Biology subjects

Cannon, M. V.

Publications and source records attributed to Cannon, M. V..

10 recordsLinked to original sources

bioRxiv2026

SCANBIT facilitates identification of tumor cell populations in scRNAseq data using pseudobulked SNV calls

MotivationSingle cell RNAseq (scRNAseq) is an ideal tool to characterize the heterogeneity within the tumor microenvironment, however, accurate identification of tumor cells can be a challenge. Reference-based methods can be inaccurate, if reference datasets are even available. Current purpose-built methods can be inaccurate, particularly with highly heterogeneous tumor types. Improved methods are needed. We explored the use of genetic variants to distinguish tumor from normal cells within scRNAseq data. ResultsWe characterized the limitations inherent to calling variants from scRNAseq data, quantifying how data sparsity precludes genetic distance calculation between single cells. As a novel workaround, we pooled data from transcriptionally similar cell clusters to call high quality variants and then calculated pairwise differences between cell populations and performed hierarchical clustering. We quantified confidence in genetic divergence between tumor and normal cell populations using bootstrapping. We performed extensive validation to assess accurate identification of tumor cells using ground-truth datasets. Application of our method to human scRNAseq samples highlighted the utility of our approach and revealed how mutational burden influences successful tumor cell identification. Improved cell type assignment in scRNAseq data will facilitate analysis of tumor samples and, in turn, accelerate our understanding of the mechanisms underlying tumor progression and reveal potential biological vulnerabilities that can be exploited to develop improved treatment options. Availability and implementationOur method is publicly available as an R package: SCANBIT (Single Cell Altered Nucleotide Based Inference of Tumor) https://github.com/kidcancerlab/scanBit.

bioinformatics↗

bioRxiv2026

Integrative Single-cell and Spatial Transcriptomic Analysis of Osteosarcoma Reveals Conserved and Distinct Ecosystems Across Sites and Species

Osteosarcoma is a heterogeneous malignancy, exhibiting significant variability among patients, individual cancer cells within a tumor, and the stromal cells that compose primary and metastatic lesions. To facilitate the study of this complex disease, we compiled a unique cross-species single-cell transcriptomic dataset totaling over a million cells/nuclei from human specimens, canine specimens, patient-derived xenografts/PDX, and syngeneic mouse models at both primary (bone) and metastatic (lung) sites. Using a rigorous process for multi-species alignment and annotation, we identified six conserved tumor cell transcriptional states organized along hierarchical differentiation trajectories from progenitor to differentiated phenotypes. Parallel analysis of tumor-associated cells identified conserved macrophage, fibroblast, and endothelial populations that exhibit species- and site-specific reprogramming. Validation by mapping cell types using spatial transcriptomics revealed structured neighborhood architectures that were reproduced across multiple samples. Cell-cell interaction analysis revealed similarities and differences in tumor-host networks across primary and metastatic sites and across species. This analysis enabled pathway-specific assessment of tumor-host communication fidelity across osteosarcoma model systems relative to humans, revealing canine osteosarcoma as a more faithful model. Metastatic lung lesions, counterintuitively, exhibited more intense and complex extracellular matrix (ECM) signaling than primary bone tumors. A key example was tumor-derived fibronectin (FN1), which engages integrin and syndecan receptors on lung epithelial cells, driving a pathological mesenchymal and profibrotic state that promotes fibrotic niche formation and metastatic lung colonization. Together, this cross-species resource delineates both conserved and divergent tumor microenvironment programs, demonstrates how model-aware analyses uncover previously unrecognized tumor-host interactions, and underscores the need for therapies that co-target tumor heterogeneity and its supportive metastatic niche. Statement of SignificanceWe show that the tumor microenvironment in human osteosarcoma patients has biological phenotypes that are conserved across both patients and species. This points towards underlying molecular mechanisms that could be therapeutically targeted.

cancer biology↗

bioRxiv2024

Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1s in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1s recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

cancer biology↗

bioRxiv2024

Metastasis-initiating osteosarcoma subpopulations establish paracrine interactions with both lung and tumor cells to create a metastatic niche

Osteosarcoma is an aggressive and deadly bone tumor, primarily afflicting children, adolescents, and young adults. Poor outcomes for osteosarcoma patients are intricately linked with the development of lung metastasis. While lung metastasis is responsible for nearly all deaths caused by osteosarcoma, identification of biologically defined, metastasis-targeting therapies remains elusive because the underlying cellular and molecular mechanisms that govern metastatic colonization of circulating tumor cells to the lung remains poorly understood. While thousands of tumor cells are released into circulation each day, very few can colonize the lung. Herein, using a combination of a novel organotypic metastasis in vitro model, single-cell RNA sequencing, human xenograft, and murine immunocompetent osteosarcoma models, we find that metastasis is initiated by a subpopulation of hypo-proliferative cells with the unique capacity to sustain production of metastasis promoting cytokines such as IL6 and CXCL8 in response to lung-epithelial derived IL1. Critically, genomic and pharmacologic disruption of IL1 signaling in osteosarcoma cells significantly reduces metastatic progression. Collectively, our study supports that tumor-stromal interactions are important for metastasis, and suggests that metastatic competency is driven, in part, by the tumor cells ability to respond to the metastatic niche. Our findings support that disruption of tumor-stromal signaling is a promising therapeutic approach to disrupt metastasis progression.

cancer biology↗

bioRxiv2024

Seclidemstat blocks the transcriptional function of multiple FET-fusion oncoproteins

Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5 portion of the FET gene with a 3 partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the in vitro potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas. SIGNIFICANCEHere, we show the noncompetitive inhibitor, seclidemstat, has in vitro activity against multiple FET fusion proteins that cause a number of rare and aggressive sarcomas. These data represent one of the largest analyses of FET fusion activity across multiple malignancies and are a valuable resource for those studying FET-rearranged sarcomas.

cancer biology↗

bioRxiv2024

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

cancer biology↗

bioRxiv2024

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells

PurposeLung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation. Experimental designUsing single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics was used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and non-tumor cells within the metastatic microenvironment. ResultsOsteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. ConclusionsFibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a pro-metastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the anti-fibrotic TKI nintedanib. Our data shed light on the non-cell autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models. Statement of translational relevanceTherapies that block metastasis have the potential to save the majority of lives lost due to solid tumors. Disseminated tumor cells must integrate into the foreign, inhospitable microenvironments they encounter within secondary organs to facilitate metastatic colonization and progression. Our study elucidated that disseminated osteosarcoma cells survive within the lung by co-opting and amplifying the lungs endogenous wound-healing response program. This osteosarcoma-induced wound response results in fibrosis of the surrounding microenvironment. Our data implicates fibrosis and abnormal wound healing as key drivers of osteosarcoma lung metastasis that can be targeted therapeutically to disrupt metastasis progression.

cancer biology↗

bioRxiv2022

Zebrafish her3 knockout impacts developmental and cancer-related gene signatures

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3s role in neurogenesis. To investigate HES3s function during development, we generated a zebrafish CRISPR/Cas9 knockout of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 knockout embryos are enriched for HOX and SOX10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts. Summary StatementHere, we generate and characterize a zebrafish her3/HES3 knockout to elucidate the functional role of her3/HES3, a transcriptional repressor, in neural development and tumorigenic processes.

developmental biology↗

bioRxiv2021

Remarkably stable copy-number profiles in osteosarcoma revealed using single-cell DNA sequencing

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNAs) are the genetic drivers of disease. Models around genomic instability conflict - it is unclear if osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little sub-clonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic time points (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance StatementChromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intra-tumoral heterogeneity and tumor evolution.

cancer biology↗

bioRxiv2021

High-throughput Identification of Eukaryotic Parasites and Arboviruses in Mosquitoes

Vector-borne pathogens cause many human infectious diseases and are responsible for high mortality and morbidity throughout the world. They can also cause livestock epidemics with dramatic social and economic consequences. Due to the high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occur after the reports of clinical cases. Here, we use high-throughput sequencing to detect and identify a wide range of parasites and viruses carried by mosquitoes from Cambodia, Guinea, Mali and Maryland. We apply this approach to individual Anopheles mosquitoes as well as pools of mosquitoes captured in traps; and compare the outcomes of this assay when applied to DNA or RNA. We identified known human and animal pathogens and mosquito parasites belonging to a wide range of taxa, insect Flaviviruses, and novel DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap is an efficient approach to monitor and identify potential vector-borne pathogens in large surveillance studies, and that analyses of RNA extracted from mosquitoes is preferable, when possible, over DNA-based analyses. Overall, we describe a flexible and easy-to-customize assay that can provide important information for vector-borne disease surveillance and research studies to efficiently complement current approaches.

microbiology↗