GeoMx Digital Spatial Profiling Research Articles

Selective vulnerability of the suprachiasmatic nucleus in progressive Alzheimer’s Disease: A human postmortem study using spatial in‐situ proteomics

AbstractBackgroundAlzheimer’s Disease (AD) patients commonly experience sleep/wake (S/W) disturbances, such as sleep fragmentation and excessive daytime sleepiness. The S/W cycle is controlled by a complex system regulated by a master regulator, the suprachiasmatic nucleus (SCN) that controls wake‐ and sleep‐promoting nuclei. Despite its importance in orchestrating sleep architecture, little is known about the molecular changes that occur in the SCN during the progression of AD and how these changes contribute to S/W disturbances.MethodWe used postmortem brain tissue of 10 controls without AD pathology and 32 subjects with progressive AD stages. Our approach included quantitative neuropathological assessments of AVP and VIP‐expressing neurons in SCN and its adjacent hypothalamic region, the supraoptic nucleus (SON). We also used an in‐situ cell‐specific spatial proteomics platform [GeoMx Digital Spatial Profiling (DSP)] to probe levels of different pathological tau species and proteins commonly dysregulated in AD.ResultPreliminary findings reveal a significant decrease in SCN volume (∼75%) (Fig.1) and a loss of ∼58% AVP neurons and ∼41% VIP neurons in Braak stage (Braak) VI compared to Braak 0. The DSP results indicate that both neurons in the SCN have twice the burden of p‐tau levels as the SON (Fig.2‐right), and neurofibrillary tanglesAdditionally, SCN shows an increase in proteins associated with glia and immune responses (Fig.2‐left). Furthermore, an increase in microglial proteins is observed in the SCN in Braak I (Fig.3‐left). In contrast, the SON displays a milder pattern of protein dysregulation in Braak I (Fig.3‐right), consistent with its lower tau expression observed in histology and DSP.ConclusionSCN is vulnerable to AD pathology even in Braak I, which is characterized by volume loss, increased immune response protein dysregulation, and later neuronal loss, as well as p‐tau pathology. This pattern supports the idea that the SCN plays a role in S/W disturbances observed from early AD stages. Interestingly, its neighboring nucleus, SON, which also has AVP neurons, remained spared, providing opportunities for studies investigating factors related to selective vulnerability. Ongoing studies are addressing changes in the SCN and SON in intermediate AD stages.

Abstract C092: Genetic profiling of high grade serous ovarian cancer among women of African ancestry

Abstract (A) AN INTRODUCTORY SENTENCE INDICATING THE PURPOSES OF THE STUDY: Ovarian cancer (OC) is a heterogeneous disease with high grade serous ovarian cancer (HGSOC) as the commonest, the most aggressive tumor, which is often diagnosed at advanced stages, and with poor overall survival. Unfortunately, like many other cancer diagnoses, women of West African ancestry (Black women) with ovarian cancer experience worse outcomes than White women. In the US, Black women have higher morbidity and mortality rates and higher un-staged or unclassified tumors compared with White women, resulting in undertreatment with subsequent compromise in progression-free survival. Genomic instability is one of the most common underlying features of ovarian cancer and defective DNA damage repair has been implicated. The goal is to uncover canonical genes associated with the aggressive behavior of HGSOC in a cohort of black women that could explain the biology of this tumor. (B) A BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES: Formalin fixed paraffin embedded (FFPE) HGSOC tissue blocks from Nigeria in West Africa (WAB), Bahama in Caribbean (CBB), and Miami in USA (USB) were identified and selected after rigorous review and confirmation with immunohistochemical (IHC) markers when needed. Integration of digital histopathology and IHC markers (estrogen receptor, progesterone receptor, Ki67 and P53), whole exome sequence, RNA sequence of the FFPE samples was used to identify the proportion of genomic instability and defective DNA damage repair genes. We use spatial genomic library on tissue microarray (TMA) slide prepared on GeoMx digital spatial profiler (DSP) Nanostring platform to study the biology of the tumor and the tumor microenvironment (TME) and proteogenomics.(C) A SUMMARY OF THE NEW, UNPUBLISHED DATA: The unsupervised principal component analysis of the tumor showed a distinct population of WAB and USB while the CBB showed admixture population. BACH2, ARG1, NAFT2 and FRA10A1 are commonly differentially expressed genes among the cohort. There is low expression of routine IHC markers among the WAB compared to the other groups. Conclusion: There is variation on gene expression by nativity and we hope to explore this variation for therapeutic purpose among black women with ovarian cancer. Citation Format: Ayodele Omotoso, Uchenna Ezenkwa, Raleigh Butler, Melissa Castillo, Alex Sanchez-Covarrubias, Matthew Schlumbrecht, Andre Pinto, Bala Audu, Sophia George. Genetic profiling of high grade serous ovarian cancer among women of African ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C092.

