Distinct Transcriptional States Research Articles

Characteristics of putative keystones in the healthy adult human gut microbiota as determined by correlation network analysis.

Keystone species are thought to play a critical role in determining the structure and function of microbial communities. As they are important candidates for microbiome-targeted interventions, the identification and characterization of keystones is a pressing research goal. Both empirical as well as computational approaches to identify keystones have been proposed, and in particular correlation network analysis is frequently utilized to interrogate sequencing-based microbiome data. Here, we apply an established method for identifying putative keystone taxa in correlation networks. We develop a robust workflow for network construction and systematically evaluate the effects of taxonomic resolution on network properties and the identification of keystone taxa. We are able to identify correlation network keystone species and genera, but could not detect taxa with high keystone potential at lower taxonomic resolution. Based on the correlation patterns observed, we hypothesize that the identified putative keystone taxa have a stabilizing effect that is exerted on correlated taxa. Correlation network analysis further revealed subcommunities present in the dataset that are remarkably similar to previously described patterns. The interrogation of available metatranscriptomes also revealed distinct transcriptional states present in all putative keystone taxa. These results suggest that keystone taxa may have stabilizing properties in a subset of community members rather than global effects. The work presented here contributes to the understanding of correlation network keystone taxa and sheds light on their potential ecological significance.

EPCO-18. DECIPHERING SPATIAL DIVERSITY OF GLIOBLASTOMA GENETIC ARCHITECTURE

Abstract Glioblastoma (GBM) is hallmarked by profound inter- and intratumoral heterogeneity. Current research has mapped key genetic drivers to distinct transcriptional profiles within GBM. Nevertheless, the intricate interactions between genetic alterations and spatial-transcriptional heterogeneity remain elusive. We utilized nestin-Tva C57BL/6 mice to induce de novo GBM via oncogene overexpression alongside shRNA-mediated silencing or deletion of tumor suppressors. We modeled critical driver mutations: PDGFB, NF1, and EGFR. Employing 10X Genomics platform for single-cell RNA sequencing, combined with spatially resolved transcriptomics and proteomics (Nanostring GeoMx), we integrated data using both horizontal (mutual nearest neighbors) and vertical (weighted nearest neighbors) approaches, analyzed through the SPATA2 software. Advanced computational techniques, including graph neural networks and single-cell deconvolution, were leveraged to unravel the genotypic and spatial variability within tumors and their microenvironments. Our findings uncover persistent cellular heterogeneity across diverse genetic contexts. However, specific genetic mutations precipitated distinct dominant transcriptional states: NF1 and EGFR mutations drove mesenchymal and astrocytic-like states, respectively, whereas PDGFB fostered a neural progenitor/cell-like state. These transcriptional shifts were closely linked to unique microenvironmental modifications, such as elevated neuronal signaling in PDGFB-driven GBMs and significant immune infiltration in NF1 and EGFR models. Through weighted co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) paired with spatial-niche deconvolution, we identified a mesenchymal transition leading to an immunosuppressive microenvironment in NF1-loss driven GBMs and increased lymphocyte presence around blood vessels in EGFR-amplified GBMs. These patterns were consistent with human spatially resolved transcriptomic data, emphasizing the genetic complexity and heterogeneity of human GBMs. Our genetically engineered mouse models serve as a robust platform for dissecting GBM with defined genetic mutations. Leveraging sophisticated analytical techniques and computational modeling, this study underscores the profound interplay between genetic drivers and the tumor microenvironment, paving the way for targeted therapeutic strategies and precision medicine in GBM.

Polyclonality overcomes fitness barriers in Apc-driven tumorigenesis.

