Type Composition Research Articles

Nonbinary 2D Distribution Tool Maps Autonomic Nerve Fiber Clustering in Lumbosacral Ventral Roots of Rhesus Macaques.

Neuromodulation of the peripheral nervous system (PNS) by electrical stimulation may augment autonomic function after injury or in neurodegenerative disorders. Nerve fiber size, myelination, and distance between individual fibers and the stimulation electrode may influence response thresholds to electrical stimulation. However, information on the spatial distribution of nerve fibers within the PNS is sparse. We developed a new two-dimensional (2D) morphological mapping tool to assess spatial heterogeneity and clustering of nerve fibers. The L6-S3 ventral roots (VRs) in rhesus macaques were used as a model system to map preganglionic parasympathetic, γ-motor, and α-motor fibers. Random and ground truth distributions of nerve fiber centroids were determined for each VR by light microscopy. The proposed tool allows for nonbinary determinations of fiber heterogeneity by defining the minimum distance between nerve fibers for cluster inclusion and comparisons with random fiber distributions for each VR. There was extensive variability in the relative composition of nerve fiber types and degree of 2D fiber heterogeneity between different L6-S3 VR levels within and across different animals. There was a positive correlation between the proportion of autonomic fibers and the degree of nerve fiber clustering. Nerve fiber cluster heterogeneity between VRs may contribute to varied functional outcomes from neuromodulation.

AxoDetect: an automated nerve image segmentation and quantification workflow for computational nerve modeling

Objective. Bioelectronic treatments targeting near-organ innervation have unprecedented clinical applications. Particularly in the spleen, the inhibition of the cholinergic inflammatory response by near-organ nerve stimulation has potential to replace pharmacological treatments in chronic and autoimmune diseases. A caveat is that the optimization of therapeutic stimulation parameters relies on in vivo experimentation, which becomes challenging due to the small nerve diameters (2 μ m), complex anatomy, and mixed axon type composition of the autonomic nerves. Effective development of in silico models requires tools which allow for fast and efficient quantification of axonal composition of specific nerves. Current approaches to generate such information rely on manual image segmentation and quantification. Approach. We developed a combined image-segmentation and model-generation software called AxoDetect: a target- and format-agnostic computer vision algorithm which can segment myelin, endo/epineurium, and both myelinated and unmyelinated fibers from a nerve image without training. Main results. AxoDetect is over 10 times faster on average when compared with current automatic methods while maintaining flexibility through the use of tunable pixel threshold filters to detect different types of tissue. When compared to a distribution-based and a manually segmented model of the splenic nerve terminal branch 1, the model generated with AxoDetect had comparable threshold prediction and was able to accurately detect an increase in activation threshold caused by the addition of surrounding fat tissue to the modeled nerve. Significance. AxoDetect contributes to the acceleration of neuromodulation treatment development through faster model design and iteration without requiring training. Furthermore, the computer vision approach and tunable nature of the filters in our method allow for its use in a variety of histological applications. Our approach will impact not only the study of nerves but also the design of implantable neural interfaces to enhance bioelectronic therapeutic options.

Subsurface architecture of the Wajrakarur Kimberlite Field, Dharwar Craton, India: Evidence from ground magnetic surveys

The Ground magnetic study in the Wajrakarur Kimberlite Field and adjoining area has unveiled prominent geological variations, including felsic doming beneath the PGC-II formation to the west of the Cuddapah Basin and mafic doming beneath the Closepet Granite zone. These doming features within the upper mantle are identified as sources of mantle upwelling, which influence the emplacement of corresponding intrusive rocks. The presence of mafic chambers enhances the magnetic response, leading to localized high signatures in magnetic data and contributes to a better understanding of the subsurface composition and distribution of rock types. The present magnetic data throw lights to study the complexity of the sub-surface structure and geological activities, have shaped the study area and serve as valuable markers for understanding the geological evolution of the area. The intersections of magnetic lineament oriented in NW-SE, NE-SW, E-W yield crucial insights into geological structures and the emplacement of Kimberlite intrusions. Through comprehensive magnetic data analysis, the study offers a unique cross-sectional view of the subsurface, reaching depth up to 60 km. This perspective unveils magnetic sources and structures, revealing shallow features, interfaces of the Archaean basement, crustal roots, and upper mantle configuration. Profound magnetic gradients observed in the study area serve as indicators of fault lines influenced by tectonic activity, kimberlite intrusion, and other fault-related mineralization if any. These features contribute to a deeper understanding of the structural configurations and geological history of the area. The present study provides information about the lateral and vertical layouts of major structures as well as the deep-seated channels. It also introduces the mafic and felsic categories of rocks, explaining the complex relationships between underlying structures, geological characteristics, and magnetic anomalies.

