GeoMxTM Digital Spatial Profiler Research Articles

Transcriptomic and proteomic spatial profiling of pediatric and adult diffuse midline glioma H3 K27-Altered.

Diffuse midline glioma, H3 K27-altered (DMG) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large scale spatial transcriptomic studies have implicated that tumor microenvironmental landscape plays an important role in determining the phenotypic differences in tumor presentation and clinical course, however, data connecting overall transcriptomic changes to the protein level is lacking. The NanoString GeoMx™Digital Spatial Profiler platform was used to determine the spatial transcriptomic and proteomic landscape in a cohort of both pediatric and adult H3 K27-altered DMG biopsy samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to establish regions of interest (ROI) for genomic and proteomic analysis. We found genetic alterations within the tumor which can be delineated across patient age and spatial location. We show that the H3 K27M mutation itself has a profound impact on tumor cells transcriptomics and interestingly we found limited fidelity between overall transcriptome and proteome. Our data also validate a previously described genomic signature at the proteomic level and reveal a special shift in the signature based on the local TME composition.

Spatial multi-omics of human skin reveals KRAS and inflammatory responses to spaceflight

Spaceflight can change metabolic, immunological, and biological homeostasis and cause skin rashes and irritation, yet the molecular basis remains unclear. To investigate the impact of short-duration spaceflight on the skin, we conducted skin biopsies on the Inspiration4 crew members before (L-44) and after (R + 1) flight. Leveraging multi-omics assays including GeoMx™ Digital Spatial Profiler, single-cell RNA/ATAC-seq, and metagenomics/metatranscriptomics, we assessed spatial gene expressions and associated microbial and immune changes across 95 skin regions in four compartments: outer epidermis, inner epidermis, outer dermis, and vasculature. Post-flight samples showed significant up-regulation of genes related to inflammation and KRAS signaling across all skin regions. These spaceflight-associated changes mapped to specific cellular responses, including altered interferon responses, DNA damage, epithelial barrier disruptions, T-cell migration, and hindered regeneration were located primarily in outer tissue compartments. We also linked epithelial disruption to microbial shifts in skin swab and immune cell activity to PBMC single-cell data from the same crew and timepoints. Our findings present the inaugural collection and examination of astronaut skin, offering insights for future space missions and response countermeasures.

Human Plasma-Like Medium Activates Immune Elements in Glioma

INTRODUCTION: Glioma Surgically eXplanted Organoids (SXOs) are ex vivo three-dimensional models demonstrated to recapitulate the parental tumor microenvironment (TME). Human plasma-like medium (HPLM), a culture medium that mirrors physiologic nutrient conditions, has been found in other cancer subtypes to induce transcriptional differences in T-lymphocyte activation. We hypothesized that SXO culture in HPLM would maintain immune cell populations and induce physiologically relevant immune-modulatory pathways. METHODS: SXOs were established and cultured in conventional Glioma Organoid Complete medium (GOC) and transitioned to HPLM for 24 or 120 hours. Formalin-fixed paraffin-embedded samples were stained for tumor cells (GFAP), immune cells (CD45), and nucleic acid (SYTO-13) and analyzed using the Nanostring GeoMx™ Digital Spatial Profiler for spatial transcriptomics. Gene expression clustering profiles were conducted using GSEA and CIBERSORT. The gene expression differences of SXOs cultured in HPLM for 24 and 120 hours compared to those in GOC were analyzed by unpaired t-tests. RESULTS: Glioma SXOs retain diverse cell populations, with 20-30% of cells belonging to the TME. CD4+ T-cells were the most common (9.7 ± 1.0), followed by CD8+ T-cells (4.4 ± 0.6) and macrophages (3.1 ± 1.2). Following the transition to HPLM for 24 or 120 hours, there were no significant differences in tumor, immune, and stromal cell fractions. HPLM culture upregulated the immune response in SXOs, encompassing enhanced cytokine secretion, as well as improved adaptive and innate immune pathways. We particularly highlighted CD4+ T-cell activation (CD69+), Treg, and TGF-β signaling pathways as significantly increased. CONCLUSIONS: Here, we present that the culture of SXOs in physiologically similar media retains the heterogeneous TME and activates innate and adaptive immune signaling pathways. Culture in HPLM enables investigation of the glioma immune microenvironment, expanding the biological relevance of experimental results from in vitro SXO models.

Spatial proteomics and cell‐type specific changes in the basal ganglia of different neuropathologic vascular dementing lesions

