Cellular Diversity Research Articles

Deciphering the prognostic landscape of osteosarcoma: Integrating the roles of hippo pathway genes, programmed cell death, and the tumor immune microenvironment.

Osteosarcoma is a highly aggressive cancer prevalent among adolescents and young adults, notorious for its tendency to metastasize to the lungs. This research delves into the molecular foundations of osteosarcoma by examining the role of the Hippo signaling pathway and its interaction with the tumor immune microenvironment (TME). Through analysis of transcriptomic data from the TARGET-OS dataset and control samples from GTEx, we identified a set of 131 genes that link high expression profiles in osteosarcoma with the Hippo pathway. A focused examination through univariate Cox regression analysis revealed eight key genes (DLG5, WNT11, TGFB2, DLG4, WNT16, ID2, WNT10B, and WNT10A) with a significant correlation to patient outcomes. Hierarchical clustering of these genes delineated two distinct patient groups with significantly different survival rates, a finding supported by Kaplan-Meier survival analysis. Further investigation into immune cell infiltration and expression profiles of immunoregulatory factors uncovered a notable pattern of immune evasion in the group with poorer prognosis, marked by reduced effector immune cell activity and lower levels of immunostimulatory factors. Single-cell sequencing highlighted the cellular diversity within osteosarcoma samples and identified markers differentiating malignant from nonmalignant cells, correlating these markers with prognostic risk scores. Our results emphasize the critical prognostic value of Hippo pathway genes and the TME in osteosarcoma, shedding light on new avenues for therapeutic intervention and patient-specific treatment strategies.

Abstract LB193: Enhanced single cell DNA methylation analysis using combinatorial indexing

Abstract The epigenetic landscape of the human brain undergoes a plethora of changes during malignant transformation. In recent years, DNA methylation-based epigenetic modifications have been widely studied using traditional techniques like bisulfite sequencing and enzymatic methyl sequencing (EM-seq). However, these methods analyze bulk cell populations and lack the granularity of single-cell analysis. Although advances in single-cell analysis have revolutionized our understanding of cellular heterogeneity and functional diversity within complex biological systems, they are still costly, low throughput, and laborious. To address these challenges, ScaleBio has pioneered combinatorial indexing technology, enabling a significant increase in cell throughput. This method utilizes the cell itself as a compartment to perform 2-3 rounds of sequential barcoding in a plate-based workflow, eliminating the need for complex instrumentation. This technology has been successfully adapted to assess DNA methylation at the single-cell level offering a robust, affordable, high-throughput protocol that enhances yield, diversity, and coverage. In this study we used ScaleBio's single-cell methylation kit to investigate DNA methylation patterns during oncogenesis using cancer cells with widespread DNA methylation changes, such as human isocitrate dehydrogenase (IDH) mutant glioma cells at the single-cell level. The IDH gene family, comprising of IDH1, IDH2, and IDH3, encodes enzymes involved in the tricarboxylic acid (TCA) cycle. IDH1/2 mutations are present in over 80% of low-grade gliomas while IDH-mutant gliomas constitute about 1/5th of all adult diffuse gliomas, thus making them one of the most common brain tumor subtypes. As such, IDH mutations have emerged as attractive therapeutic targets for glioma treatment. By uncovering DNA methylation patterns at the single cell level, we provide here an epigenetic map to better understand this complex disease and better inform clinical discoveries. We achieved high cell recovery and robust cytosine coverage throughout our analysis of single cell methylomes isolated from human glioma tumor tissue. Using this data we generated a ranked list of the top hypo- and hypermethylated genomic regions and identified cell type specific clusters seen in different pathological states by looking at Differentially Methylated Regions (DMR) uncovering unique single-cell methylation profiles that may be obscured by bulk or pseudo-bulk analysis. These data show that the ScaleBio single-cell methylation workflow offers increased sensitivity, specificity, and accuracy in identifying DNA methylation sites when compared to other techniques while offering a comprehensive view of methylomes and providing insights into cellular heterogeneity and trajectories. Citation Format: Sanika Khare, Dominic Skinner, Maggie Nakamoto, Hosu Sin, Ashley Woodfin, Eric Pu, Lin Lin, Jason Koth, Beth Walczak. Enhanced single cell DNA methylation analysis using combinatorial indexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB193.