Group composition of income types and the absolute-relative framing of public good contributions

This paper examines the effect of absolute-relative framing on contributions to a public good. Group members have high and low endowments and the number of high-income members in each group varies between treatments. They make contributions without and with a minimum contribution level. We express the contribution metric either in absolute amount or as a relative proportion of a member's initial endowment. We propose that these institutional designs affect contribution behavior through shifting reference points in decisions. First, as the number of high-type members increases, the average contribution increases, especially among the low-type members in absolute framing. Second, relative framing makes the contribution proportions between high and low types move closer than in absolute framing. Third, the difference in contributions between absolute and relative conditions mostly disappears when the minimum contribution level is introduced. We show that the effects of group members’ endowments, relative framing, and the MCL policy represent the internal, external and the institutional reference point respectively with increasing strength.

Incorporation of spray-dried encapsulated bioactive peptides from coconut (Cocos nucifera L.) meal by-product in bread formulation.

This study aimed to stabilize and mask the bitterness of peptides obtained from the enzymatic hydrolysis of coconut-meal protein with maltodextrin (MD) and maltodextrin-pectin (MD-P) as carriers via spray-drying. Essential (~35%), hydrophobic (~32%), antioxidant (~15%), and bitter (~45%) amino acids comprised a significant fraction of the peptide composition (with a degree of hydrolysis of 33%). The results indicated that the peptide's production efficiency, physical and functional properties, and hygroscopicity improved after spray-drying. Morphological features of free peptides (fragile and porous structures), spray-dried with MD (wrinkled with indented structures), and MD-P combination (relatively spherical particles with smooth surfaces) were influenced by the process type and feed composition. Adding free and microencapsulated peptides to the bread formula (2% W/W) caused changes in moisture content (35%-43%), water activity (0.89-0.94), textural properties (1-1.6 N), specific volume (5.5-6 cm3/g), porosity (18%-27%), and color indices of the fortified product. MD-P encapsulated peptides in bread fortification resulted in thermal stability and increased antioxidant activity (DPPH and ABTS+ radical scavenging: 4.5%-39.4% and 31.6%-46.8%, respectively). MD-P (as a carrier) could maintain sensory characteristics and mask the bitterness of peptides in the fortified bread. The results of this research can be used to produce functional food and diet formulations.

Abstract 1517: Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers

Abstract Background: Microsatellite instability (MSI-h) is a strong biomarker to predict response to immune checkpoint therapy and patient’s outcome in colorectal cancer. Although enrichment of distinct T-cell subpopulations, such as type 1 cytotoxic T-cells (Tc1) or type 1 helper T-cells (Th1), have been identified to impact patient’s outcome and response to immune checkpoint therapy, only little is known about the underlying changes in the composition of the immune tumor microenvironment. Material and Methods: To assess the density, composition, degree of functional marker expression, and spatial interplay of T-cell subpopulations in 93 MSI and 1153 microsatellite stable (MSS) colorectal cancers, a tissue microarray as well as large sections were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, CD27, CD56, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis. Results: The composition of Type 1 (T-bet+), Type 2 (GATA3+), Type 17 (RORyT+), NKT-like (CD56+), regulatory (FOXP3+), and follicular (BCL6+) cytotoxic (CD3+CD8+) or helper (CD3+CD4+) T-cells showed marked differences between MSI and MSS patients. For instance, the density and proportion of Tc1 as well as Th1 was significantly higher (p<0.001 each) in MSI compared to MSS patients. In contrast, the density and composition of Tregs, Th2, Th17 T-cells was significantly lower in MSI compared to MSS patients in the intraepithelial tumor compartment. In addition, among the MSI colorectal cancers, NKT-cells showed the highest level of Granzyme B that was significantly higher than in MSS patients (p<0.05). The degree of immune checkpoint expression (i.e., TIM3, PD-1, CTLA-4) was significantly higher in MSI compared to MSS patients for most T-cell subpopulations (p<0.05 each). The additional analysis of 10 large sections confirmed the observations from the TMA analysis and revealed that the alterations in the T-cell composition seen in the center of the tumor largely reflect the T-cell composition at the invasive margin in both MSI and MSS patients. Conclusion: This study identified a higher proportion of Tc1 cytotoxic T-cells and Th1 helper T-cells accompanied by a paucity of regulatory T-cell subpopulations, Th17 and Th2 T- helper cells in MSI colorectal cancers compared to MSS patients. Citation Format: Tim Mandelkow, Zhihao Huang, Elena Bady, Jan H. Müller, Guido Sauter, Julia Ebner, Maximilian Lennartz, Natalia Gorbokon, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Niclas C. Blessin. Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1517.