AbstractBackgroundVascular dementia (VD) is the second most common cause of dementia behind Alzheimer’s disease. VD has been associated with various cardiovascular maladies, which are thought to contribute to the white matter abnormalities associated with dementia. Cerebrovascular impairment disrupts CNS blood supply resulting in inflammation, oxidative stress, and hypoxia. The aim of this project is to study the impact of the cytotoxic environment created by disruptions to the CNS vasculature, which could be detrimental to glial (microglia, astrocyte) viability and function. We examined age and sex‐matched VD and control human autopsy brain from the Honolulu Asia Aging Study.MethodUtilizing Nanostring’s GeoMx™ Digital Spatial Profiling (DSP), we analyzed autopsy brain to compare neuropathologic vascular dementia lesions (lacunar, macro, and chronic microinfarcts) in the basal ganglia across the disease spectrum (absent/mild, moderate, severe). DSP enables spatial analysis of expression levels for 73 proteins in multiple regions of interest on formalin‐fixed paraffin‐embedded sections. Morphology markers were IBA1 (microglia) and GFAP (astrocytes) (Figure 1a‐b).Indica Labs’ HALO object co‐localization module was used to determine astrocyte, microglia, and total nuclei counts.ResultFor chronic microinfarct severity, astrocyte and microglial counts decreased as the number of microinfarcts increased. By contrast, for macroinfarcts, astrocyte counts increased in addition to microglia steadily decreasing as the number of macroinfarcts increased. Analyses comparing increasing chronic microinfarct severity revealed numerous proteins with differential expression in both microglia and astrocytes, including pTauS404, IDE, p62, ATG5 and P2RX7 (Figure 2 microglia; Figure 3 astrocytes).ConclusionUsing a spatial proteomic technique, we demonstrated that various vascular dementing lesions have unique glial autophagy and inflammatory profiles at different disease stages. Overall, lower levels of vascular dementia lesions display lower levels of inflammation and increased upregulation of protective pathways that may contribute to maintenance of cognitive function. We also demonstrated basal ganglia cell population changes across different disease severities for chronic microinfarcts and macroinfarcts. Findings suggest that there is progressive astrocyte and microglial loss in the setting of chronic microinfarcts and increased astrogliosis in the setting of macroinfarcts. Whether there is a definitive inverse relationship in astrocyte counts between chronic microinfarcts and macroinfarcts remains unclear.

Spatial proteomics and cell‐type specific changes in the basal ganglia of different neuropathologic vascular dementing lesions

AbstractBackgroundVascular dementia (VD) is the second most common cause of dementia behind Alzheimer’s disease. VD has been associated with various cardiovascular maladies, which are thought to contribute to the white matter abnormalities associated with dementia. Cerebrovascular impairment disrupts CNS blood supply resulting in inflammation, oxidative stress, and hypoxia. The aim of this project is to study the impact of the cytotoxic environment created by disruptions to the CNS vasculature, which could be detrimental to glial (microglia, astrocyte) viability and function. We examined age and sex‐matched VD and control human autopsy brain from the Honolulu Asia Aging Study.MethodUtilizing Nanostring’s GeoMx™ Digital Spatial Profiling (DSP), we analyzed autopsy brain to compare neuropathologic vascular dementia lesions (lacunar, macro, and chronic microinfarcts) in the basal ganglia across the disease spectrum (absent/mild, moderate, severe). DSP enables spatial analysis of expression levels for 73 proteins in multiple regions of interest on formalin‐fixed paraffin‐embedded sections. Morphology markers were IBA1 (microglia) and GFAP (astrocytes) (Figure 1a‐b).Indica Labs’ HALO object co‐localization module was used to determine astrocyte, microglia, and total nuclei counts.ResultFor chronic microinfarct severity, astrocyte and microglial counts decreased as the number of microinfarcts increased. By contrast, for macroinfarcts, astrocyte counts increased in addition to microglia steadily decreasing as the number of macroinfarcts increased. Analyses comparing increasing chronic microinfarct severity revealed numerous proteins with differential expression in both microglia and astrocytes, including pTauS404, IDE, p62, ATG5 and P2RX7 (Figure 2 microglia; Figure 3 astrocytes).ConclusionUsing a spatial proteomic technique, we demonstrated that various vascular dementing lesions have unique glial autophagy and inflammatory profiles at different disease stages. Overall, lower levels of vascular dementia lesions display lower levels of inflammation and increased upregulation of protective pathways that may contribute to maintenance of cognitive function. We also demonstrated basal ganglia cell population changes across different disease severities for chronic microinfarcts and macroinfarcts. Findings suggest that there is progressive astrocyte and microglial loss in the setting of chronic microinfarcts and increased astrogliosis in the setting of macroinfarcts. Whether there is a definitive inverse relationship in astrocyte counts between chronic microinfarcts and macroinfarcts remains unclear.

Hippocampal subregion‐specific proteomic differences between Alzheimer disease (AD) and primary age‐related tauopathy (PART)

AbstractBackgroundAlthough Alzheimer disease (AD) and primary age‐related tauopathy (PART) both harbor 3R/4R‐tau immunopositive Alzheimer‐type neurofibrillary degeneration, they differ in the spatial development of neurofibrillary tangles (NFTs) in the hippocampus. PART displays an early predilection for neurofibrillary degeneration in the CA2 subregion of the hippocampus, whereas AD typically develops NFTs in the entorhinal cortex and CA1 subregion initially (Figures 1‐2).MethodUsing Nanostring’s GeoMx™ Digital Spatial Profiling (DSP), we compared hippocampal subregion protein expression differences between AD and PART. The DSP panel allows for spatial analysis of the level of expression of 73 different proteins in multiple regions of interest (ROIs) on formalin‐fixed paraffin embedded sections. Our ROIs were NFT‐bearing neurons and non‐NFT‐bearing neurons in the entorhinal cortex, CA1 and CA2 hippocampal subregions, as well as the neuronal microenvironments in these regions.ResultAnalyses identified several proteins displaying differential expression in tangle‐bearing neurons when comparing AD to PART. These included higher levels of synaptic and neuronal markers in PART, such as Synaptophysin, MAP2, Calbindin and NRGN, as well as higher levels of proteins involved in degradation pathways, such as CTSD, GPNMB, Park7 and FUS. PART also displayed higher levels of Neprilysin in CA2, and higher IDE in CA1, which are proteins that help degrade β‐amyloid (Figure 3). Many of these proteins were also differentially expressed in the non‐tangle bearing neurons. In addition, AD demonstrated higher levels of p‐tau T231, S214, S199, and S396 in non‐tangle bearing neurons of CA1, as well as higher expression of Aβ1‐40. In the microenvironment, NEFL, NRGN, CLEC7a, GPNMB, and CTSD were higher in PART, while p‐tau S396 and BACE1 were higher in AD.ConclusionIn conclusion, PART cases displayed higher levels of neuronal and synaptic markers, suggesting better maintenance of neuronal and synaptic integrity than AD, as well as higher levels of proteins important for degradation of detrimental proteins. AD cases expressed higher levels of certain p‐tau epitopes, especially in and around CA1 non‐tangle bearing neurons. In addition, AD displayed higher levels of BACE1, whereas PART displayed higher levels of CTSD, Neprilysin and IDE, which could explain the differences in β‐amyloid deposition in these cases.