Epigenetics in Cancer Therapy

Epigenetics regulates parts of the genetic information accessible to cellular machinery, thus contributing to cellular diversity. Disruption of epigenetic harmonisation can lead to dysregulation of signaling pathways and contribute to diseases like cancer. Epigenetic processes are crucial in coordinating the normal growth and preservation of gene activity unique to mammal tissues. Epigenetic studies in current cancer research are vital for early detection through biomarker identification, understanding tumor heterogeneity, predicting treatment responses, and uncovering resistance mechanisms. This knowledge informs targeted therapies, enhances personalized medicine, and improves overall outcomes in cancer patients. This review comprehensively explores epigenetic processes in healthy cells, emphasizing histone modification-lysine acetylation and DNA methylation. The intricate mechanisms governing genetic expression and their dysregulation in cancer progression are also reviewed. Examining prevalent cancer types, this review stresses the pivotal role of epigenetics in understanding and potentially targeting these processes for therapeutic interventions.

Intestinal cell diversity and treatment responses in a parasitic nematode at single cell resolution

BackgroundParasitic nematodes, significant pathogens for humans, animals, and plants, depend on diverse organ systems for intra-host survival. Understanding the cellular diversity and molecular variations underlying these functions holds promise for developing novel therapeutics, with specific emphasis on the neuromuscular system’s functional diversity. The nematode intestine, crucial for anthelmintic therapies, exhibits diverse cellular phenotypes, and unraveling this diversity at the single-cell level is essential for advancing knowledge in anthelmintic research across various organ systems.ResultsHere, using novel single-cell transcriptomics datasets, we delineate cellular diversity within the intestine of adult female Ascaris suum, a parasitic nematode species that infects animals and people. Gene transcripts expressed in individual nuclei of untreated intestinal cells resolved three phenotypic clusters, while lower stringency resolved additional subclusters and more potential diversity. Clusters 1 and 3 phenotypes displayed variable congruence with scRNA phenotypes of C. elegans intestinal cells, whereas the A. suum cluster 2 phenotype was markedly unique. Distinct functional pathway enrichment characterized each A. suum intestinal cell cluster. Cluster 2 was distinctly enriched for Clade III-associated genes, suggesting it evolved within clade III nematodes. Clusters also demonstrated differential transcriptional responsiveness to nematode intestinal toxic treatments, with Cluster 2 displaying the least responses to short-term intra-pseudocoelomic nematode intestinal toxin treatments.ConclusionsThis investigation presents advances in knowledge related to biological differences among major cell populations of adult A. suum intestinal cells. For the first time, diverse nematode intestinal cell populations were characterized, and associated biological markers of these cells were identified to support tracking of constituent cells under experimental conditions. These advances will promote better understanding of this and other parasitic nematodes of global importance, and will help to guide future anthelmintic treatments.

Impact of Micro- and Nano-Plastics on Human Intestinal Organoid-Derived Epithelium.

The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids.

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparationand sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

Single-cell profiling transcriptomic reveals cellular heterogeneity and cellular crosstalk in choroidal neovascularization model

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD.

Abstract 3649: A novel spatial multi-omic approach for biological discoveries in colonic diseased tissues using a comprehensive Immuno-Oncology Proteome Atlas and Whole Transcriptome Atlas