Differential spatial transcriptome profile of the hippocampal formation of protected PSEN1 E280A familial Alzheimer’s disease cases

AbstractBackgroundWe have described two cases with ∼ 30 years of delayed disease onset among carriers of PSEN1 mutation E280A, suffering from familial Alzheimer’s disease (FAD). One case was homozygous for the ApoE Christchurch (hApoEch) mutation. This case showed sparse hyperphosphorylated tau (pTau) pathology in several cortical areas. The second case was heterozygous for the RELN‐COLBOS mutation and was globally affected by severe pTau and amyloid beta pathology, with evidence of localized cellular protection in the entorhinal cortex (EC). Here we used digital spatial profiling (DSP) transcriptomics to characterize the hippocampal formation of FAD protected cases and identify possible molecular mechanisms of protection.MethodsIn depth morphological characterization of the EC together with identification of thirteen different regions of interest (ROIs) were performed in Formalin‐fixed paraffinized 3 mm thick slides taken from the hippocampal formation (Fig. 1A) of the RELN‐COLBOS FAD case, the hApoEch FAD case, three FAD cases, and three sporadic Alzheimer’s cases (SAD). Slides were immunostained for NeuN and GFAP for morphological ROIs identification and processed for localized transcriptome analyses using the GeoMX DSP platform from Nanostring. Obtained data was processed for quality control, dimensionality reduction and differential gene expression (DGE) analyses.ResultsDimensionality reduction of transcriptomic data obtained from all ROIs and studied cases showed a distinctive distribution of ROIs belonging to both protected cases (Fig. 1B). Furthermore, DGE was observed in all sampled ROIs. ROIs such as hippocampal white matter or dentate gyri showed a higher number of differentially expressed genes due to a more homogeneous cellular composition of the ROIs (Fig. 1C). Also, EC and dentate gyrus from FAD protected cases showed distinctive molecular signatures compatible with excitatory neuronal pathways (Fig. 1D). Complementary morphological analyses showed increased neuronal density in the supragranular region of the EC in the RELN‐COLBOS FAD protected case, specifically in neuronal layer II.ConclusionOur results suggest that regardless of the protective gene conferring protection to FAD, the EC is one key protected structure with distinctive regional transcriptomic profiles suggesting the preservation of neuronal function in this region and hinting towards a possible therapeutic target in the protection against Alzheimer’s disease.

Abstract A074: Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade

Abstract Background: IL-12 is a pleotropic inflammatory cytokine with broad stimulatory effects on immune cells and robust antitumor activity in syngeneic murine tumor models, making it an attractive target for cancer immunotherapy. Unfortunately, systemic administration of recombinant human IL-12 causes severe toxicity in patients. With the goal of improving the therapeutic index for IL-12 therapy, we have engineered an IL-12 prodrug referred to as an INDUKINETM molecule that is dosed systemically but designed to be selectively activated in tumors. Methods: Mice bearing MC38, CT26, B16F10, and EMT-6 syngeneic tumors were treated with surrogate mWTX-330 (a chimeric IL-12 containing INDUKINETM molecule), and tumor growth and body weight were monitored over time. Tumor tissues were harvested at various timepoints and analyzed by flow cytometry, NanoString GeoMx Digital Spatial Profiling (DSP), and high-plex immunofluorescence to characterize the mechanisms associated with response. Results: mWTX-330 is comprised of a half-life extension domain and an inactivation domain tethered to chimeric IL-12 by tumor protease-sensitive linkers. These linkers were chosen for their selective processing by a majority of human tumors in vitro as well as mouse tumors in vitro and in vivo. The linkers have been shown in preclinical studies to remain stable in normal human tissues including serum. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, including those resistant to checkpoint blockade, and preferentially activated tumor-infiltrating immune cells versus peripheral cells. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy, although NK and CD4+ T cells also contributed to tumor growth inhibition. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DCs), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. Conclusion: Based on the strong biological rational and preclinical studies of surrogate mWTX-330, we created WTX-330, a fully human IL-12 INDUKINETM molecule that is stable in human serum and is reliably and selectively processed by human tumor samples in vitro. A first-in-human clinical trial to evaluate the safety profile, pharmacokinetics, and preliminary antitumor activity of WTX-330 in patients with relapsed/refractory advanced or metastatic solid tumors or non-Hodgkin lymphoma is now underway (NCT05678998). Citation Format: Christopher J Nirschl, Heather R Brodkin, Sameer S Chopra, Connor J Dwyer, Daniel J Hicklin, Randi Isaacs, Nesreen Ismail, Cynthia Seidel-Dugan, Zoe Steuert, Jenna M Sullivan, William M Winston, Andres Salmeron. Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A074.