Loss-of-function mutations in the tumour suppressor APC are an initial step in intestinal tumorigenesis1,2. APC-mutant intestinal stem cells outcompete their wild-type neighbours through the secretion of Wnt antagonists, which accelerates the fixation and subsequent rapid clonal expansion of mutants3-5. Reports of polyclonal intestinal tumours in human patients and mouse models appear at odds with this process6,7. Here we combine multicolour lineage tracing with chemical mutagenesis in mice to show that a large proportion of intestinal tumours have a multiancestral origin. Polyclonal tumours retain a structure comprising subclones with distinct Apc mutations and transcriptional states, driven predominantly by differences in KRAS and MYC signalling. These pathway-level changes are accompanied by profound differences in cancer stem cell phenotypes. Of note, these findings are confirmed by introducing an oncogenic Kras mutation that results in predominantly monoclonal tumour formation. Further, polyclonal tumours have accelerated growth dynamics, suggesting a link between polyclonality and tumour progression. Together, these findings demonstrate the role of interclonal interactions in promoting tumorigenesis through non-cell autonomous pathways that are dependent on the differential activation of oncogenic pathways between clones.

Abstract B076: Morphological diversity predicts functional traits in pancreatic cancer

Abstract Cell morphologies represent a phenotypic integration of intrinsic cellular processes, changes in cell state, and interactions with neighboring cells. While analyses of large cell line collections such as the Cancer Dependency Map (DepMap) have greatly enhanced discovery of novel molecular biomarkers and therapeutic targets, morphological analysis of these models has not yet been performed. Hence, there is a rich opportunity to discover novel regulators and functional properties of cell morphologies by linking image-based profiling to -omics based assays. Here, we studied the transcriptional and functional properties of 16 human pancreatic cancer cell lines, associations with distinct cell morphologies, and treatment-induced remodeling. At baseline, we observed extensive morphological heterogeneity across and within cell lines, as well as differences in multicellular organization. We categorized cell line morphologies into epithelial, mesenchymal, and spheroid, and organizational patterns into tightly aggregated, multilayered, and dispersed. We performed differential expression analysis across morphological subtypes utilizing DepMap transcriptomics data and queried functional correlates including CRISPR dependency, drug sensitivity, and proclivity for metastasis. For example, we discovered that the mesenchymal morphology and multilayered organizational pattern [WH3] were associated with metastatic potential, while expressing reduced levels of tight junction and cell adhesion genes. We then explored treatment associated morphological changes and their potential as cost-effective biomarkers for cell-intrinsic resistance to chemotherapy (e.g., 5-FU and gemcitabine) and KRAS inhibitors (e.g., MRTX1133 and RMC- 6236). Cells were treated continuously and monitored with live cell imaging (Incucyte SX5) followed by Cell Painting at the three-day endpoint. We observed conserved morphological responses to therapy across cell lines including a shift towards spindle-like cell shapes with neurite-like projections in chemotherapy-treated conditions, and a cell bloating phenomena characterized by an increase in cell area in KRAS inhibitor treated conditions. To directly link transcriptional state to morphology, we performed 1000-plex spatial molecular imaging (Nanostring CosMx) at subcellular resolution to pools of cell lines, demonstrating that morphological diversity within cell lines corresponded to distinct transcriptional states. In conclusion, this study highlights the potential of harnessing cell morphological information in a rapid, cost-effective phenotyping assay to aid precision oncology efforts leveraging patient- derived in vitro models. Citation Format: Dennis Gong, William L Hwang. Morphological diversity predicts functional traits in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B076.

Multi-omic lineage tracing predicts the transcriptional, epigenetic and genetic determinants of cancer evolution

Cancer is a highly heterogeneous disease, where phenotypically distinct subpopulations coexist and can be primed to different fates. Both genetic and epigenetic factors may drive cancer evolution, however little is known about whether and how such a process is pre-encoded in cancer clones. Using single-cell multi-omic lineage tracing and phenotypic assays, we investigate the predictive features of either tumour initiation or drug tolerance within the same cancer population. Clones primed to tumour initiation in vivo display two distinct transcriptional states at baseline. Remarkably, these states share a distinctive DNA accessibility profile, highlighting an epigenetic basis for tumour initiation. The drug tolerant niche is also largely pre-encoded, but only partially overlaps the tumour-initiating one and evolves following two genetically and transcriptionally distinct trajectories. Our study highlights coexisting genetic, epigenetic and transcriptional determinants of cancer evolution, unravelling the molecular complexity of pre-encoded tumour phenotypes.