Abstract The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. While spatial transcriptomics has traditionally led the way in plex, multiple studies have demonstrated a poor correlation between RNA expression and protein abundance, owing to transcriptional and translational regulation, target turnover, and most critically, post-translational protein modifications. Therefore, a more holistic, ultra-high-plex, and high-throughput proteomic atlas approach becomes critical for the next phase of discovery biology. Here, we present a barrier-breaking, spatial proteomics panel that was designed to accelerate scientific discoveries. A Digital Spatial Profiler platform is uniquely suited to support high-plex proteomics, allowing for the simultaneous analyses of proteins from discrete regions of interest (ROIs) in FFPE tissue sections while preserving spatial context. The assay relies upon abcam antibodies coupled to photocleavable DNA barcodes readout with NGS sequencing, allowing for theoretically unlimited plex. Here we introduce the Human Immuno-Oncology Proteome Atlas (IPA), a 570+ antibody-based proteomic discovery panel, compatible with immunohistochemistry on FFPE tissues with NGS readout. IPA is the highest-plex, most comprehensive, antibody-based multi-omic panel to date focusing on key areas of immuno-oncology, oncology, immunology, epigenetics, metabolism, cell death, and signaling pathway regulation. Here we demonstrate the performance of IPA on various cell lines and tissue. Additionally, we show the power of IPA, using the spatial multi-omic assay along with the GeoMx® Whole Transcriptome Atlas (> 18,000 transcripts), a 40-plex custom antibody panel and microbiome-curated RNA custom spike-in (~220 transcripts) to evaluate 70 different colon disease samples across 5 pathologies including adenocarcinoma, hyperplasia, and chronic inflammation. This is the highest-plex multi-omic (~610-plex proteins and >18,220 genes) study ever implemented for spatial biology. When we compared the diseased tissue to normal tissue, we observed an upregulation of specific pathways associated with tumorigenesis and inflammation. Furthermore, we observed distinct differences in proteomic and transcriptomic landscape between pathologies. The cutting-edge, data-driven, expert-curated IPA panel is at the forefront of spatial proteomics, empowering the researcher for the acceleration of biological discoveries. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Alyssa Rosenbloom, Shilah Bonnett, Mark Conner, Christine Kang, Erin Piazza, Brian Filanoski, Rhonda Meredith, Hye Son Yi, Lori Hamanishi, Eduardo Ignacio, Nadine Nelson, Michael Prater, Vik Devgan, Rudy Van Eijsden, Melanie Moon, Lesley Isgur, Terence Theisen, Margaret Hoang, Gary Geiss, Joseph M. Beechem. A novel spatial multi-omic approach for biological discoveries in colonic diseased tissues using a comprehensive Immuno-Oncology Proteome Atlas and Whole Transcriptome Atlas [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 3649.

Abstract 5633: Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA)

Abstract IMA is a distinct subtype of lung adenocarcinoma (LUAD), marked by a cellular identity shift from a pulmonary to a gastric lineage with the loss of the pulmonary lineage specifier NKX2-1. This distinctive transformation underscores the complexity of lung cancer heterogeneity and highlights the critical role of lineage-specifying transcription factors in dictating tumor behavior. Notably, both human and murine models of IMA overexpress HNF4α, a known lineage specifier for gastric differentiation in the adult mouse mucosa. Also, concomitant deletion of Hnf4a and Nkx2-1 at tumor initiation impairs tumor growth and the extent of HNF4α in regulating IMA tumor growth, cellular identity and drug response remains unknown at large. Here, we utilized genetically engineered mouse models (GEMM) that relies on a sequential recombination strategy to delete Hnf4a in established tumors. We induce tumor formation through intratracheal delivery of Flp-O virus, activating the expression of mutant KRASG12D and Cre in lung epithelial cells. Tamoxifen administration recombines either Nkx2-1 alone (KN) or in combination with Hnf4a (KNH). We have extensively studied the phenotypic role of HNF4α in GEMM and organoids and identified gene sets regulated by HNF4α in IMA at the bulk level. To complement our findings, we profiled both the tumor and stromal compartments via scRNAseq analysis on our GEMM to gain insights into the cellular diversity within the tumor microenvironment (TME) and the role HNF4α plays. Additionally, we performed ChIPSeq in organoids to elucidate the genome occupancy of HNF4α and determine the mechanisms of gene regulation in IMA. We find that deletion of Hnf4a impairs tumor growth in GEMM and this is phenocopied in organoids. We identified numerous differentially expressed genes (DEGs) in KNH compared to KN and validated several DEGs in vivo. GSEA analysis reveals a significant involvement in regulating cellular identity and suggests a potential impact on anti-tumor immune responses. To this, our scRNAseq data revealed an enrichment of N2-TAN (tumor associated neutrophils) associated genes in KNH compared to KN, suggesting a nuanced interplay between HNF4α and the TME. Additionally, HNF4α exhibited dual functionality, acting as an activator and indirectly as a repressor by modulating the activity of other transcription factors like FOXA1. FOXA1 was found to bind de novo target genes following the loss of HNF4α. These findings so far collectively illuminate the multifaceted role of HNF4α in IMA, primarily driving tumorigenesis in a cell autonomous manner and secondarily skewing the TME towards a protumor via N2-TAN following loss of HNF4α in tumors. Citation Format: Headtlove Essel Dadzie, Gabriela Fort, Katherine Gillis, Pengshu Fang, Walter Orellana, Eric L. Snyder. Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA) [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 5633.