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of >1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR <0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

Spatially resolved, high-dimensional transcriptomics sorts out the evolution of biphasic malignant pleural mesothelioma: new paradigms for immunotherapy

BackgroundMalignant Pleural Mesothelioma (MPM) is a dreadful disease escaping the classical genetic model of cancer evolution and characterized by wide heterogeneity and transcriptional plasticity. Clinical evolution of MPM is marked by a progressive transdifferentiation that converts well differentiated epithelioid (E) cells into undifferentiated and pleomorphic sarcomatoid (S) phenotypes. Catching the way this transition takes place is necessary to understand how MPM develops and progresses and it is mandatory to improve patients’ management and life expectancy. Bulk transcriptomic approaches, while providing a significant overview, failed to resolve the timing of this evolution and to identify the hierarchy of molecular events through which this transition takes place.MethodsWe applied a spatially resolved, high-dimensional transcriptomic approach to study MPM morphological evolution. 139 regions across 8 biphasic MPMs (B-MPMs) were profiled using the GeoMx™Digital Spatial Profiler to reconstruct the positional context of transcriptional activities and the spatial topology of MPM cells interactions. Validation was conducted on an independent large cohort of 84 MPMs by targeted digital barcoding analysis.ResultsOur results demonstrated the existence of a complex circular ecosystem in which, within a strong asbestos-driven inflammatory environment, MPM and immune cells affect each other to support S-transdifferentiation. We also showed that TGFB1 polarized M2-Tumor Associated Macrophages foster immune evasion and that TGFB1 expression correlates with reduced survival probability.ConclusionsBesides providing crucial insights into the multidimensional interactions governing MPM clinical evolution, these results open new perspectives to improve the use of immunotherapy in this disease.

Tumor lineage-specific immune response in brain metastatic disease: opportunities for targeted immunotherapy regimen?

Metastases in the brain are the most severe and devastating complication of cancer. The incidence of brain metastasis is increasing. Therefore, the need of finding specific druggable targets for brain metastasis is demanding. The aim of this study was to compare the brain (immune) response to brain metastases of the most common tumor lineages, viz., lung adenocarcinoma and breast cancer. Targeted gene expression profiles of 11 brain metastasis of lung adenocarcinoma (BM-LUAD) were compared to 11 brain metastasis of breast cancer (BCBM) using NanoString nCounter PanCancer IO 360™ Panel. The most promising results were validated spatially using the novel GeoMx™ Digital Spatial Profiler (DSP) Technology. Additionally, Immune cell profiles and expression of drug targets were validated by multiplex immunohistochemistry. We found a more active immune response in BM-LUAD as compared to BCBM. In the BM-LUAD, 138 genes were upregulated as compared to BCBM (adj. p ≤ 0.05). Conversely, in BCBM 28 genes were upregulated (adj. p ≤ 0.05). Additionally, genes related to CD45 + cells, T cells, and cytotoxic T cells showed to be expressed higher in BM-LUAD compared to BCBM (adj. p = 0.01, adj. p = 0.023, adj. p = 0.023, respectively). The spatial quantification of the immune cells using the GeoMx DSP technique revealed the significantly higher quantification of CD14 and CD163 in tumor regions of BM-LUAD as compared to BCBM. Importantly, the immune checkpoint VISTA and IDO1 were identified as highly expressed in the BM-LUAD. Multiplex immunohistochemistry confirmed the finding and showed that VISTA is expressed mainly in BM-LUAD tumor cells, CD3 + cells, and to fewer levels in some microglial cells in BM-LUAD. This is the first report on differences in the brain immune response between metastatic tumors of different lineages. We found a far more extensive infiltration of immune cells in BM-LUAD as compared to BCBM. In addition, we found higher expression of VISTA and IDO1 in BM-LUAD. Taken together, targeted immune therapy should be considered to treat patients with BM-LUAD.