Abstract 6249: Single-cell RNA sequencing investigation of multi-stage NASH-related HCC mouse model

Abstract Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and a leading cause of cancer-related death worldwide. Its incidence is also one of the most rapidly increasing in the West. Most (>80%) of HCCs are diagnosed at advanced stage and therefore not operable. Even after surgical resection, the long-term prognosis of HCC remains unsatisfactory due to high recurrence rates. It is also an extremely difficult-to-treat cancer. Most HCC patients have a background of chronic liver disease, including chronic viral infection, excessive alcohol consumption or non-alcoholic fatty liver disease NAFLD)/steatohepatitis (NASH). In June 2023, new nomenclature of NAFLD and NASH have been internationally announced as metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis, respectively. Due to the enforcement of neonatal HBV vaccination program and the availability of effective antiviral treatments on HBV and HCV, the global incidence of viral hepatitis-related HCC has been declining, whereas NAFLD/NASH-related incidence of HCC has been increasing. We established NASH-related HCC mouse model using Western diet (WD) plus carbon tetrachloride (CCl4) method. This model demonstrated steatosis, fibrosis, obesity and HCC with high incidence. It has also been reported to accurately portrait the key pathological features of the patients. Next, we collected liver tissue samples at different timepoints (normal; Week 12: NAFLD/NASH; Week 18: fibrosis/cirrhosis; Week 32: HCC) and analyzed them using single-cell RNA sequencing (scRNA-seq). Single-cell sequencing offers an unprecedented platform to perform systematic and in-depth investigation of the admixed tumor microenvironment (TME) in HCC tumor. We have taken advantage of the unique multi-dimensional capacity of scRNA-seq to delineate multifaceted landscapes and cell-cell communication in multi-stage NASH-related HCC mouse model. After removing the low-quality cells, our multi-stage dataset consisted of >130,000 cells. Using typical cell type markers, we identified major cell types, namely T cells, B cells, myeloid cells, liver sinusoidal endothelial cells, hepatic stellate cells, hepatocytes and HCC tumor cells. We observed a change in the cell type composition during disease progression. Throughout our study, we closely examined the communication level between different cell types to identify any irregularities or disturbances that could lead to NASH-related HCC. Our findings concluded that hepatic stellate cells, despite existing in low proportion at all stages, exhibited the strongest level of interaction with other cell types. Moreover, our data also established the comprehensive multi-stage cellular landscape of NASH-relative HCC. Citation Format: Tina Suoangbaji, Vanilla Xin Zhang, Daniel Wai-Hung Ho, Irene Oi-Lin Ng. Single-cell RNA sequencing investigation of multi-stage NASH-related HCC mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6249.

Abstract 7044: Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients

Abstract Human telomerase reverse transcriptase (hTERT) vaccine has found to be effective for most of the participated de novo metastatic prostate cancer patients in clinical studies. Nevertheless, the molecular mechanisms behind the immune response induced by the hTERT vaccine are not fully elucidated. Among the cohort of 21 patients treated with androgen deprivation therapy, radiotherapy and hTERT vaccine, we used RNA-sequencing and TCR-sequencing (20 and 14 pre-treatment and 20 and 21 post-treatment samples, respectively) to inspect the changes in transcriptome and TCR-repertoire after the hTERT vaccine treatment and differences between patients that had early response and patients that had late or no response. We found multiple hallmarks being shifted after the treatment and being different between early and late responders. Also, a change in activity of multiple gene set signatures was detected after the treatment and activity of part of the gene set signatures predicted survival. Deconvolution of cell type composition from RNA-sequencing data revealed changes in immune cell abundances after the treatment. TCR-sequencing revealed changes in composition and diversity of TCR repertoires after the treatment and these differed between early and late responders. The results of this study will help to predict patients’ responses to the cancer vaccine and to further understand the mechanisms behind successful cancer vaccines. Citation Format: Reetta Nätkin, Julia Elisabeth Simensen, Nikolai Engedal, Espen Basmo Ellingsen, Marita Westhrin, Ingunn Westgaard, Eivind Hovig, Wolfgang Lilleby, Matti Nykter, Alfonso Urbanucci. Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7044.