Histone H3.3 lysine 9 and 27 control repressive chromatin at cryptic enhancers and bivalent promoters

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

1468: Hypoxia drives spatially distinct epigenetic and transcriptional cell states in Glioblastoma

1468: Hypoxia drives spatially distinct epigenetic and transcriptional cell states in Glioblastoma

Decoding spatiotemporal transcriptional dynamics and epithelial fibroblast crosstalk during gastroesophageal junction development through single cell analysis

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract 6925: Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC)

Abstract SCLC is the deadliest type of lung cancer, which is mostly diagnosed at late stages and accounts for 15% of pulmonary tumors. Even though chemotherapy is initially effective, patients with SCLC relapse quickly and develop resistant tumors. Genome studies revealed a universal loss of TP53 and RB1; recent transcriptional studies categorized SCLC based on the expression of 4 lineage transcription factors (TFs). We aimed to dissect how genomic cues impact transcriptional phenotypes, tumor cell plasticity and the dynamics of tumor and immune cell interactions, to thus decipher molecular mechanisms of phenotypic divergence and therapy resistance in SCLC. We performed genome sequencing and single cell transcriptome profiling of 55 tumors from patients with SCLC, including primary tumors, local and distant metastases and of paired relapsing tumors acquired after chemotherapy. Single cell sequencing was performed with the 10x Genomics platform and yielded > 200.000 cells. Transcriptome profiles at single cell levels revealed co-expression of at least two of the lineage TFs ASCL1, NEUROD1 or POU2F3 in each tumor. Copy number (cn) estimation for transcriptome data pointed to genomic events impacting transcriptional heterogeneity within the tumor. Furthermore, our data pointed to a marked intra- and interpatient, as well as to inter-metastatic heterogeneity. To specifically investigate how underlying genetic alterations affect molecular phenotypes, integrative studies across all patients were performed. This revealed shared transcriptional programs in tumors with amplifications of MYC family members; trajectory inference for cases with low and high-level copy gains of MYCN pointed to distinct transcriptional states. We performed multi-regional and longitudinal studies of matched patient cases to reconstruct the genomic patterns of clonal evolution, and projected genomic subsets to transcriptional profiles at single cell level, thus analyzing the effect of genome diversity on the transcriptional landscape. Additionally, tumor intrinsic profiles of heterogeneity were mapped to single cell data of patient tumors to elucidate the effects on tumor-immune cell interactions. All together our data points to a concerted regulation of a multitude of lineage factors and transcriptional programs in each SCLC tumor and provides a first comprehensive framework for the study of underlying genetic alterations that shape the transcriptional landscape and phenotypic plasticity in SCLC. Citation Format: Marcel Schmiel, Laura Kaiser, Maria Cartolano, Martin Peifer, Roman K. Thomas, Julie George. Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC) [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 6925.