Abstract 5558: Understanding the molecular mechanism of tumor budding and its relationship with tumor microenvironment in colorectal cancer

Abstract Background: Tumor budding (TB), a single or cluster of up to 4 cells at the tumor-invasive front, is a well-established prognostic marker and an independent predictor of metastasis in colorectal cancer (CRC). Despite that, the underlying molecular mechanism that drives this phenotype and its relationship with the tumor microenvironment (TME) remains unclear. Methods: The relationship between TME and TB was investigated using multiplex immunofluorescence (mIF) in tissue microarrays (TMAs) of the tumor core and full sections. GeoMxTM digital spatial profiling (DSP) was employed to identify the mechanism underlying TB and its surrounding TME, using the GeoMx Immune-oncology pathway panel in full CRC sections. Results: In the current study, TB was independently associated with decreased cancer-specific survival (OR 1.856, 95% CI 1.360-2.533, P<0.001) in a cohort of 650 CRC patients. Additionally, TB was significantly associated with the Glasgow microenvironment score (GMS) (P=0.017). mIF performed in TMAs of the tumor core (n=650) showed that high CD3+ (OR0.374, 95% CI0.263-0.533, P<0.001) associated with good prognosis, whereas high CD68+ (OR2.902, 95% CI1.728-4.873, P<0.001) associated with poor prognosis. In addition, eighteen full sections were stained with an immune-related multiplex panel. Interestingly, in the invasive area, CD3+CD8+ was less frequent, while CD3+FOXP3+ was significantly higher compared to the stromal area in a patient with high TB (P=0.06, P<0.01, respectively). Moreover, though no significant difference was found, tumors with high buds showed an increase in neutrophil marker (CD66b) compared to low buds. No association is also found with a myeloid marker (CD68, CD163). The proliferation index of the tumor in the invasive area showed that the tumor with low buds is likely to be more proliferative. Besides, we found that a small subpopulation of budding is KI67+, suggesting the heterogeneity among TB. Furthermore, the mechanism underlying TB was observed using GeoMxTM. Twelve cases, selected from mIF stained, with low (n=6) or high buds (n=6) were utilized, and 96 areas of interest (AOI) were identified from the tumor core, invasive front, and stroma. The results showed the differential expression of genes between the tumor core and the invasive front in patients with high TB. A significant decrease in immune-related genes (e.g., CD3, NKG7, IL6, CXCR6, CD47, IFNAR1 and VSIR) compared to the stromal area at the invasive front was also observed (P<0.001). Conclusion: TB is associated with poor prognosis and showed a distinct gene expression characteristic compared to the tumor mass. It may also affect the inflammatory response at the invasive tumor. The underlying mechanism of TB and its relationship with TME warrants future studies to confirm this finding. Citation Format: Phimmada Hatthakarnkul, Holly Leslie, Hester Van Wyk, Leah Officer-Jones, Silvia Cusumano, Aula Ammar, Kathryn A.F. Pennel, Jean A. Quinn, Jennifer Hay, James Park, Noori Maka, John Le Quesne, Chanitra Thuwajit, Nigel Jamieson, Joanne Edwards. Understanding the molecular mechanism of tumor budding and its relationship with tumor microenvironment in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5558.

Abstract 2052: High-plex spatial multiomics of pediatric and adult diffuse midline glioma, H3 K27-altered

Abstract Background: Diffuse midline glioma (DMG), H3 K27-altered are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative landscapes between pediatric and adult DMGs is not known. Methods: The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult DMG, H3 K27-altered tissue samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the Illumina sequencing platform to assess transcriptomic and proteomic data for both cohorts of samples. Results: Our data revealed the immune and genetic expression seen within both populations across multiple regions of interest within the samples. Differences were seen specific to each population that may have clinical significance and warrant further exploration including a significant increase in genes associated with RTK pathways in the pediatric samples. Conclusion: The digital spatial analysis of pediatric and adult DMG, H3 K27-altered shows differences within the molecular landscape which may contribute to the clinical differences between both populations. These observed differences may play a role in future treatments and further research is needed to explore this. Citation Format: Sudarshawn Damodharan, Alyssa Rosenbloom, Mahua Dey. High-plex spatial multiomics of pediatric and adult diffuse midline glioma, H3 K27-altered [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2052.

MP69-03 EXAMING THE LANDSCAPE OF CONCURRENT UPPER TRACT AND LOWER TRACT UROTHELIAL CARCINOMA USING SPATIAL TRANSCRIPTOMIC ANALYSIS