Spatial profiling of cancer-associated fibroblasts of sporadic early onset colon cancer microenvironment

The incidence of sporadic early-onset colon cancer (EOCC) has increased worldwide. The molecular mechanisms in the tumor and the tumor microenvironment (TME) in EOCC are not fully understood. The aim of this study is to unravel unique spatial transcriptomic and proteomic profiles in tumor epithelial cells and cancer-associated fibroblasts (CAFs). Here, we divide the sporadic colon cancer tissue samples with transcriptomic data into patients diagnosed with EOCC (<50 yrs) and late-onset colon cancer (LOCC, ≥50 yrs) and then, analyze the data using CIBERSORTx deconvolution software. EOCC tumors are more enriched in CAFs with fibroblast associated protein positive expression (FAP(+)) than LOCC tumors. EOCC patients with higher FAP mRNA levels in CAFs have shorter OS (Log-rank test, p < 0.029). Spatial transcriptomic analysis of 112 areas of interest, using NanoString GeoMx digital spatial profiling, demonstrate that FAP(+) CAFs at the EOCC tumor invasive margin show a significant upregulation of WNT signaling and higher mRNA/protein levels of fibroblast growth factor 20 (FGF20). Tumor epithelial cells at tumor invasive margin of EOCC tumors neighboring FAP(+) CAFs show significantly higher mRNA/protein levels of fibroblast growth factor receptor (FGFR2) and PI3K/Akt signaling activation. NichNET analysis show a potential interaction between FGF20 and FGFFR2. The role of FGF20 in activating FGFR2/pFGFR2 and AKT/pAKT was validated in-vitro. In conclusion, we identify a unique FAP(+) CAF population that showed WNT signaling upregulation and increased FGF20 levels; while neighbor tumor cells show the upregulation/activation of FGFR2-PI3K/Akt signaling at the tumor invasive margin of EOCC tumors.

StandR: spatial transcriptomic analysis for GeoMx DSP data.

To gain a better understanding of the complexity of gene expression in normal and diseased tissues it is important to account for the spatial context and identity of cells in situ. State-of-the-art spatial profiling technologies, such as the Nanostring GeoMx Digital Spatial Profiler (DSP), now allow quantitative spatially resolved measurement of the transcriptome in tissues. However, the bioinformatics pipelines currently used to analyse GeoMx data often fail to successfully account for the technical variability within the data and the complexity of experimental designs, thus limiting the accuracy and reliability of the subsequent analysis. Carefully designed quality control workflows, that include in-depth experiment-specific investigations into technical variation and appropriate adjustment for such variation can address this issue. Here, we present standR, an R/Bioconductor package that enables an end-to-end analysis of GeoMx DSP data. With four case studies from previously published experiments, we demonstrate how the standR workflow can enhance the statistical power of GeoMx DSP data analysis and how the application of standR enables scientists to develop in-depth insights into the biology of interest.

TMIC-75. SPATIALLY-RESOLVED TRANSCRIPTOME ANALYSIS OF BRAIN METASTATIC BREAST CANCER REVEAL KEY MEDIATORS OF BRAIN-TROPIC METASTATIC POTENTIAL

Abstract BACKGROUND In women with metastatic breast cancer, 30% (HER2+) to 50% (triple negative) develop brain metastases. Breast-to-brain metastasis carries a poor median survival of only 10 months and is accompanied by severe neurologic decline. Halting the progression of breast cancer brain metastases requires modulation of the tumor microenvironment (TME), yet little is known about how the primary breast TME affects brain tropism, or how, once there, metastatic cancer cells interact with normal brain cells (e.g., neurons, astrocytes, microglia). METHODS We constructed a large (n = 253 cores), diverse, clinically-annotated tissue microarray (TMA) of breast cancer brain metastases with surrounding brain (n = 127), associated primary breast tumors (n = 67, with 30 matched), normal brain (n = 23), and staining control (n = 18) tissue samples, from patients (n = 140) treated at Stanford Hospital between 2008 – 2018. We then leveraged a combination of genome-wide transcriptome analysis and high resolution spatial imaging (using GeoMx Digital Spatial Profiling) to interrogate the distinct, spatially-driven gene signatures of tumor, immune, and normal brain cells within the breast cancer brain metastasis TME. RESULTS We revealed novel gene regulatory programs that are associated with metastatic potential from the primary breast to the brain. We also identified not only diverging features of the breast and brain TMEs, but equivalent features which may contribute to preferential honing and outgrowth of metastatic breast cells in the brain microniche. Additionally, we uncovered significant cellular crosstalk between tumor, immune, and brain cell populations, including putative cell-cell interactions and intercellular signaling pathways. CONCLUSION These results served as a starting point for identifying key tumor-immune-brain interactions responsible for priming immune and brain tolerance of malignant cells, allowing outgrowth and progression of breast cancer brain metastases. Further investigation focused on tumor-brain crosstalk, specifically tumor-microglia interactions, will provide promising targets for therapeutic advancements.