Abstract 5486: Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma

Abstract Background:Previous studies have shown that intratumoral immune aggregates called tertiary lymphoid structures (TLS) are associated with exceptional response to immune checkpoint blockade (ICB) in several cancer types. However, the specific mechanisms through which TLS contribute to ICB-response remain poorly characterized in part due to technical limitations with isolating TLS. Here, we use the NanoString GeoMx digital spatial profiler to interrogate TLS with deep spatial and transcriptional resolution and identify cellular and molecular features of response-associated TLS in melanoma. Methods:Twelve neoadjuvant ICB-treated high-risk resectable melanoma patients [NCT02519322] with formalin-fixed paraffin embedded (FFPE) surgical resections were included in this study. Major pathologic response (MPR) defined as <10% viable tumor at time of surgery was used to evaluate response (R – responder; NR – non-responder). TLS were sampled (via H&E) using 1mm core punches and assembled into a tissue microarray (TMA). The TLS TMA was probed using the human whole transcriptome atlas (huWTA) and the human T-cell receptor (huTCR) atlas. Melanoma (S100B) and immune markers (CD45 and CD20) were used to guide region-of-interest (ROI) selection of classical TLS. Differential gene expression was calculated using a linear mixed model and corrected with Benjamini-Hochberg. Immune deconvolution was performed using CIBERSORTx with FDR cutoff of < 0.25. Results:Fifty percent (6/12) of patients included in this study demonstrated MPR following treatment with neoadjuvant ICB. Principal component analysis of huWTA probes revealed distinct transcriptional states of R TLS vs NR TLS, with PC1 strongly correlating with response status (r=0.88, p<0.0001). Differential immune analysis showed that R TLS harbored more CD8+ T-cells (p<0.001), NK cells (p=0.004), γδ T-cells (p=0.017) and memory B-cells (p=0.026) and preferentially upregulated inflammatory genes such as pattern recognition receptors (TLR8 and KLRC2) and immune-stimulatory markers (CD27 and CD103). Immune-repertoire analysis revealed a trend towards increased TCR diversity in R TLS (p = 0.3) and distinct TCR signatures in R TLS underscored by elevated expression of TRDJ4 (p<0.001), TRGJ1/2(p<0.001), TRGV4(p<0.001) and TRAV1-2(p=0.018) genes amongst others. Conclusions:These findings demonstrate that, although melanomas from both R and NR patients contain TLS, the immune-phenotype of R TLS is more cytotoxic and characterized by a more diverse TLS-resident T-cell population. These findings present new insights into the therapeutic underpinnings of TLS in cancer and will inform future investigations evaluating their formation/function in solid tumors. Citation Format: Manoj Chelvanambi, Brenda Melendez, Elise F. Nassif, Rossana N. Lazcano, Matthew J. Lastrapes, Bharat S. Bhushan, Sarah B. Johnson, Khalida Wani, Davis R. Ingram, Y David Seo, Beth A. Helmink, Michael G. White, Russell G. Witt, Laurence P. Diggs, Golnaz Morad, Monika Zelazowska, Somnath Paul, Florentia Dimitriou, Ashish V. Damania, Matthew C. Wong, Neeta Somaiah, Nadim J. Ajami, Emily Z. Keung, Wolf H. Fridman, James P. Allison, Padmanee Sharma, Kevin M. McBride, Tina Cascone, Christina L. Roland, Alexander J. Lazar, Jennifer A. Wargo. Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in 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 5486.

Abstract A010: Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations

Abstract Over half of BRAF-mutant melanoma patients with initial response to targeted therapy will recur with resistant disease. It is thought that recurrence arises from the ability of resistant clonal populations to enter and exit a slow-cycling persister state, evading treatment in quiescence before returning to a proliferative state. However, clonal transcriptional profiles and their spatial relationships in the tumor environment are not well characterized. Through longitudinal profiling of tumors from pre-treatment through the growth of resistant tumors, we track clonal lineages and transcriptional state changes to identify recurrent evolutionary patterns. BRAF mutant patient-derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) and H&E histological staining were performed at five timepoints across 94 and 133 days of PDX models WM4237 and WM4007 treatment, tracking clonal populations with intact tissue structure. Deep learning feature extraction on high-resolution H&E-stained images allowed the detection of histological features that co-localize with expression levels. A novel computational workflow performed integration of samples across time points, pathway enrichment analysis, and pseudotemporal ordering, allowing spatial recreation of copy number variation based clonal phylogenies and cell fate trajectory inference during the treatment course. We characterize the clonal populations by the burden of accumulated copy number variations and establish clonal phylogeny. With the added temporal resolution, ST profiling during melanoma treatment enables observation of a global shift where all lineages begin the transition into the persister transcriptional state. The drug-sensitive cells are eliminated due to treatment, and distinct transcriptional states arise due to metabolic plasticity regardless of the lineage of origin. The clones of the transient persister state constitute the minimal residual disease, where melanoma cells have an increase in invasive capacity and a slowing of the cell cycle. The ability to survive through the persister state and re-emerge into a proliferating clone where the cell cycle is upregulated varies by lineage. We observe that the transcriptional changes during emergence from dormancy show decreased invasiveness and continued reliance on oxidative respiration versus glycolysis. We compare the re-emerged resistant cell populations and pre-treatment samples to pinpoint the changes in the transcriptional state of the MAPK signaling pathway and find that sets of upregulated genes differ by the model, WM4237 and WM4007. We connect deep learning imaging features extracted from longitudinal histology profiling to ST profiles to associate histology data with the cell cycle activation, invasiveness, hypoxia state, and metabolic mode across the timepoints. We observe that the imaging features alone can be potentially used to track the evolution of the tumor cell population phenotypes during drug treatment. Citation Format: Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang, Jill C. Rubinstein. Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A010.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions

BackgroundDespite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets.MethodsWe collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis.ResultsThe bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption.ConclusionsThese results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Overview of Gene Expression Dynamics during Human Oogenesis/Folliculogenesis.