You have accessJournal of UrologyCME1 Apr 2023MP69-03 EXAMING THE LANDSCAPE OF CONCURRENT UPPER TRACT AND LOWER TRACT UROTHELIAL CARCINOMA USING SPATIAL TRANSCRIPTOMIC ANALYSIS Shreyas Naidu, Facundo Davaro, Xuefeng Wang, Elizabeth Davaro, Heather Huelster, Kyle Rose, Gustavo Borjas, Aram Vosoughi, and Roger Li Shreyas NaiduShreyas Naidu More articles by this author , Facundo DavaroFacundo Davaro More articles by this author , Xuefeng WangXuefeng Wang More articles by this author , Elizabeth DavaroElizabeth Davaro More articles by this author , Heather HuelsterHeather Huelster More articles by this author , Kyle RoseKyle Rose More articles by this author , Gustavo BorjasGustavo Borjas More articles by this author , Aram VosoughiAram Vosoughi More articles by this author , and Roger LiRoger Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003332.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite histologic similarities between upper-tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB), there are differences in the clonal types, mutations, and immune landscape between these tumors. In this study, we performed multiplex spatial profiling of RNA on tumor samples from patients with concomitant UTUC and UCB in the evaluation of the tumors using the GeoMx™ Digital Spatial Profiling platform. METHODS: Three patients with concomitant high grade non-invasive UTUC and UCB were identified. Six FFPE samples from cystectomy and nephroureterectomy for the three patients were identified. A pathologist selected 18 regions of interest (ROIs), including tumor, tumor-stroma interface, and stroma area on each specimen. Multiplex IHC was performed on each sample with stains for DNA, Pan-CK, CD20 (B cells), and CD45 (immunocytes). The GeoMx™ Digital Spatial Profiling platform was used to sequence the RNA from the ROIs on each FFPE slide. Spatial deconvolution was used to determine ROI cellular composition. RESULTS: Multiplex IHC images demonstrated the increased infiltration of CD20 and CD45 immune cells in the UCB in compared to UTUC tumors; however, the proportion of inflammatory cells (i.e. T-cells, B-cells, plasma cells, T-regulatory cells, macrophages) are similar in different ROIs within each tumor and in UCB and UTUC tumors in each patient and even in samples from same sites among all patients. From the 11000+ genes identified through GeoMx analysis, we identified variable expression among UTUC and UCB samples. Notably, UNC5B, a gene that codes for a netrin receptor that is correlated with bladder cancer recurrence, was highly expressed in UCB. Additionally, UTUCs showed higher expression of histone regulatory genes (H1-3, H2BC5, H2BC8), which may lead to tumorigenesis and progression to muscle-invasive UC. CONCLUSIONS: UTUC demonstrates lower immune cells infiltration, consistent with previous UTUC studies; however, the proportion of inflammatory cells component is similar in different foci of the tumor and in concomitant UCB and UTUC tumors in each patient and even among all tumor samples from the same site. UNC5B, which has been associated with tumor recurrence, is highly expressed in UCB. UTUC patients showed higher expression of histone regulatory genes. Source of Funding: Moffitt Cancer Center © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e964 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shreyas Naidu More articles by this author Facundo Davaro More articles by this author Xuefeng Wang More articles by this author Elizabeth Davaro More articles by this author Heather Huelster More articles by this author Kyle Rose More articles by this author Gustavo Borjas More articles by this author Aram Vosoughi More articles by this author Roger Li More articles by this author Expand All Advertisement PDF downloadLoading ...

Spatial genomics reveals a high number and specific location of B cells in the pancreatic ductal adenocarcinoma microenvironment of long-term survivors.

Only 10% of pancreatic ductal adenocarcinoma (PDAC) patients survive longer than five years. Factors underlining long-term survivorship in PDAC are not well understood. Therefore, we aimed to identify the key players in the tumor immune microenvironment (TIME) associated with long-term survivorship in PDAC patients. The immune-related gene expression profiles of resected PDAC tumors of patients who survived and remained recurrence-free of disease for ≥36 months (long-term survivors, n=10) were compared to patients who had survived ≤6 months (short-term survivors, n=10) due to tumor recurrence. Validation was performed by the spatial protein expression profile of immune cells using the GeoMx™ Digital Spatial Profiler. An independent cohort of samples consisting of 12 long-term survivors and 10 short-term survivors, was used for additional validation. The independent validation was performed by combining qualitative immunohistochemistry and quantitative protein expression profiling. B cells were found to be significantly increased in the TIME of long-term survivors by gene expression profiling (p=0.018). The high tumor infiltration of B cells was confirmed by spatial protein profiling in the discovery and the validation cohorts (p=0.002 and p=0.01, respectively). The higher number of infiltrated B cells was found mainly in the stromal compartments of PDAC samples and was exclusively found within tumor cells in long-term survivors. This is the first comprehensive study that connects the immune landscape of gene expression profiles and protein spatial infiltration with the survivorship of PDAC patients. We found a higher number and a specific location of B cells in TIME of long-term survivors which emphasizes the importance of B cells and B cell-based therapy for future personalized immunotherapy in PDAC patients.

Spatial proteomics in the hippocampi of resistant and resilient individuals as compared to Alzheimer’s disease (AD) and primary age‐related tauopathy (PART).