EPCO-27. RECONSTRUCTION OF THE SPATIAL ECOSYSTEM OF GLIOBLASTOMA REVEALS RECURRENT RELATIONSHIPS BETWEEN TUMOR CELL STATES AND TUMOR MICROENVIRONMENT

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options. Multiple studies have characterized at single cell resolution the GBM as being composed of transcriptional cell states interconnected with components in the tumor immune microenvironment (TME). Our group proposed and validated the first single cell guided functional classification of GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, single cell technologies lack the spatial relationships among the cell states of GBM and between GBM cell states and TME. Spatially resolved transcriptomic technologies are emerging as powerful tools to reconstruct the spatial architecture of a tissue. We performed spatial transcriptomics of multicellular regions of interest (ROI) in 8 IDH wild-type GBM with both CosMx Spatial Molecular Imager, which analyzes 1,000 RNA probes and 64 proteins at single cell resolution, and GeoMx Digital Spatial Profiler which profiles the whole transcriptome (~18,000 genes) at ROI resolution. We integrated the two platforms to define single cell states and non-malignant cells and developed a computational deconvolution approach for CosMx spatial data which utilized GeoMx ROI resolved transcriptomic profiles as a-priori information to predict cell type abundances. Spatial deconvolution of CosMx derived single cells revealed spatial segregation of the tumor cell clones and cellular states and highlighted recurrent patterns of cell states, distinct TME cell types associated with coherent histopathological features across multiple samples. Our studies established a scalable approach to resolve the transcriptional heterogeneity of GBM and reconstruct the architecture of GBM cell states and tumor microenvironment.

Single Cell Multi Omics and Spatial Analysis Reveals Plasmablast Signature Malignant Cells As a Key of Intratumor Heterogeneity in Primary Central Nervous System Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) constitutes approximately 5% of extranodal lymphomas. More than 90% of PCNSL are diagnosed as diffuse large B-cell lymphomas (DLBCL), mainly classified as the activated B cell (ABC) subtype. Recent genetic analyses have identified key molecular drivers of PCNSL, including MYD88, CARD11 and CD79B. However, molecular analyses using bulk samples have provided limited insights into the heterogeneity in lymphoma cells of PCNSL. To improve our understanding in the biology of PCNSL, we conducted a multimodal single-cell analyses and integrated single-cell and spatial analyses to characterize the molecular properties of PCNSL tumors. Methods:Our study included seven PCNSL patients and two patients with reactive lymph nodes (RLN). We performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) combined with B-cell and T-cell receptor (BCR and TCR) analysis on samples from 3 PCNSL and 2 RLN patients, while the remaining PCNSL patients' samples were subjected to single-cell transcriptomic sequencing. Additionally, six formalin-fixed and paraffin-embedded (FFPE) tissues from the patients analyzed in the single-cell sequencing (except for one patient) were performed with GeoMx digital spatial profiling (DSP). Result: To ensure data quality, we filtered out low-quality cells and doublets, resulting in a dataset of 41,556 single cells. B cells represented the most abundant cluster, followed by T cells and monocytes. Copy number alterations (CNAs) data derived from InferCNV analysis showed distinct CNAs patterns in lymphoma cells of each patient. UMAP plot projection analysis revealed the presence of a distinct subtype of malignant cells within the B-cell population, which was characterized as high expression of plasmablast (PBL)-associated surface proteins, including SLAMF7, CD39, and CD38. Enrichment analysis based on germinal center (GC) gene expression signatures (Holmes AB et al. J Exp Med. 2020) revealed a broader spectrum of B-cell differentiation states in malignant B cells. Single-cell BCR sequencing further demonstrated that different GC signature state cells harbored the same BCR repertoire within each patient, suggesting a common origin of malignant cells. Non-negative matrix factorization (NMF) analysis confirmed the enrichment of PBL signature scores in the distinct malignant cell population observed in the UMAP plot, consistent with DPE analysis (Figure A). Interestingly, the proportion of PBL signature cells varied among patients. Signaling interaction analysis highlighted CD70-CD27 and SPP1-CD44 pathways as enriched in tumor cells. PBL signature cells received TNFSF13B and TNFSF13 signaling through TNFRSF17 (BCMA), whereas other tumor cells expressed TNFRSF13C (BAFF-R) and received TNFSF13B signaling alone. We further analyzed two public bulk RNA-seq data (Bruno A et al. Neuro Oncol 2018, n = 6; Radke J et al. Nat Commun 2022, n=38) in PCNSL to decompose tumor components. Tumor PBL components were identified in most PCNSL cases, although several cases exhibited a low presence of tumor PBL population. Of note, analysis including these public datasets with our single-cell cohort showed that PCNSL samples with high tumor PBL proportion had unfavorable prognosis . Additionally, we performed DSP to integrate the single-cell and spatial multi-omics analyses. We deconvoluted the cell composition by using transcriptomic data derived from each region of interest (ROI) and found that the cell proportions in ROI were consistent with the corresponding single-cell results. However, there were differential profiles of cell composition according to patients rather than ROIs, suggesting that PCNSL tumors may display spatial homogeneity (Figure B). Conclusion: Through the application of single-cell and spatial transcriptomic analysis, we identified a distinct subtype expressing the PBL signature gene. These findings have important therapeutic implications and provide new insights into the mechanisms of relapse in PCNSL.