Abstract 1596: Disrupting local immunosuppression by combined myCAF/myeloid targeting in pancreatic cancer

Abstract Immunotherapy has revolutionized clinical care for many patients, but some cancers, such as pancreatic ductal adenocarcinoma (PDAC) remain stubbornly resistant. Local immunosuppression (LIS) is one of the striking hallmarks of PDAC due to the combined effects of multiple different immunosuppressive cell types in the tumor microenvironment (TME). There is great need for an increased understanding of the cellular crosstalk within tumors to understand how stromal cells coordinate in their suppression of immune responses. Oncogenic KRAS activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. Prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the contributions of multiple distinct cell types. Here we examine the interactions of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs) and how they coordinate to suppress immune responses in the PDAC TME. We interrogate PDAC stromal cell biology by using targeted therapies to perturb various cell populations both in vivo using genetically engineered mouse models, and ex vivo using PDAC tumor explants. Explants are short-term slice cultures that enable experimental study of intact tumor sections with a full complement of cell types. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases the proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Dafydd Thomas, Lukas Vlahos, Alvaro Curiel-Garcia, Amanda R. Decker-Farrell, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Disrupting local immunosuppression by combined myCAF/myeloid targeting in pancreatic cancer [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 1596.

Abstract 6945: Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma

Abstract The five-year survival rate of glioblastoma multiforme (GBM) is 5.8%, and its standard of care has not changed since 2005. This stagnation in therapeutic development is due, in part, to its characteristic heterogeneity–multiforme–highlighting a critical need to understand the biological processes underlying this GBM hallmark. Despite the well-documented individual relationships between extrachromosomal DNA (ecDNA), GBM, and heterogeneity, the interplay between the three is not well understood. The Furnari lab has generated a library of cancer avatars, or human induced pluripotent stem cells (hiPSCs) engineered to harbor cancer driving mutations. Single cell RNA sequencing (scRNA-seq) was used to characterize the longitudinal evolution of the first avatars engineered to have TP53−/−;PDGFRAΔ8-9 and PTEN−/−;NF1−/− genotypes. The TP53−/−;PDGFRAΔ8-9 avatar acquired ecDNA, while the PTEN−/−; NF1−/− avatar did not. Re-analysis of the scRNA-seq data identified a subpopulation of cells in the TP53−/−;PDGFRAΔ8-9 avatar that had high expression of CDK4 and PDGFRA, two genes that are commonly co-amplified, found on ecDNA, and drive cellular states. This subpopulation of cells also had highly expressed genes near the CDK4 locus indicating a structural variant and possibly ecDNA. This population diverged transcriptionally from other cells and was associated with the OPC-like cellular state, which has been shown to be associated with high levels of PDGFRA and, at times, co-expression with CDK4. While TP53−/−;PDGFRAΔ8-9 tumors contained cells across all four GBM cellular states, PTEN−/−;NF1−/− tumors had a significantly less diverse composition of cellular states suggesting that ecDNA may facilitate cellular state diversity. With the eventual goal to directly test whether ecDNA enables transcriptional state heterogeneity compared to chromosomally inherited amplifications, CRISPR-Cas9 and CRISPR-C were implemented to engineer EGFRvIII ecDNA in U87. While inverse PCR confirmed the successful generation of ecDNA in U87, ecDNA was lost following long term in vitro cell passaging. Current follow up work aims to identify different intrinsic (oncogene amplified, genetic background, cell type, cell line) and extrinsic (in vitro conditions, orthotopic engraftment) contexts that promote the selection and amplification of engineered ecDNA, as it would be would be valuable to have genotypically identical cancer models with different classes of amplifications (i.e., engineered ecDNA vs. lentiviral overexpression, which is chromosomally inherited). Together, these results highlight the potential to use hiPSC-derived models and single cell methods to investigate gliomagenesis and cell identity. Citation Format: Brett Taylor, Brandon Jones, Daisuke Kawauchi, Yohei Miyake, Raghav Vadla, Nathan Jameson, Shunichiro Miki, Tomoyuki Koga, Bing Ren, Frank Furnari. Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma [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 6945.