AbstractBackgroundAn intriguing group of individuals, termed “resilient,” demonstrate neuropathologic changes consistent with Alzheimer disease (AD), yet display no cognitive impairment. In addition, there are older individuals who resist the development of AD neuropathologic change despite their age, termed “resistant.” Herein, we analyzed protein expression in the hippocampi around normal and neurofibrillary tangle (NFT)‐bearing neurons in these resistant and resilient individuals as compared to those with AD and primary age‐related tauopathy (PART) with cognitive decline.MethodUtilizing Nanostring’s GeoMx™ Digital Spatial Profiling (DSP) technology, we compared individuals with AD neuropathologic change and dementia to individuals with AD neuropathologic change and no dementia (resilient), as well as PART with dementia and resistant (PART without dementia) cases. The DSP panel allows for spatial analysis of the level of expression of 73 different proteins in multiple regions of interest (ROIs) on formalin‐fixed paraffin‐embedded sections. We analyzed hippocampal sections, with our ROIs being NFT‐bearing neurons, non‐NFT‐bearing neurons, and their surrounding microenvironments (non‐neuronal cells and neuropil), including a comparison of entorhinal cortex, CA1 and CA2 subregions (Figures 1 and 2).ResultAnalyses comparing NFT‐bearing neurons to non‐NFT‐bearing neurons, revealed 22 proteins with differential expression, including various p‐tau epitopes, Neprilysin, ADAM10, IDE and GPNMB (Figure 3). Comparing resilient and AD NFTs, we identified 17 proteins with differential expression, including Park 5, HSC70 and VPS35 (Figure 4). The microenvironment of the resistant compared to others highlighted several proteins with differential expression including GFAP, NRGN and NEFL (Figure 5). A comparison of NFT‐bearing neurons in non‐demented cases displayed regional protein expression differences between entorhinal and CA1 including alpha‐synuclein and CSF1R. Distinct regional protein expression differences in NFT‐bearing neurons in demented cases were also identified including EMP1 and ATG12 (Figure 6).ConclusionIn conclusion, using a novel spatial proteomic technique, we have demonstrated that resistant and resilient individuals display lower levels of inflammation and upregulation of protective pathways, that in turn lead to maintenance of neuronal integrity. In addition, although the resistant and resilient harbor PART and AD pathology (respectively), we have identified protein expression differences that may further our understanding of the mechanisms by which the resistant and resilient evade cognitive decline.

Investigation of differentially expressed proteins through neurofibrillary tangle maturation in Alzheimer’s disease using NanoString’s GeoMx Digital Spatial Profiler

AbstractBackgroundNeurofibrillary tangles are dynamic entities with a lifespan encompassing three maturity levels: intracellular pretangles, intracellular mature tangles, and extracellular ghost tangles. Proteins are suggested to be differentially expressed throughout the tangle lifespan; however, past studies may be limited by spatial expression or number of proteins investigated. We sought to assess differences in protein expression across the tangle lifespan using GeoMxTM Digital Spatial Profiler (DSP) by NanoString, a new technology allowing for spatially derived multiplex investigation.MethodWe used DSP to measure protein expression in hippocampi from Alzheimer’s disease (n=6) and nondemented controls (n=2) using the “Human Neural Cell Profiling Core” and “Alzheimer’s Pathology” module. Regions of interest (ROIs) were classified by the major tangle maturity level recognized by a primary fluorescent conjugated tau antibody. Protein expression was normalized to the geomean of the three housekeeping genes. The tau‐positive segment was specifically analyzed for comparisons. ANOVA was used for group‐wise comparisons and t‐test using the Benjamini‐Yekutieli adjustment for multiple tests was used for pair‐wise comparisons.ResultAs expected, cytoskeletal (e.g., MAP2, Tau) and neuronal (e.g., NeuN) markers generally decreased through the neurofibrillary tangle lifespan (Figure). Markers profiled as neurodegenerative (e.g., amyloid‐β1‐42, APOE, APP) generally increased through tangle maturity levels, except for TDP‐43 which decreased. Interestingly, there was no significant difference in ubiquitin expression. Astrocytic GFAP and S100B expression was significantly increased through each maturity level. Expression of microglial‐specific markers P2RY12 and TMEM119 was significantly elevated in ghost tangles compared to mature tangles. Additional microglial/macrophage markers including Iba‐1 and CD68 were generally increased as neurofibrillary tangles matured. Interestingly, APOE expression remained low in pretangles and mature tangles before dramatically increasing in ghost tangles (Figure).ConclusionOur findings suggest that proteins are differentially expressed throughout the tangle lifespan and the majority followed a monotonically directed pattern. Non‐monotonically directed protein patterns may reflect the death of the neuron during the ghost tangle maturity level. Future studies will test the hypothesis that the microenvironment of the hippocampus is altered with increasing vulnerability to tangle pathology.

Spatial Transcriptomics of Tissue-Based Immune Response to Acute Graft-Versus-Host Disease of the Lower Gastrointestinal Tract