Expression of IDO1 in Tumor Microenvironment Significantly Predicts the Risk of Recurrence/Distant Metastasis for Patients With Esophageal Squamous Cell Carcinoma

In this study, we aimed to explore immune markers predicting locoregional recurrence/distant metastasis (R/M) for patients with esophageal squamous cell carcinoma (ESCC) post-surgical intervention by using a novel high-throughput spatial tool to quantify multiple immune proteins expressed in ESCC and lymphocytes in tumor microenvironment (TME-L). First, formalin-fixed paraffin-embedded tissues from surgical patients with ESCC (n = 94) were constructed on a microarray, which was then divided into discovery (n = 36) and validation cohorts (n = 58). Using a newly developed GeoMx digital spatial profiling tool, 31 immune proteins in paired ESCC and TME-L, morphologically segmented by PANCK and CD45, respectively, from the discovery cohort were quantified, releasing 2,232 variables. Next, the correlation matrix was analyzed using the Corrplot package in R Studio, resulting in 6 closely correlated clusters. The Least Absolute Shrinkage and Selection Operator regression scoring model predictive of R/M risk with superior specificity was successfully established based on the 3 following hierarchically clustered immune proteins: ARG1 in ESCC/PANCK+, STING, and IDO1 in TME-L/CD45+. Moreover, the expression of IDO1 in TME-L, rather than in ESCC, significantly predicted the R/M risk score with an area under curve of 0.9598. In addition, its correlation with R/M status was further validated by dual immunohistochemistry staining of IDO1 and CD45 in discovery and validation cohorts. Above all, our findings not only provide a more accurate scoring approach based on quantitative immune proteins for the prediction of R/M risk, but also propose that IDO1 in TME-L potentially plays a driving role in mediating R/M in ESCC.

Spatial proteomics of hippocampal subfield-specific pathology in Alzheimer's disease and primary age-related tauopathy.

Alzheimer's disease (AD) and primary age-related tauopathy (PART) both harbor 3R/4R hyperphosphorylated-tau (p-tau)-positive neurofibrillary tangles (NFTs) but differ in the spatial p-tau development in the hippocampus. Using Nanostring GeoMx Digital Spatial Profiling, we compared protein expression within hippocampal subregions in NFT-bearing and non-NFT-bearing neurons in AD (n=7) and PART (n=7) subjects. Proteomic measures of synaptic health were inversely correlated with the subregional p-tau burden in AD and PART, and there were numerous differences in proteins involved in proteostasis, amyloid beta (Aβ) processing, inflammation, microglia, oxidative stress, and neuronal/synaptic health between AD and PART and between definite PART and possible PART. These results suggest subfield-specific proteome differences that may explain some of the differences in Aβ and p-tau distribution and apparent pathogenicity. In addition, hippocampal neurons in possible PART may have more in common with AD than with definite PART, highlighting the importance of Aβ in the pathologic process. Synaptic health is inversely correlated with local p-tau burden. The proteome of NFT- and non-NFT-bearing neurons is influenced by the presence of Aβ in the hippocampus. Neurons in possible PART cases share more proteomic similarities with neurons in ADNC than they do with neurons in definite PART cases.

Abstract PO-082: Antigen presentation and inflammatory effects on immune surveillance: Window of opportunity metformin monotherapy trial for squamous cell carcinoma of the head and neck (HNSCC)

Abstract Introduction: The tumor microenvironment frequently displays dysregulated cellular metabolism, which contributes to aggressive behavior of the tumor. Though the mechanism of effect is unclear, clinical data suggest an association between metformin and improved outcomes in head and neck squamous cell carcinoma (HNSCC). The aim of this study is to investigate post-metformin treatment effects on the tumor microenvironment (TME) of HNSCC by using spatial RNA sequencing data from a metformin monotherapy window-of-opportunity clinical trial. Methods: This was a single-center prospective clinical trial of metformin in patients with HNSCC and a plan for definitive surgical resection. Primary lesions were biopsied prior to treatment and compared to matched post-treatment surgical specimens. Following biopsy-proven HNSCC diagnosis, metformin was initiated at 500 mg/day and increased to 1,000 mg twice daily by day 6. Spatial RNA sequencing of 18,000+ genes in CD45, CD163, and PanCK segments was conducted using Nanostring’s GeoMx digital spatial profiling (DSP). Sequencing data was analyzed by using unbiased gene set enrichment analysis (GSEA) and reported as a rank in gene list from 18,000+ total genes for post- vs pre-treatment analyses. Results: In all HPV positive, post-treatment samples in all three segments, GSEA demonstrated enrichment of antigen presenting cell types including NK cells, monocytes, and dendritic cells (q&amp;lt; 0.001). Inflammatory and metabolism signatures (IFN-y, IFN-a, oxidative phosphorylation) were enriched after metformin monotherapy in HPV+ samples within the PanCK positive segment (q&amp;lt; 0.001). Epithelial mesenchymal transition (EMT) was noted to be positively enriched while plasma cell signatures were negatively enriched within in HPV- post-treatment samples in the CD163 segment (q&amp;lt; 0.001). Within the CD45 positive segment, enriched expression of mRNAs for ribosomal-protein subsets were noted in T-cell gene sets and not in B-cell gene sets; in the CD45 segment, cytoplasmic ribosomal proteins is the second-most enriched gene set from C2 canonical pathways gene sets. Conclusion: Antigen presentation, inflammation, and ribosomal protein encoding genes were enriched with metformin monotherapy in the window of opportunity setting for HNSCC. Ribosomal proteins may also play a role in antigen presentation and immune surveillance in the post-treatment setting. Further research is required to describe the TME effects of metformin monotherapy and its effects on treatment response. Citation Format: Derek S. Mann, Sruti Tekumalla, Amiti Jain, Jacob Riordan, Alban Linnenbach, Diana Menezes, Ubaldo Martinez-Outschoorn, Joseph M. Curry. Antigen presentation and inflammatory effects on immune surveillance: Window of opportunity metformin monotherapy trial for squamous cell carcinoma of the head and neck (HNSCC) [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-082.