Graded FGF activity patterns distinct cell types within the apical sensory organ of the sea anemone Nematostella vectensis

Bilaterian animals have evolved complex sensory organs comprised of distinct cell types that function coordinately to sense the environment. Each sensory unit has a defined architecture built from component cell types, including sensory cells, non-sensory support cells, and dedicated sensory neurons. Whether this characteristic cellular composition is present in the sensory organs of non-bilaterian animals is unknown. Here, we interrogate the cell type composition and gene regulatory networks controlling development of the larval apical sensory organ in the sea anemone Nematostella vectensis. Using single cell RNA sequencing and imaging approaches, we reveal two unique cell types in the Nematostella apical sensory organ, GABAergic sensory cells and a putative non-sensory support cell population. Further, we identify the paired-like (PRD) homeodomain gene prd146 as a specific sensory cell marker and show that Prd146+ sensory cells become post-mitotic after gastrulation. Genetic loss of function approaches show that Prd146 is essential for apical sensory organ development. Using a candidate gene knockdown approach, we place prd146 downstream of FGF signaling in the apical sensory organ gene regulatory network. Further, we demonstrate that an aboral FGF activity gradient coordinately regulates the specification of both sensory and support cells. Collectively, these experiments define the genetic basis for apical sensory organ development in a non-bilaterian animal and reveal an unanticipated degree of complexity in a prototypic sensory structure.

Abstract 4335: High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors

Abstract This study is aimed at testing the hypothesis that increased patient age fosters conditions that are favorable for head and neck squamous cell carcinomas (HNSCC) of the oral cavity development. There is a well-documented increase in cancer incidence with age observed across diverse cancer types, where elderly patients exhibit diminished survival outcomes compared to their younger counterparts. While the convergence of aging and cancer hallmarks offers valuable insights, further work is needed to elucidate the age-specific mechanisms influencing HNSCC, beyond the shared characteristics of these biological processes. To this end, we have assembled a high-quality human single-cell RNA-sequencing HNSCC atlas profiling more than 230,000 cells across more than 50 patients with ages ranging between 18 and 89, which provides a resource to investigate age-associated changes in the disease heterogeneity. For this library, we integrated six publicly available single-cell RNAseq datasets from 54 HPV-negative patients to create the atlas. Cells were clustered, classified, characterized by gene set enrichment analysis, both in the epithelial cell compartment and in the tumor microenvironment (TME). Differential cell type proportion analysis was performed to identify cell types enriched in young (<49 years) or old (>70 years) patients. Cell-cell communication analysis was performed to identify signaling events occurring between different populations, and how these and specific ligand-receptor pairs differed in patients of different ages. CNV analyses were performed to identify cancer subclones and assess level of variation across tumors. Interestingly, we identified distinct cell populations and signaling events that associate with age, and we were able to compare, validate and recapitulate these observations in the 4MOSC1 OSCC isograft tumor model in old and young mice. Additional notable findings include a differential enrichment for cytotoxic CD8 T cells over dysfunctional CD8 T cells in young patients when compared to old, suggesting younger patients have a more effective immune TME. We also found an enrichment for cancer-associated fibroblast populations in old patients (myCAFs) that send collagen signals to malignant epithelial cells, a result we have recapitulated using the 4MOSC1 model, and hypothesize may play a role in ECM stiffening, EMT, and metastasis. Further analyses are ongoing, and we plan to validate the hypotheses generated, specifically the presence of differential abundance of specific cell populations, and age-specific ligand-receptor signaling events that lead to tumor growth. Citation Format: Lina Kroehling, Anthony Spinella, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4335.

Abstract 2468: Phase II study of cryoablation and post-progression immune checkpoint inhibition in metastatic melanoma