Abstract 78: Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells

Abstract Measuring functional signatures of immune cells in a comprehensive manner, spanning the inflammatory (Th1/Th17) and immunosuppressive (Th2/Treg) lineages, would provide key insights into several facets of cancer research and therapy. Determining mechanisms underlying success/failure of checkpoint blockade, defining immunosuppressive activity of the tumor-resident cell subsets, and identifying immunological biomarkers that predict clinical outcomes, could all be achievable if detection of cytokines spanning many lineages among individual cells was in reach. To achieve this goal, powerful cytometric research tools are required. Cell phenotyping panels are expanding and often now include measurement of intracellular markers of functional potential, such as cytokines, phosphorylation events, and transcription factors. Many of these readouts, including cytokines of the Th2/Treg lineages (IL-5, IL-10, and IL-13), have been notoriously difficult to detect in human cells using fluorescence-based cytometry. Large panels with multiplex intracellular markers are subject to challenges with autofluorescence and spillover spread, thus limiting fluorochrome capacity and reducing resolving power for rare subsets even with full spectrum flow cytometers. For such applications, mass cytometry may overcome such limitations and enable better signal resolution. To address this, we evaluated three small (13-plex) panels using a full spectrum flow cytometer and the CyTOF XT™ mass cytometer. Each panel was comprised of surface and intracellular analytes (cytokines, phospho-epitopes, or transcription factors) and designed to minimize potential impact of spectral overlap on the resolution of spectral flow data. PBMCs were stimulated, split, and stained with either fluorochrome- (Cytek® Aurora) or metal-conjugated antibodies (CyTOF XT). Data sets were analyzed by PhenoGraph clustering and visualized with opt-SNE to determine cellular functional diversity. Overall, data collected on the CyTOF XT demonstrated superior resolution for many intracellular readouts, including cytokines IL-5, IL-10, and IL-13. Also, when better signal/noise enabled accurate clustering, a higher number of immune cell clusters possessing distinct functional signatures resulted from mass cytometry datasets as compared with fluorescent counterparts. In summary, the CyTOF XT mass cytometry platform offers superior signal resolution compared to spectral flow for a range of intracellular targets. Use of mass cytometry translates to the identification of more diverse functional subsets and thus a clearer understanding of the immune signatures present in a sample in the context of unbiased analysis. Our findings indicate that the CyTOF XT platform could serve as a catalyst for seminal discoveries in immune profiling to drive therapeutic design and advanced disease monitoring in cancer. Citation Format: Erika L. Smith-Mahoney, Michael J. Cohen, Anna C. Belkina, Thirumahal Selvanantham, David King, Christina Loh, Amedeo J. Cappione, Jennifer E. Snyder-Cappione. Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells [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 78.

Abstract 3385: Modeling evolutionary fitness in resistant cancers based on a common adaptability trait

Abstract Cancer is composed of a major subpopulation of proliferative cells and a minor subpopulation of highly adaptable cells. Although it is evident that cancer is an evolution like process driven by rare adaptable cancer cells that persist under various selection pressures, most of our knowledge about cancer comes from studying proliferative cancer cells. To overcome the hurdle in modeling adaptable cancer cell state that defeats all currently offered therapies, we are exploiting a linkage that exists between different adaptability substates such as metabolic, regulatory, and structural substates. Our studies, thus far carried out with aggressive triple-negative breast cancer and melanoma cell lines, suggest that it is feasible to apply severe metabolic challenges as realistic selection pressures/bottlenecks for modeling highly abnormal and highly adaptable cancer cells that drive cancer evolution and therapy resistance. The most significant aspect of our approach is an ability to model rare resistant cells (approximately 0.01% of cells) that survive in reversible quiescence under selection pressures. Monitoring of cells under microscope for several weeks under a severe and prolonged metabolic challenge, for example, a lack of glutamine in culture medium, revealed perhaps most interesting and cancer-relevant cells that survive in quiescence for weeks and then advance to generate therapy-resistant cells. Such microscopic monitoring revealed a significant heterogeneity among surviving cells. As examples, 1) surviving cells differ in the depth of quiescence, some cells trying to proliferate sooner than others; 2) as cells progress from quiescence to proliferation, their fate is far from certain: cells may progress to yield few cells, tiny colony, or large colony and then stop proliferating and in most instances die (abortive attempts at evolution). Of significance, the cell lines derived inflammatory breast cancer (IBC), an aggressive subtype of breast cancer, possessed a higher evolutionary fitness than non-IBC cell lines. This approach also eliminates 99.99% of cells that proliferate in cell culture but would not survive selection pressures in the body. The adaptable cells that survived the bottleneck and then proliferated indefinitely were highly resistant and metastatic. Molecular analyses of adaptable cells, including gene expression microarrays, CGH microarrays, and whole genome sequencing revealed mechanisms for their plasticity (exemplified by markers of high EMT), and for generating cellular diversity by altering epigenome (exemplified by low TET2) and by structural modifications in transcriptome (exemplified by high FTO). We conclude that the phenotype-based approach described here is good at modeling deep intrinsic resistance and may be useful in developing strategies for improving outcomes in resistant cancers. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Balraj Singh, Vanessa N. Sarli, Anthony Lucci. Modeling evolutionary fitness in resistant cancers based on a common adaptability trait [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 3385.