Spatial transcriptomics reveals distinct tissue niches linked with steroid responsiveness in acute gastrointestinal GVHD

AbstractSevere acute graft-versus-host disease (aGVHD) is associated with significant mortality and morbidity, especially in steroid-resistant (SR) cases. Spatial transcriptomic technology can elucidate tissue-based interactions in vivo and possibly identify predictors of treatment response. Tissue sections from 32 treatment-naïve patients with biopsy-confirmed lower gastrointestinal (GI) aGVHD were obtained. The GeoMx digital spatial profiler was used to capture transcriptome profiles of >18 000 genes from different foci of immune infiltrates, colonic epithelium, and vascular endothelium. Each tissue compartment sampled showed 2 distinct clusters that were analyzed for differential expression and spatially resolved correlation of gene signatures. Classic cell-mediated immunity signatures, normal differentiated epithelial cells, and inflamed vasculature dominated foci sampled from steroid-sensitive cases. In contrast, a neutrophil predominant noncanonical inflammation with regenerative epithelial cells and some indication of angiogenic endothelial response was overrepresented in areas from SR cases. Evaluation of potential prognostic biomarkers identified ubiquitin specific peptidase 17–like (USP17L) family of genes as being differentially expressed in immune cells from patients with worsened survival. In summary, we demonstrate distinct tissue niches with unique gene expression signatures within lower GI tissue from patients with aGVHD and provide evidence of a potential prognostic biomarker.

JS07.4.A REGIONAL B-CELL MEDIATED IMMUNOSUPPRESSION IDENTIFIED THROUGH SPATIAL AND SINGLE-CELL TRANSCRIPTOMIC ANALYSIS OF CHORDOMA

Abstract BACKGROUND Chordomas, rare indolent neoplasms, mainly occur in the skull base or lower spine. Therapies, including surgery and radiotherapy, often fail to impede local recurrence. Though checkpoint inhibition therapy shows promise, recent immunotherapy trials failed to reach endpoints. Here, we aim to investigate the immunological landscape of chordomas using spatial and single-cell transcriptomics, and TCR/BCR profiling, elucidating cellular interplay and the intricate mechanisms governing the immunosuppressive tumor microenvironment. MATERIAL AND METHODS We generated a comprehensive single-cell atlas of the chordoma neoplastic landscape and its associated microenvironment, incorporating paired single-cell transcriptomic samples from blood and tumor cells of six patients, as well as single-cell BCR and TCR sequencing data. Utilizing scGEN, we integrated pre-annotated gene expression datasets and identified both established and novel antigen binding patterns by employing TCR Bidirectional Encoder Representations from Transformers (BERT). Moreover, we conducted GeoMx Digital Spatial Profiler (DSP) analysis by selecting regions of interest (ROIs) that encompass critical immunological and malignant niches. We also curated and quantified cell distributions in immunohistochemical staining for a total of 107 chordomas. RESULTS Utilizing pseudo-temporal mapping, we delineated the trajectories of CD8 and CD4 T cells in chordomas, spanning from blood to tumor stroma and encompassing naïve, memory, cytotoxic, and T cell exhaustion profiles. Through an integrated TCR profiling methodology, we observed extratumoral clonal expansion of cytotoxic and memory T cells in the blood, whereas the majority of intratumoral T cells with shared motifs exhibited terminal exhaustion. Leveraging TCR-BERT, we identified binding patterns exclusively enriched in exhausted T cell receptors' binding sites and uncovered shared motifs within the reactive tumor-infiltrating compartment, implying the existence of potential reservoirs for anti-tumor immunity. Investigating the cell-cell interactions driving intratumoral exhaustion, we identified M2 tumor-associated macrophages and a plasma cell phenotype-like subset upregulating TGFB and IL10 expression, referred to as regulatory B cells (Bregs). Spatial profiling substantiated the proximity of regulatory B cells and exhausted T cells within immunosuppressive niches, suggesting an underexplored mechanism contributing to tumor-derived T cell exhaustion. CONCLUSION Regulatory B-cells have been identified in the TME of various solid tumors, including colorectal, breast, cervical, and ovarian carcinomas. Our study emphasizes the role of regulatory B cells in the chordoma immune environment, providing crucial insights into novel immunosuppressive mechanisms and pathways leading to T cell exhaustion in chordoma.

Use of high-plex data provides novel insights into the temporal artery processes of giant cell arteritis.