The oocyte transcriptome follows a tightly controlled dynamic that leads the oocyte to grow and mature. This succession of distinct transcriptional states determines embryonic development prior to embryonic genome activation. However, these oocyte maternal mRNA regulatory events have yet to be decoded in humans. We reanalyzed human single-oocyte RNA-seq datasets previously published in the literature to decrypt the transcriptomic reshuffles ensuring that the oocyte is fully competent. We applied trajectory analysis (pseudotime) and a meta-analysis and uncovered the fundamental transcriptomic requirements of the oocyte at any moment of oogenesis until reaching the metaphase II stage (MII). We identified a bunch of genes showing significant variation in expression from primordial-to-antral follicle oocyte development and characterized their temporal regulation and their biological relevance. We also revealed the selective regulation of specific transcripts during the germinal vesicle-to-MII transition. Transcripts associated with energy production and mitochondrial functions were extensively downregulated, while those associated with cytoplasmic translation, histone modification, meiotic processes, and RNA processes were conserved. From the genes identified in this study, some appeared as sensitive to environmental factors such as maternal age, polycystic ovary syndrome, cryoconservation, and in vitro maturation. In the future, the atlas of transcriptomic changes described in this study will enable more precise identification of the transcripts responsible for follicular growth and oocyte maturation failures.

Single-Cell RNA-Seq Analysis Reveals Distinct Tumor and Immunosuppressive T Cell Phenotypes in CLL Patients Treated with Ibrutinib

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKIs) and their introduction into clinical practice represents a major advance in the treatment of chronic lymphocytic leukemia (CLL). Monotherapy with ibrutinib or other BTKIs generally do not induce complete remissions or undetectable minimal residual disease (uMRD) even with extended therapy. The reason why some patients relapse, and some have minimal residual disease is unknown. Therefore, there is a need to understand the differences between ibrutinib sensitive and resistant CLL cells along with the immune microenvironment to identify novel therapeutic targets. Methods: Here, we investigate the cellular heterogeneity of peripheral blood mononuclear cells from patients with CLL treated with ibrutinib using single-cell RNA sequencing. To understand ibrutinib resistance mechanisms, samples were collected from 8 patients at baseline (before treatment) (1), where BTK C481S is present with low variant allele frequency (VAF) (2) and at time of relapse (3). Data from these were compared to ibrutinib sensitive patient samples at baseline (1) (before treatment), 3yr (2) and 5yr (3) on treatment timepoints. Single cells were isolated using the 10X Genomics 5' immune profiling kit. Data were processed with CellRanger v3.1.0. Poor quality cells were filtered out based on a high proportion of mitochondrial reads and low overall feature count. Sample variation was reduced using Batchelor to aid clustering. Clustering was performed using the partitioning and Leiden methods. Cluster top markers were calculated using functions from monocle3. Identity mapping was performed using Seurat and a published PBMC reference dataset. Results: Single-cell RNA sequencing revealed transcriptional heterogeneity within the B cell cluster (Fig 1A). Three subpopulations of B cells were defined based on differential representation in the ibrutinib-sensitive and resistant patients. Ibrutinib sensitive cells showed enrichment of B cell populations with upregulation of MHC I molecules and TNF family members. We also identified that inflammatory response and metabolic-related pathways were decreased, whereas cellular response to stress and DNA repair programs were increased in the ibrutinib resistance samples. Gene module analysis showed that ibrutinib sensitive samples had an increase in T cell activation, RNA splicing, cell adhesion and migration programs during therapy. We hypothesized that the emergence of BTK C481S mutation in ibrutinib-resistant cells would lead to a distinct transcriptional profile. Long-read sequencing was performed using Oxford Nanopore technology. BTK-mutant cells were identified based on the presence of two or more unique RNA molecules detected with the C481S mutation and these were predominantly identified in the low-VAF and relapse samples. However, there was no significant difference between BTK-mutant cells and the remaining B cells. To see if T cell phenotypes reflected the differences observed in B cell transcriptional state, we characterized and quantified T cell populations according to ibrutinib response and investigated the changes in the T cell population. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in both resistant and sensitive CLL patients. However, there was a significant (p<0.05) enrichment of Tregs in the ibrutinib-resistant samples. We found a connection between the immunosuppressive phenotype in the microenvironment demonstrating tumor's intrinsic immunological reactive program with exhausted T cells. Also, we found significant difference in the expression of checkpoint molecules and suppressive genes in T cells (Fig 1B) and NK cells. Conclusion: Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma

AbstractTumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3− Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

Distinct stage-specific transcriptional states of B cells derived from human tonsillar tissue.

B cells within secondary lymphoid tissues encompass a diversity of activation states and multiple maturation processes that reflect antigen recognition and transition through the germinal center (GC) reaction, in which mature B cells differentiate into memory and antibody-secreting cells (ASCs). Here, utilizing single-cell RNA-seq, we identify a range of distinct activation and maturation states of tonsil-derived B cells. In particular, we identify what we believe is a previously uncharacterized CCL4/CCL3 chemokine-expressing B cell population with an expression pattern consistent with B cell receptor/CD40 activation. Furthermore, we present a computational method that leverages regulatory network inference and pseudotemporal modeling to identify upstream transcription factor modulation along a GC-to-ASC axis of transcriptional maturation. Our data set provides valuable insight into diverse B cell functional profiles and will be a useful resource for further studies into the B cell immune compartment.

Abstract 631: Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions

Abstract Background: Renal cell carcinoma (RCC) is the most common renal tumor in adults, and among these clear cell RCC (ccRCC) is most common subtype and accounts for most renal cancer-related deaths. One-third of patients with advanced ccRCC present with bone metastases at the time of diagnosis. Despite therapeutic advances, once cancer has metastasized to the bone, it represents a highly morbid and ultimately lethal disease. Comprehensive genomic studies in ccRCC have provided important insights into the somatic alterations and intra-tumor heterogeneity. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined. Methods: We collected fresh patient samples directly from the operating room and performed single-cell transcriptomic profiling of human ccRCC tumors (ccRCC primary), solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow stroma cells from patients undergoing hip replacement surgery (Benign) as control. We provide a comparative single cell transcriptomic analysis between ccRCC primary tumors and bone metastatic lesions. Results: Comparison of malignant cells from primary and metastatic sites reveal a distinct transcriptional signature that is predictive of metastatic potential and patient survival. The bone marrow in metastatic patients has sustained immune-suppressive microenvironment, featuring increased, exhausted CD8+ cytotoxic T-cells, T-regulatory cells and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival, markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. Conclusions: These results provide insights into mechanisms underlying of ccRCC tumors progression leading bone metastasis. We also highlight potential communication channels and therefore therapeutic targets within both the immune and the tumor cell populations. Citation Format: Shenglin Mei, Adele M. Alchahin, Ioanna Tsea, Youmna Kfoury, Taghreed Taghreed, Hirak Sarkar, Shulin Wu, Alexander O. Subtelny, Yida Zhang, Keyan Salari, Chin-Lee Wu, Mark A. Randolph, David T. Scadden, Douglas Dahl, John Shin, Peter Kharchenko, Philip Saylor, David Sykes, Ninib Baryawno. Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 631.

Abstract 2229: Cell state transitions shape the intratumoral composition of small cell lung carcinoma