Abstract Introduction: Efforts to overcome resistance to immune checkpoint inhibition (ICI) are paramount, and novel combination strategies are in development. Image-guided percutaneous cryoablation is an established minimally invasive oncologic treatment that has demonstrated immune modulatory effects. We hypothesized that cryoablation may prime the tumor microenvironment (TME) through direct modulation of the tumor, thereby generating an anti-tumor response in ICI refractory tumors. Methods: In this non-randomized phase II single-center study, subjects with unresectable melanoma progressing on ICI underwent cryoablation of an enlarging metastasis, and ICI was continued for a minimum of two additional cycles. The primary endpoint was safety and feasibility, with objective response rate (ORR) and disease control rate (DCR) in non-ablated lesions a key secondary endpoint. To better understand the inflammatory state of TME as well as factors determining response to cryoablation, we performed immunohistochemistry (PD-L1, CD8+ TIL) as well as single-cell RNA-sequencing (ScRNAseq) on evaluable pre- and post-cryoablation biopsies obtained from metastatic sites of 15 patients. Results: From May 2018 through July 2020, 17 patients were treated. This regimen was feasible with all patients ongoing cryoablation with no unexpected safety signal. The ORR and DCR was 23.5% (95% CI 6.81-49.9) and 41.2% (95% CI 18.44-67.08), respectively, with four patients experiencing PR and three patients SD. Durable control, defined as tumor control ≥6 months, was achieved in 35%. Median progression-free survival (PFS) was 59 days (95% CI 42, 179) with a 6-month PFS rate of 23.5% (95% CI 7.3%, 44.9%). Median overall survival (OS) was 387 days (95% CI 106, 608) with a 1-year OS rate of 53% (95% CI (27.6%, 73%). Correlative analysis demonstrated pre-ablation tumors were uniformly associated with a “cold” TME, as all samples were PD-L1 negative and the majority had low levels of CD8+ TIL, defined as ˂5% of tumor cells associated with CD8+ TILs. ScRNAseq revealed no clear trends of cell type composition to predict best response to cryoablation, however an increase in B/plasma cells was seen in patients with disease control, suggesting these cells may play a role in mounting a durable anti-tumor response after cryoablation. Conclusion: The anti-tumor efficacy of combining cryoablation and post-progression ICI was superior to what would be expected with the use of post-progression ICI alone with an acceptable safety profile. Our data support further study of this novel, synergistic therapeutic approach. Citation Format: Meghan J. Mooradian, Florian J. Fintelmann, Thomas J. LaSalle, Judit Simon, Alexander Graur, Alona Muzikansky, Mari Mino-Kenudson, Sophia Shalhout, Howard L. Kaufman, Russell W. Jenkins, Donald Lawrence, Aleigha Lawless, Tatyana Sharova, Raul N. Uppot, Jacy Fang, Emily M. Blaum, Anna L. Gonye, Irena Gushterova, Genevieve M. Boland, Nir Hacohen, Moshe Sade-Feldman, Ryan J. Sullivan. Phase II study of cryoablation and post-progression immune checkpoint inhibition in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2468.

Abstract 7433: CGC-hosted comprehensive pipeline for spatial transcriptomics analysis

Abstract Spatial transcriptomics has been experiencing significant growth in the past years. Inspired by two widely adopted methodologies, particularly in situ hybridization and next-generation sequencing, with an emphasis on the single-cell RNA sequencing (scRNA-seq), this hybrid method facilitates whole transcriptome profiling while preserving spatial context at high resolutions, thus allowing the exploration of novel insights in cancer research. We introduce the highly configurable solution for sequencing-based technologies that allow comprehensive spatial analysis. The pipeline is available on NCI-funded Cancer Genomics Cloud (CGC) platform, powered by Seven Bridges. The CGC platform provides a collaborative cloud base computation infrastructure that collocates computation, over 1000 bioinformatics workflows, and 4+ PB data to researchers, making the analysis of the Cancer Research data Commons (CRDC) datasets accessible from any environment. This pipeline is developed using reliable, widely adopted packages and can process datasets generated by several leading technologies. It comprises a set of common steps including quality control, data preprocessing, dimensionality reduction, cluster identification, detection of spatially variable features, and integration with scRNA-Seq reference. The pipeline is highly configurable, allowing for various settings to be modified and optimized for result refinement. Additionally, it allows the selection of only specific components of the pipeline to be executed. The key steps of the pipeline are visually represented, allowing researchers to get a detailed insight into results. Here, we demonstrate a typical flow of spatial transcriptomics analysis on publicly available datasets using the developed pipeline. We illustrate the impact of pipeline settings on the analysis outcomes. Furthermore, we identify spatially variable genes whose expressions show a distinct localization within the tissue. Finally, we perform the data integration to predict the cell type composition within the determined spatial domains. Spatial transcriptomics analysis is a powerful method that has highly improved critical aspects of cancer research, such as the characterization of tumor microenvironment, the discovery of novel biomarkers and the clarification of drug resistance mechanisms. The ongoing evolution of this method is expected to play vital role in our deep understanding of complex spatial relationships within tissues. This CGC-hosted workflow is developed to contribute to the promising advancements. Citation Format: Miona Rankovic, Nevena Vukojicic, Nevena Ilic Raicevic, Vida Matovic, Divya Sain, Jack DiGiovanna, Brandi Davis-Dusenbery. CGC-hosted comprehensive pipeline for spatial transcriptomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7433.