To identify the key coding genes underlying the biomarkers and pathways associated with giant cell arteritis (GCA), we performed an in situ spatial profiling of molecules involved in the temporal arteries of GCA patients and controls. Furthermore, we performed pharmacogenomic network analysis to identify potential treatment targets. Using human formalin-fixed paraffin-embedded temporal artery biopsy samples (GCA, n = 9; controls, n = 7), we performed a whole transcriptome analysis using the NanoString GeoMx Digital Spatial Profiler. In total, 59 regions of interest were selected in the intima, media, adventitia, and perivascular adipose tissue (PVAT). Differentially expressed genes (DEGs) (fold-change > 2 or < -2, p-adjusted < 0.01) were compared across each layer to build a spatial and pharmacogenomic network and to explore the pathophysiological mechanisms of GCA. Most of the transcriptome (12,076 genes) was upregulated in GCA arteries, compared to control arteries. Among the screened genes, 282, 227, 40, and 5 DEGs were identified in the intima, media, adventitia, and PVAT, respectively. Genes involved in the immune process and vascular remodeling were upregulated within GCA temporal arteries but differed across the arterial layers. The immune-related functions and vascular remodeling were limited to the intima and media. This study is the first to perform an in situ spatial profiling characterization of the molecules involved in GCA. The pharmacogenomic network analysis identified potential target genes for approved and novel immunotherapies.

Spaceflight-Induced Gene Expression Profiles in the Mouse Brain Are Attenuated by Treatment with the Antioxidant BuOE.

The demands of deep space pose a health risk to the central nervous system that has long been a concern when sending humans to space. While little is known about how spaceflight affects transcription spatially in the brain, a greater understanding of this process has the potential to aid strategies that mitigate the effects of spaceflight on the brain. Therefore, we performed GeoMx Digital Spatial Profiling of mouse brains subjected to either spaceflight or grounded controls. Four brain regions were selected: Cortex, Frontal Cortex, Corunu Ammonis I, and Dentate Gyrus. Antioxidants have emerged as a potential means of attenuating the effects of spaceflight, so we treated a subset of the mice with a superoxide dismutase mimic, MnTnBuOE-2-PyP 5+ (BuOE). Our analysis revealed hundreds of differentially expressed genes due to spaceflight in each of the four brain regions. Both common and region-specific transcriptomic responses were observed. Metabolic pathways and pathways sensitive to oxidative stress were enriched in the four brain regions due to spaceflight. These findings enhance our understanding of brain regional variation in susceptibility to spaceflight conditions. BuOE reduced the transcriptomic effects of spaceflight at a large number of genes, suggesting that this compound may attenuate oxidative stress-induced brain damage caused by the spaceflight environment.

Identifying prognostic markers in spatially heterogeneous breast cancer microenvironment

To gain deeper insights into the microenvironment of breast cancer, we utilized GeoMx Digital Spatial Profiling (DSP) technology to analyze transcripts from 107 regions of interest in 65 untreated breast cancer tissue samples. Our study revealed spatial heterogeneity in the expression of marker genes in tumor cell enriched, immune cell enriched, and normal epithelial areas. We evaluated a total of 55 prognostic markers in tumor cell enriched regions and 15 in immune cell enriched regions, identifying that tumor cell enriched regions had higher levels of follicular helper T cells, resting dendritic cells, and plasma cells than immune cell enriched regions, while the levels of resting CD4 memory in T cells and regulatory (Treg) T cells were lower. Additionally, we analyzed the heterogeneity of HLA gene families, immunological checkpoints, and metabolic genes in these areas. Through univariate Cox analysis, we identified 5 prognosis-related metabolic genes. Furthermore, we conducted immunostaining experiments, including EMILIN2, SURF4, and LYPLA1, to verify our findings. Our investigation into the spatial heterogeneity of the breast cancer tumor environment has led to the discovery of specific diagnostic and prognostic markers in breast cancer.

Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment.

Cancer drug development is currently limited by a paradigm of preclinical evaluation that does not adequately recapitulate the complexity of the intact human tumor microenvironment (TME). To overcome this, we combined trackable intratumor microdosing (CIVO) with spatial biology readouts to directly assess drug effects in patient tumors in situ. In a first-of-its-kind phase 0 clinical trial, we explored the effects of an investigational stage SUMOylation-activating enzyme (SAE) inhibitor, subasumstat (TAK-981) in 12 patients with head and neck carcinoma (HNC). Patients scheduled for tumor resection received percutaneous intratumor injections of subasumstat and vehicle control 1 to 4 days before surgery, resulting in spatially localized and graded regions of drug exposure (∼1,000-2,000 μm in diameter). Drug-exposed (n = 214) and unexposed regions (n = 140) were compared by GeoMx Digital Spatial Profiler, with evaluation at single-cell resolution in a subset of these by CosMx Spatial Molecular Imager. Localized regions of subasumstat exposure revealed SUMO pathway inhibition, elevation of type I IFN response, and inhibition of cell cycle across all tumor samples. Single-cell analysis by CosMx demonstrated cell-cycle inhibition specific to the tumor epithelium, and IFN pathway induction commensurate with a TME shift from immune-suppressive to immune-permissive. Pairing CIVO with spatial profiling enabled detailed investigation of response to subasumstat across a diverse sampling of native and intact TME. We demonstrate that drug mechanism of action can be directly evaluated in a spatially precise manner in the most translationally relevant setting: an in situ human tumor.