Background: Corticosteroid resistant acute graft-versus-host disease (GVHD) of the lower gastrointestinal (GI) tract is a leading cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We hypothesized that distinct gene expression profiles exist within affected GI tissue at GVHD diagnosis and may be associated with treatment outcomes. Methods: Unstained tissue sections from 33 patients with biopsy-confirmed acute lower GI GVHD were used for spatial transcriptomics using the NanoString GeoMxTM Digital Spatial Profiler (DSP) platform. Fluorescent morphology markers (PanCK, CD45 and CD31) were used to visualize the epithelial, immune and endothelial compartments and guide capture of RNA expression profiles using the GeoMxTM Human Whole Transcriptome Atlas (18,676 genes). A total of 739 areas of interest (AOIs) were sampled across the 33 patients with a total of 57,960 immune, 37,610 epithelial, and 2298 vascular endothelial cells analyzed (average of 1756.4 immune, 1139.7 epithelial and 69.6 endothelial cells per case). The gene specific oligos were quantified using Illumina's i5 x i7 dual-indexing system and sequenced on the Illumina NextSeq 500 using a 2 x 75 MidOutput v2.5 flow cell. The FASTQ files were processed to generate target probe-specific count files and saved in DCC format. Gene expression files were normalized per NanoString protocol. All unsupervised clustering and differential expression were done in Bioconductor in the R statistical computing space. Results: The gene expression data for each AOI of immune, epithelial, and endothelial cells were normalized and the top 500 most variable genes were used for unsupervised hierarchical clustering to obtain an unbiased classification of different subtypes. There were two clear major AOI clusters for each of the cellular compartments with immune clustering shown in Figure 1A. The left immune cluster (IC1) and the right immune cluster (IC2) were then evaluated for association with clinical GVHD outcomes. IC1 had an over-representation of patients with steroid sensitive GVHD (Fisher exact p value = <0.0001) and AOIs with low grade pathology (Fisher exact p value = 0.031). To obtain gene level differences between IC1 and IC2, differential expression was performed using a Wilcoxon Rank Sum test and volcano plots were generated to illustrate the most differentially expressed genes across the visual AOI-based clusters (Figure 1B). Genes with an adjusted p-value of 0.05 for association with the particular arm of the volcano plot and at least a 2-fold change over the other cluster were considered significant. Gene set enrichment analysis (GSEA) using curated datasets from MSigDB was performed and identified that IC1 genes were enriched for IFNγ response (q value = 2.99e-5), TNFα signaling (q value = 2.99e-5) and inflammatory response (q value = 0.0063). These IC1 differentially expressed genes included CD74 (835 vs. 361.6 mean normalized gene transcripts), LYZ, β2-microglobulin, C1QB and CD68. IC2, on the other hand, had a prominent exhausted/immunosuppressive signature significantly enriched for Th17 cell genes (q value = 0.027) by GSEA. Notable genes that were statistically significant were FCF1 (152.3 vs. 74 mean normalized transcripts) and USP17L11 (78.3 vs. 36.4 mean normalized transcripts). Extension of this analysis to the epithelial (EC) and vascular endothelial (VC) cell compartments also identified distinct clustering with both having an inflamed (EC1 and VC1) and a basal-like state (EC2 and VC2). Interestingly, the inflamed epithelial cells (EC2) associated with immunosuppressive IC2 immune cells (Fisher exact p-value = 0.006), but inflamed vascular endothelium (VC1) associated with the pro-inflammatory IC1 immune cells (Fisher exact p-value = 0.0005). Conclusion: Spatial transcriptomics provides high resolution gene expression profiles in distinct cellular compartments in human acute GI GVHD samples and provides a better understanding of the relationship of the immune response with epithelial and vascular endothelial cell phenotypes. Initial analysis demonstrates that an inflammatory immune signature is associated with steroid responsive acute GI GVHD and an exhausted phenotype with steroid resistance. Future studies are needed to confirm the predictive value of specific gene expression profiles as biomarkers of corticosteroid response at diagnostic biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPCO-32. HIGHLY MULTIPLEXED PROTEOGENOMIC SPATIAL PROFILING OF PEDIATRIC AND ADULT H3 K27-ALTERED DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND H3 K27-altered diffuse midline gliomas (DMG) are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative landscapes between pediatric and adult DMGs is not known. METHODS The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult H3 K27-altered DMG tissue samples. 5 samples from each population were assessed in triplicates for a total of 30 samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the NanoString spatial profiler and Illumina sequencing system to assess proteomics and gene expressions for both cohorts of samples. RESULTS Our data revealed that there were immune and genetic changes that were seen within both populations across multiple regions of interest within the tumor tissues. A number of immune cell types and protein markers exhibited significant difference between the pediatric and adult tissue cohorts. CONCLUSION The proteogenomic spatial analysis of pediatric and adult H3 K27-altered DMG shows different immune and genetic profiles which are proposed to contribute to the clinical differences between both populations. These observed differences may play a role in future treatments for H3 K27-altered DMG and further research is needed to explore this.