Abstract 4898: Highly accurate profiling of leukocyte composition from bulk peripheral blood with targeted digital cytometry

Abstract Background: Cell profiling methods such as flow/mass cytometry and single-cell RNA-sequencing are powerful tools for quantifying immune composition from healthy and neoplastic tissues. However, only a modest number of markers can be interrogated by the former and the latter remains cost prohibitive for large-scale analysis. Here we demonstrate robust, accurate, and reproducible enumeration of immune cell subsets from 36 whole blood samples using SureSelect XT HS2 RNA sequencing combined with CiberMed’s iSortTM digital cytometry solution. Enrichment of genes in the LM22 signature matrix, a well-established collection of reference profiles for deconvolving 22 human immune subsets, was achieved with two new targeted sequencing panels—Agilent SureSelect CD CiberMed Heme and Agilent SureSelect CD CiberMed Heme + HiRes. Both panels were assessed for their ability to profile leukocyte subsets with CiberMed’s iSortTM Fractions software, an optimized and standardized version of CIBERSORTx with novel enhancements. Methods: Whole blood samples were freshly collected from 36 healthy donors and split into two fractions—one was immediately processed for complete blood count (CBC) and flow cytometric enumeration of major leukocyte populations; the other was stored in PAXgene Blood RNA tubes for subsequent RNA sequencing. A Sysmex system was employed for CBC quantification and a Becton Dickinson 6-color TBNK MultiTest in vitro diagnostic (IVD) assay was employed for enumerating B cells, CD8 T cells, CD4 T cells, NK cells. These data were used as ground truth to assess iSortTM deconvolution performance from targeted and whole-transcriptome bulk RNA sequencing data. RNA-seq expression values were used as input to iSortTM Fractions to deconvolve 22 immune subsets in each sample. Results: Across 7 major populations, cell type fractions determined by iSortTM Fractions were highly concordant with ground truth fractions determined by clinical grade standards (r≥0.96) and exhibited strong reproducibility across technical replicates (r≥0.98). Furthermore, targeted enrichment using the SureSelect CD CiberMed Heme panel reduced the sequencing requirement by nearly 50-fold compared to whole-transcriptome sequencing, while also improving accuracy. Conclusion: These new panels for digital cytometry are being released through the Agilent Community Design program to enable focused, reliable, and high-throughput analysis of cell type composition from peripheral blood samples. Citation Format: Aki Nakao, Aaron M. Newman, Ash A. Alizadeh, Maximilian Diehn, Mistuni Ghosh, Manjula Aliminati, Jayati Ghosh, Kristi Stephenson, Ashutosh Ashutosh. Highly accurate profiling of leukocyte composition from bulk peripheral blood with targeted digital cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4898.

Abstract 5498: Multi-modal examination of spatial heterogeneity in the astrocytoma microenvironment

Abstract Astrocytoma is a diffuse tumor that infiltrates the brain. Despite post-operative therapy including radiation and chemotherapy, 52% of patients’ tumors recur within five-years of surgery. These tumors are characterized by mutations in the IDH1 or IDH2 gene that confer metabolic alterations, most notably the production of the onco-metabolite 2-hydroxyglutarate. To reveal the metabolic alterations and their upstream regulation across the tumor microenvironment, we applied spatial metabolomics and spatial transcriptomics to sequential sections from resected tumor samples of patients with low grade astrocytoma. This necessitated extensive optimization of atmospheric-pressure MALDI-TOF, including the establishment of a custom automated computational pipeline with abilities to identify metabolic niches. Additionally, using sequential sections of the same tumor, we also compared all commercially available imaging-based spatial transcriptomic technologies (10x Genomics Xenium, Nanostring CosMx, Vizgen MERSCOPE) to evaluate their suitability with regards to building a multi-modal model of metabolic activity and regulation across the tumor microenvironment. We found that 10x Genomics Xenium was most suited for this task as image registration across sequential sections was comparatively straight-forward. Combining spatial transcriptomics generated with 10x Genomics Xenium and spatial metabolomics across 8 matched sequential slices of the same tumor, we identified metabolic niches associated with distinct cell type composition and transcriptional regulation. Citation Format: Joel Moffet, Jurgen Kriel, Tianyao Lu, Lutz Freytag, James Whittle, Sarah Best, Saskia Freytag. Multi-modal examination of spatial heterogeneity in the astrocytoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5498.