PIK3CA mutation subtype delineates distinct immune profiles in gastric carcinoma.

PIK3CA mutations in cancer regulate tumour immunogenicity. Given that PIK3CA mutation subtypes influence therapeutic responses to AKT inhibitor and that H1047R mutation confers selective growth advantages after immunotherapy, we hypothesised that immune phenotypes may depend on PIK3CA mutation subtypes. We investigated 133 gastric cancers (GCs) harbouring PIK3CA mutation [21 E542K (15.8%), 36 E545X (27.1%), 26 H1047X (19.5%), and 46 others (34.6%)].Four patients (3.0%) had a combination of mutations (E542K + E545K in 3 patients and E545K + H1047R in 1 patient). Epstein-Barr virus (EBV) and microsatellite instability (MSI) status, PD-L1 (programmed death-ligand 1) combined positive score (CPS), and stromal tumour-infiltrating lymphocytes (TILs) were assessed. Concurrent genomic alterations, GeoMx digital spatial profiling (DSP), and OPAL multiplex immunohistochemistry (mIHC) were analysed, and correlation between the two assays was investigated. Of the 133 PIK3CA-mutant (PIK3CAm ) GCs, MSI-high GC was significantly frequent in the H1047X mutation subtype (p = 0.005), while EBV positivity did not affect the mutation subtypes. There was no significant survival difference between the E542K, E545X, and H1047X subgroups. However, in the subgroup analysis for EBV-positive GC, H1047Xm GC showed a trend towards shorter survival than E542K and E545Xm GC (p = 0.090 and 0.062). With DSP analysis, H1047Xm GC showed elevated VISTA (p = 0.0003), granzyme B (p < 0.0001), CD4 (p = 0.0001), and CD45 (p < 0.0001) expression compared with the E542Km or E545Xm GC subgroups, and only VISTA expression remained significant (p < 0.0001) using OPAL mIHC. DSP and OPAL analyses showed a moderate correlation of CD4 (ρ = 0.42, p = 0.004) and CD8 (ρ = 0.62, p < 0.001) expression levels in a comparison of six antibodies. Immune-related protein expression levels were evident when classified by the three PIK3CA hotspot mutations, and H1047Xm GC showed the highest immune-related protein expression compared with E542Km or E545Xm GC. Our results demonstrated distinct immune profiles in GC with PIK3CA hotspot mutations using GeoMx DSP and OPAL mIHC, and there was a correlation between the two multiplex platforms. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Deep multi-omic analyses to identify targetable pathways in desmoplastic small round cell tumor (DSRCT) with opportunities for clinical intervention.

e22006 Background: DSRCT is an extremely rare and highly aggressive soft-tissue sarcoma affecting adolescents and young adults with male predominance. The 5-year overall survival is &lt; 25% despite intensive multi-modality treatment including aggressive surgical resection, radiotherapy, chemotherapy, and targeted therapy. Therefore, more effective and innovative therapeutic options are urgently needed. Recent advances in genomic, epigenomic, proteomic, and immune profiling have uncovered a large number of therapeutic targets that are transforming the field of cancer medicine. This study aimed to identify novel targetable pathways in DSRCT using a deep multi-omics-based platform. Methods: Three patients with biopsy proven metastatic DSRCT were enrolled under an IRB-approved observational study within the “Serial Measurements of Molecular and Architectural Responses to Therapy” (SMMART) program at OHSU. Biopsies taken at enrollment were analyzed with targeted DNA sequencing, whole transcriptome sequencing, immunohistochemistry (IHC), NanoString GeoMx digital spatial profiling (DSP) multiplexed proteomics, single cell cyclic immunofluorescence (Cyc-IF), and reverse phase protein array (RPPA). Results: Multiple clinically relevant targets were identified, including HER2, androgen receptor (AR), elevated DNA damage response, and MAPK and mTOR signaling pathways. All three DSRCTs expressed HER2: clinical IHC identified HER2 low (1+) to HER2 equivocal (2+) expression; DSP analysis showed high HER2 and phospho-HER2 expression; and HER2 RNA expression was elevated compared to the TCGA sarcoma cohort. Two of the three patients displayed high AR protein expression, assessed by clinical IHC (90-100% positive cells) and DSP (top quartile compared to a 20 mixed-subtype breast cancer cohort). Strong evidence of DNA damage response was demonstrated at both RNA and protein levels, and RPPA revealed high PARP1, PAR, p-ATM, p-RPA32, and RAD51. Within cell signaling pathways, one DSRCT exhibited high total and phospho-ERK1/2 protein across all clinical and exploratory assays, while another patient displayed evidence of elevated mTOR pathway signaling by RPPA. Conclusions: Overall, this multi-omic analysis provided new insights into possible therapeutic targets for this rare and difficult to treat sarcoma subtype. Results from this study will be further validated in vivo using patient-derived xenograft (PDX) models, providing support for development of multiple early phase trials.