Abstract Introduction: Small cell lung carcinoma (SCLC) is characterized by rapid growth, early metastases, and initial response followed by almost invariable resistance to therapy. Studies to date have not determined the extent that diverse transcriptional programs drive SCLC and contribute to its lethality. We sought to characterize the intra-tumoral transcriptional heterogeneity of SCLC. We identify multivalent, distinct, and commutable transcriptional states that confer discrete functions in individual SCLC tumors. Methods: We developed a biorepository of patient-derived xenografts (PDX) (n = 64) and matched PDX-derived ex vivo lines. We used multi-omic profiling (RNAseq, scRNAseq, and ATAC seq), single-cell fluorescence tracking of fate-defining transcription factor (TF)-driven states, and mathematical and statistical models (Markov chain) to study the topology of the SCLC transcriptional landscape and its plasticity. Human tumor material and associated clinical data were obtained after informed written consent on an IRB-approved prospective registry. Results: We show that individual SCLC tumors are more heterogenous than previously appreciated, displaying distinctive equilibria in the proportion of cells within well-delimited cellular states (ASCL1, NEUROD1 and YAP1). We also show that transcriptional states undergo transitions, which we identified as a mechanism for maintaining cell state diversity. We measured the kinetics of state transitions using single-cell fluorescence tracking of ex vivo cultures and found that these measure were associated significantly with transition estimates using stochastic transition theory (i.e. Markov chains). ATAC-seq profiling indicated a role for the epigenome in the state diversity of SCLC. Namely, there was preferential promoter accessibility to Ascl1, NeuroD1, and Yap1 in a manner consistent with gene and protein expression in the respective subpopulations. Our results indicate that the transition rates between cell types in individual tumors were largely governed by tendencies to reach an equilibrium state that are critical for configuring intratumoral cell state proportions. Conclusion: In conclusion, we demonstrate that TF driven cell states can transition to maintain an equilibrium in cell state proportions. Our work advances a model of cellular states and program diversity in SCLC and nominates new therapeutic strategies designed to limit the plasticity of this lethal cancer. Citation Format: Priyanka Gopal, Aaron Petty, Kevin Rogacki, Titas Bera, Rohan Bareja, Craig Peacock, Mohamed Abazeed. Cell state transitions shape the intratumoral composition of small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2229.

Abstract 1256: Transglutaminase-2 is elevated in the invasive cell state and modulates the tumor immune microenvironment in cutaneous melanoma

Abstract Cutaneous melanoma is the most fatal skin cancer; resistance to targeted therapies contributes to poor prognosis. In some cases, resistance arises from pre-existing tumor heterogeneity, which allows subpopulations of drug-tolerant cells with distinct transcriptional states to survive in the presence of drug. The loss of SOX10, a lineage-specific transcription factor, is known to drive melanoma phenotype switching from a proliferative to an invasive drug-tolerant state. Here, we found that knocking out SOX10 in human and mouse melanoma cell lines leads to the upregulation of transglutaminase-2 (TGM2), a calcium-dependent cross-linking enzyme. Analysis of publicly available bulk and single-cell RNA-sequencing data showed that TGM2 is highly expressed in the invasive cell state in melanoma cell lines and patient-derived melanoma cultures, respectively. Furthermore, knockdown of SOX10 in patient-derived melanoma cultures increases TGM2 mRNA expression. 3D spheroid assays and extracellular matrix production/remodeling assays showed that knockdown of TGM2 has no effect on the invasiveness of SOX10-deficient cells. However, we found that TGM2 is secreted by SOX10-deficient cells and hypothesized that it could modulate the tumor immune microenvironment (TIME) given that TGM2 has been associated with increased immune infiltration in melanoma patient samples. To investigate the effect of TGM2 on the TIME, we stably overexpressed TGM2/Tgm2 in syngeneic mouse melanoma cell lines and allowed tumors to grow in immune-competent C57BL/6 mice. As compared to empty vector, TGM2/Tgm2-overexpression led to an increase in the percentage and number of CD4+ T cells and B cells, and a decrease in the number of myeloid cells, in the tumor immune compartment by flow cytometry analysis. Future studies will assess which subsets of CD4+ T cells and B cells infiltrate TGM2/Tgm2-overexpressing tumors, how these changes to the TIME affect melanoma invasiveness, and if TGM2 expression affects response to targeted therapy in vivo. Overall, our data suggest that TGM2 is highly expressed in the invasive melanoma cell state, is regulated by the SOX10 transcription factor, and can modulate adaptive immune cells in the TIME. Citation Format: Signe Caksa, Nicole A. Wilski, Erica L. Kitterman, McKenna Q. Glasheen, Jacob S. Heilizer, Timothy J. Purwin, Claudia Capparelli, Andrew E. Aplin. Transglutaminase-2 is elevated in the invasive cell state and modulates the tumor immune microenvironment in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1256.