Abstract 5485: Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level

Abstract We characterized immune cell exclusion in estrogen receptor positive (ER+) breast cancer through the study of spatial relationships among various subsets of cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and other immune cells using a novel in-situ spatial transcriptomic technology. ER+ breast cancer is the most common breast cancer subtype. While endocrine therapy and immune checkpoint inhibitors have considerably reduced relapse and mortality for breast cancer patients, tumor resistance to treatment remains a major challenge. The tumor microenvironment (TME) plays an important role in this mechanism of resistance. Our previous spatial proteomic analysis of ER+ breast FFPE samples demonstrated the intricate relationship between tumor and immunosuppressive cells such as CAFs and TAMs via the CXCR4-CXCL12 axis associated with the exclusion of CD8 T cells from the TME. However, the limited number of antibodies in the panel made it challenging to investigate the diversity of those CAF and TAM populations and better understand their contribution to T cell exclusion. For this purpose, we further characterized the TME of 4 human ER+ breast cancer FFPE cores using the Xenium in-situ transcriptomic platform with a breast cancer-focused panel of 280 genes from 10x Genomics plus 100 additional genes selected from literature and publicly available single cell datasets to delineate CAF and TAM sub-populations. Image analysis enabled the quantification and spatial mapping of the target genes within each cell boundary. Cells were manually annotated through unsupervised clustering of over 1 million cells. The cell phenotyping analysis allowed us to annotate major cell types including tumor cells, macrophages, T cells and fibroblasts. With our curated panel, we further characterized CAF and TAM sub-populations by identifying myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs) in addition to M1 and M2-like macrophages as reported in the literature. We then performed spatial neighborhood analysis based on the similarity of the cell type composition in the vicinity of each cell. The results showed that myCAFs and M2-like macrophages are found in proximity to tumor cells and may play a role in T-cell exclusion. In addition, iCAFs mainly express CXCL12, a chemokine known to attract immunosuppressive cells such as regulatory T cells and M2-like macrophages as well as repel cytotoxic T cells from infiltrating the tumor via the CXCR4-CXCL12 axis. Overall, our work shows the potential of spatial transcriptomics to elucidate the spatial distribution of cells and their contribution to the immunosuppressive TME, which may enable researchers to improve understanding of tumor biology and drug target discovery efforts. Citation Format: Julien Tessier, Sandra Curras-Alonso, Joon Sang Lee, Fabien Delahaye, Hong Ma, Katie Malley, Dinesh Bangari, Donald Jackson, Angela Hadjipanayis. Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5485.

Abstract 3668: Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients

Abstract Plasma cell-free DNA (cfDNA) originates from apoptotic cells in the blood, providing a real-time insight into the biology of various cell types. The fragment coverage pattern of cfDNA across the genome is known to reflect the distinct chromatin status of originating cells. While extensive research has focused on fragments derived from cancer, known as circulating tumor DNA (ctDNA), for inference about cancer biology, cfDNA fragments from healthy cells have received little attention. Our study explores the potential of cfDNA to decipher the relative abundance of distinct blood cell types, particularly immune system cell types. We collected a cohort of 273 bladder (BC) and colorectal cancer (CRC) patients and 45 healthy controls for plasma whole genome sequencing and assessed the fragment coverage profile at transcription start sites (TSS) of protein coding genes. A significant negative correlation was observed between TSS coverage and whole blood gene expression (Spearman’s rho = -0.65). Furthermore, TSS coverage revealed a bimodal distribution, characterized by modes corresponding to high and low gene expression levels in blood cells. This may suggest the cfDNA coverage profile as a binary expression status classifier to distinguish between expressed and unexpressed genes in blood cells. We further evaluated TSS with differential coverage between age groups of healthy individuals and relapse status of cancer patients. Notably, out of the TSS that significantly differentiated middle-aged (aged 40-65) from older individuals (aged 65+), 31 were also able to distinguish relapse from non-relapse patients. Among these TSS were CD36, SLAMF7, TABBP and PDCD1, known to be involved in immune related pathways. Finally, we evaluated coverage at cell-type specific TSS and found dendritic cells (P-value: CRC = 8e-06, BC = 5.1e-3), monocytes (CRC = 1.6e-04, BC = 2.5e-3), and NK cells (CRC = 8.5e-04, BC = 0.026) to exhibit a significant increase in coverage in the cancer cohorts compared to healthy controls. Conversely, T-cells showed a decrease in coverage (CRC = 0.041, BC = 0.062). In conclusion, cfDNA coverage profile may identify TSS with differential coverage between patient groups and characterize a shift in blood cell type composition as a response to cancer. Future work may show if fragment coverage quantifies the relative contribution of cfDNA from distinct immune cell types. We anticipate our analysis to illuminate the utility of plasma cfDNA as a biomarker for cancer patient’s immune competency, enabling improved patient stratification for treatment, leveraging their distinct immune capabilities. Citation Format: Laura Andersen, Amanda Frydendahl, Tenna Vesterman Henriksen, Christina Demuth, Mads Heilskov Rasmussen, Iver Nordentoft, Karin Birkenkamp-Demtröder, Jakob Skou Pedersen, Søren Besenbacher, Lars Dyrskjøt, Claus L. Andersen, Nicolai J. Birkbak. Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3668.

