autonomous and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Consequently, GOF mutp53-MCMs-CIN-cytosolic DNA-cGAS-STING-NC-NF-κB signaling promotes tumor cell metastasis and an immunosuppressive tumor microenvironment through antagonizing interferon signaling and regulating genes associated with pro-tumorigenic inflammation. Our findings have important implications for understanding not only the GOF activities of TP53 mutations but also the genome-guardian role of p53 and its inactivation during tumor development and progression.

Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While induction chemotherapy leads to remission in most patients, a significant number will experience relapse. Therefore, there is a need for novel therapies that can improve remission rates in patients with relapsed and refractory AML. CD70 is the natural ligand for CD27 (a member of the TNF superfamily) and appears to be a promising therapeutic target. Consequently, there is considerable interest in developing chimeric antigen receptor (CAR) T-cell therapy products that can specifically target CD70 in various neoplasms, including AML. In this study, we employed routine diagnostic techniques, such as immunohistochemistry and flow cytometry, to investigate the expression of CD70 in bone marrow samples from treatment-naïve and relapsed AML patients after hypomethylating agents (HMA). Also, we evaluated the impact of HMA on CD70 expression and examined CD70 expression in various leukemic cell subsets and normal hematopoietic progenitors.

Increased expression of CD70 in relapsed acute myeloid leukemia after hypomethylating agents.

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.

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.

Tumor-infiltrating bacteria are increasingly recognized as modulators of cancer progression and therapy resistance. We describe a mechanism by which extracellular intratumoral bacteria, including Fusobacterium, modulate cancer epithelial cell behavior. Spatial imaging and single-cell spatial transcriptomics show that these bacteria predominantly localize extracellularly within tumor microniches of colorectal and oral cancers, characterized by reduced cell density, transcriptional activity, and proliferation. In vitro, Fusobacterium nucleatum disrupts epithelial contacts, inducing G0-G1 arrest and transcriptional quiescence. This state confers 5-fluorouracil resistance and remodels the tumor microenvironment. Findings were validated by live-cell imaging, spatial profiling, mouse models, and a 52-patient colorectal cancer cohort. Transcriptomics reveals downregulation of cell cycle, transcription, and antigen presentation genes in bacteria-enriched regions, consistent with a quiescent, immune-evasive phenotype. In an independent rectal cancer cohort, high Fusobacterium burden correlates with reduced therapy response. These results link extracellular bacteria to cancer cell quiescence and chemoresistance, highlighting microbial-tumor interactions as therapeutic targets.

ArticleTotal : 2	Year
Publication title	2025
Publication title	2025

Article	Year
Spatial Transcriptomics Depict Ligand-Receptor Cross-talk Heterogeneity at the Tumor-Stroma Interface in Long-Term Ovarian Cancer Survivors. Cancer research\| PubMed ID: 36787106	2023
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
Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential. Cancer discovery\| PubMed ID: 37285225	2023
Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response. Nature communications\| PubMed ID: 38167338	2024
Increased expression of CD70 in relapsed acute myeloid leukemia after hypomethylating agents. Virchows Archiv : an international journal of pathology\| PubMed ID: 38388965	2024
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
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
Tumor-infiltrating bacteria disrupt cancer epithelial cell interactions and induce cell-cycle arrest. Cancer cell\| PubMed ID: 41106380	2025

Javier A. Gomez

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