P799: SPATIAL ANALYSIS IDENTIFIES A SPECTRUM OF IMMUNE DYSREGULATION IN ACQUIRED BONE MARROW FAILURE CONDITIONS

Background: Poor Graft Function (PGF), manifested by multilineage cytopenias with complete donor chimerism post allogeneic stem cell transplantation, and acquired Aplastic Anaemia (AA) are acquired bone marrow failure syndromes. Both are thought to result from T cell activation resulting in excessive interferon-γ (IFNγ) production, which in turn suppresses haematopoiesis. In AA, this can promote clonal stem cell selection and potentially progression to myeloid malignancy. Despite similarities in the clinical presentation and proposed mechanism, no studies to date have compared the immunobiology of the bone marrow (BM) microenvironment in these conditions. Aims: To examine the immune microenvironment of the BM in archival AA and PGF BM trephines and identify common immunopathologies. Methods: Archival BM trephines were sourced from patients with PGF (n=20), AA at Diagnosis (AA_DX) (n=15), AA at Progression (AA_PROG) to MDS/AML (n=15) and normal controls (NC) (n=20). 6 x 300µm regions identified by dual CD3/CD45 immunofluorescent staining were analysed per BM trephine using NanoString GeoMX™ digital spatial profiling for the expression of 57 proteins with an immunology focused panel. Data was analysed using a Limma-Voom bioinformatics pipeline. Results: Significant dysregulation was identified across multiple proteins. Overall, 22 proteins had significantly different expression in AA_DX samples (13 up, 9 down) compared to NC with AA_PROG and PGF having 8 (4 up, 4 down) and 14 (7 up, 7 down) respectively. As would be expected given the hypocellular BM, expression of CD45 was significantly reduced compared to NC across AA_DX (adj P = 5.413e-10), AA_PROG (adj P = 8.299e-8) and PGF (adj P = 8.634e-9) groups. When AA_DX patients were stratified into those who progressed (n=6) vs those who did not (n=9), or when AA_DX vs AA_PROG was compared in patients with matched samples (n=6) there were no significantly differentially expressed proteins identified. This suggests that while AA provides an immune microenvironment that is permissive to MDS/AML pathogenesis, it may not directly impact on clonal evolution. Total monocytes were unchanged by CD11c expression. However, AA and PGF samples exhibited upregulation of CD163 compared to NC (AA_DX adj P = 4.5e-6; AA_PROG adj P = 1.2e-5; PGF adj P = 1.58e-8) suggesting an increase in monocyte/macrophage lineage cells. Expression of CD66b was also downregulated in AA_DX (adj P = 8.459e-24), AA_PROG (adj P = 1.193e-10) and PGF (adj P = 2.244e-14) suggesting a reduction in non-classical monocytes, neutrophils or granulocytic MDSCs. Importantly, VISTA was downregulated across AA_DX (adj P = 6.84e-27), AA_PROG (adj P = 1.1e-5) and PGF (adj P = 1.58e-8) and STING was downregulated in AA_DX compared to NC (adj P = 6.139e-13) with a trend towards reduced expression in PGF. Decreased expression of these key immunoregulatory proteins may have wide ranging effects on BM resident macrophages, dendritic cells, MDSCs and T cells leading to reduced immunoregulation and increased T cell activation/IFNγ production, ultimately resulting in stem cell depletion. Data on cell specific expression is currently being obtained to refine this model. Summary/Conclusion: Spatial analysis demonstrated that patients with AA and PGF exhibit similar patterns of protein expression likely resulting in a BM immune microenvironment of decreased immunoregulation. AA_DX samples exhibited a greater degree of dysregulation of multiple markers compared to PGF suggesting that these diseases represent a spectrum of immune dysregulation.

Abstract 1116: Spatial distribution of granzyme B to improve the predictive power of response to immunotherapy in operable lung cancer

Abstract Purpose Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. There is a strong rationale for incorporating immunotherapy into the treatment of early-stage NSCLC, given the breakthrough results with PD-1 checkpoint inhibitors in advanced-stage NSCLC. How immunotherapy should be implemented in patients who are operable is still unclear. Most of the efforts so far to identify clinically useful biomarkers do not preserve spatial information and leave us blind to the critical source of information revealed in the cell-to-cell biology of the tumor microenvironment (TME). In order to overcome these limitations, we used spatial biomarkers assays that preserve this critical information about which cells are influencing treatment response. Method Frozen sections from retrospectively collected surgically resected NSCLC (adenocarcinoma and squamous cell carcinoma) tumors treated with adjuvant pembrolizumab therapy were used. Patients were classified in two groups according to their Objective Response Rate (ORR): Complete Response (CR) and Progression Disease (PD) for spatial transcriptomic and proteomics assays. Associated to RNA In situ hybridization (ISH) technology, the NanoString GeoMx™ Digital Spatial Profiling (DSP) technology was used to determine immune markers within compartment specific defined by fluorescence localization [tumor (panCK) and leucocytes (CD45)]. Wilcoxon, Linear Mixed Model and Mann-Whitney U statistic tests were used to evaluate differences between ORR groups. Results Using the spatial analysis, we identified 4 protein markers from immune cell profiling and immune activation status modules independently associated with benefit from single-agent PD-1 checkpoint blockade in spatial context. High expression of CD4, CD40, Granzyme B and HLA-DR were significantly associated with all favorable clinical outcome, whereas upregulation of extracellular matrix (ECM) proteins related to pro-tumoral reprogramming of the stroma were associated with immunotherapy resistance. We also validated the DSP finding that high granzyme level in the stroma compartment was predictive for response to check point inhibitor in the same set of patients using ISH immunofluorescence, strengthening this marker relevance in antitumor immunity and as a predictive biomarker candidate for immunotherapy in NSCLC. Conclusion This work identifies a number of relevant spatial candidate predictors of immunotherapy outcome in spatial context for operable lung cancer that show promise for future validation in larger independent cohorts. Citation Format: Corinne Ramos, Ophelie Lemieux, Adele Ponzoni, Jonathan Stauber. Spatial distribution of granzyme B to improve the predictive power of response to immunotherapy in operable lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1116.

DIPG-29. Highly multiplexed digital spatial profiling of the tumor immune environment of pediatric and adultH3 K27M-mutant diffuse midline gliomas

Abstract BACKGROUND: H3 K27M-mutant diffuse midline gliomas (DMGs) are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative immune landscape between pediatric and adult DMGs is not known. METHODS: The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult H3 K27M-mutant DMG tissue samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the Illumina sequencing system to assess gene expression and proteomics for both cohorts of samples. RESULTS: Our data revealed that there were immune and genetic marker changes that were seen within both populations across multiple regions of interest within the tumor tissues. A number of immune cell types and protein markers exhibited significant difference between the pediatric and adult tissue cohorts. CONCLUSION: The digital spatial analysis of pediatric and adult H3 K27M-mutant DMGs show different immune and genetic profiles which are proposed to contribute to the clinical differences between both populations. These observed differences may play a role in future treatments for H3 K27M-mutant DMGs and further research is needed to explore this.