Abstract 1146: Pan-cancer characterization of cancer cell state and plasticity using spatially resolved transcriptomics

Abstract Cancer cell state heterogeneity constitutes a foundational characteristic of cancer, posing a formidable barrier to the discovery of biomarkers and the efficacy of therapeutic interventions. However, current understanding of cancer cell heterogeneity and plasticity remains limited, especially in the spatial context. Previous studies underscored that cancer cell states are not strictly governed by genetics, but rather display a remarkable degree of plasticity. Recent pan-cancer single-cell studies have unveiled dozens of recurrent cancer cell states. Nevertheless, the relationship between these cancer cell states and tumor microenvironment (TME) remains poorly understood, especially when considering the diversity across different tumor types. Recent advancements in spatial transcriptomics (ST) have paved the way for a novel approach to spatially profile cell location, organization, and interaction within the tumor microenvironment. In this study, we curated ST data (Visium, 10x Genomics) from public repositories and in-house datasets, encompassing a total of >200 tissue sections across 11 cancer types. By incorporating histological annotations and inferred copy number variations, we systematically investigated the spatial heterogeneity of cancer cell states and inferred clonal architectures of cancer cell-enriched spots. Non-negative matrix factorization was applied to identify highly recurrent cancer states and transcriptional hallmarks associated with intra-tumor heterogeneity. By employing this approach, we uncover a diverse curated gene set of meta-programs. Notably, we observe a high level of reproducibility of these meta-programs when compared to the findings from single-cell level. Furthermore, we observe significant variations of cell states across different cancer types, even among two cancer subtypes within a specific tissue. We noted a unique distribution of 'hypoxia' and 'epithelial-mesenchymal transition' in the basal lineage tumor. Following this, we perform single-cell and ST co-embedding using available tools, including CellTrek and CytoSPACE. We noticed the cell type composition of tumor’s neighbor spots play a significant role in influencing tumor states, especially for myofibroblastic CAFs and the tumor state in epithelial-mesenchymal transition. These findings contribute valuable insights into the relationship between cancer cell state plasticity and interactions with the TME, offering potential targets for further exploration and therapeutic intervention. Citation Format: Guangsheng Pei, Kyung S. Cho, Yunhe Liu, Serrano Alejandra, Rossana Lazcano, Enyu Dai, Guangchun Han, Fuduan Peng, Daiwei Zhang, Yanshuo Chu, Ansam F. Sinjab, Jiahui Jiang, Mingyao Li, Cassian Yee, Andrew Futreal, Alex Lazar, Humam Kadara, Jianjun Gao, Luisa M. Soto, Anirban Maitra, Jaffer Ajani, Linghua Wang. Pan-cancer characterization of cancer cell state and plasticity using spatially resolved transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1146.

Generation of synthetic whole-slide image tiles of tumours from RNA-sequencing data via cascaded diffusion models.

Training machine-learning models with synthetically generated data can alleviate the problem of data scarcity when acquiring diverse and sufficiently large datasets is costly and challenging. Here we show that cascaded diffusion models can be used to synthesize realistic whole-slide image tiles from latent representations of RNA-sequencing data from human tumours. Alterations in gene expression affected the composition of cell types in the generated synthetic image tiles, which accurately preserved the distribution of cell types and maintained the cell fraction observed in bulk RNA-sequencing data, as we show for lung adenocarcinoma, kidney renal papillary cell carcinoma, cervical squamous cell carcinoma, colon adenocarcinoma and glioblastoma. Machine-learning models pretrained with the generated synthetic data performed better than models trained from scratch. Synthetic data may accelerate the development of machine-learning models in scarce-data settings and allow for the imputation of missing data modalities.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Molecular Profiling of Axial Spondyloarthritis Patients Reveals an Association between Innate and Adaptive Cell Populations and Therapeutic Response to Tumor Necrosis Factor Inhibitors.

This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.