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and generation of cellular diversity. In the Caenorhabditis elegans (C. elegans) zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains, and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, while in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

Transcriptomic analysis of rat brain response to alternating current electrical stimulation: unveiling insights via single-nucleus RNA sequencing.

Electrical brain stimulation (EBS) has gained popularity for laboratory and clinical applications. However, comprehensive characterization of cellular diversity and gene expression changes induced by EBS remains limited, particularly with respect to specific brain regions and stimulation sites. Here, we presented the initial single-nucleus RNA sequencing profiles of rat cortex, hippocampus, and thalamus subjected to intracranial alternating current stimulation (iACS) at 40Hz. The results demonstrated an increased number of neurons in all three regions in response to iACS. Interestingly, less than 0.1% of host gene expression in neurons was significantly altered by iACS. In addition, we identified Rgs9, a known negative regulator of dopaminergic signaling, as a unique downregulated gene in neurons. Unilateral iACS produced a more focused local effect in attenuating the proportion of Rgs9+ neurons in the ipsilateral compared to bilateral iACS treatment. The results suggested that unilateral iACS at 40Hz was an efficient approach to increase the number of neurons and downregulate Rgs9 gene expression without affecting other cell types or genes in the brain. Our study presented the direct evidence that EBS could boost cerebral neurogenesis and enhance neuronal sensitization to dopaminergic drugs and agonists, through its downregulatory effect on Rgs9 in neurons.

Integration Analysis of Single-Cell Multi-Omics Reveals Prostate Cancer Heterogeneity.

Prostate cancer (PCa) is an extensive heterogeneous disease with a complex cellular ecosystem in the tumor microenvironment (TME). However, the manner in which heterogeneity is shaped by tumors and stromal cells, or vice versa, remains poorly understood. In this study, single-cell RNA sequencing, spatial transcriptomics, and bulk ATAC-sequence are integrated from a series of patients with PCa and healthy controls. A stemness subset of club cells marked with SOX9high ARlow expression is identified, which is markedly enriched after neoadjuvant androgen-deprivation therapy (ADT). Furthermore, a subset of CD8+ CXCR6+ T cells that function as effector T cells is markedly reduced in patients with malignant PCa. For spatial transcriptome analysis, machine learning and computational intelligence are comprehensively utilized to identify the cellular diversity of prostate cancer cells and cell-cell communication in situ. Macrophage and neutrophil state transitions along the trajectory of cancer progression are also examined. Finally, the immunosuppressive microenvironment in advanced PCa is found to be associated with the infiltration of regulatory T cells (Tregs), potentially induced by an FAP+ fibroblast subset. In summary, the cellular heterogeneity is delineated in the stage-specific PCa microenvironment at single-cell resolution, uncovering their reciprocal crosstalk with disease progression, which can be helpful in promoting PCa diagnosis and therapy.

Bulk RNA Sequencing of Human Pediatric Lung Cell Populations Reveals Unique Transcriptomic Signature Associated with Post-natal Pulmonary Development.

Post-natal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human post-natal lung development are yet to be fully understood. In this study we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and non-endothelial mesenchymal cells, along with lung leukocytes) from one-day-old to eight-year-old organ donors with no known lung disease. For analysis, we considered the donors in 4 age groups [less than 30 days old neonates, 30 days to < 1 year old infants, toddlers (1 to < 2 years) and children 2 years and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared to number of DEG found during infant to toddler- or toddler to older children- transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.

Applications of multiphoton microscopy in imaging cerebral and retinal organoids.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

The Drosophila neuroblast polarity cycle at a glance.

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure.