The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intra-tumor transformation process is poorly understood.We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stressresponsive DTC cells, inflammatory ATC cells (iATCs), mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Further, our analysis identified two important milestones: 1) a diploid stage, where iATC cells were diploids with inflammatory phenotypes, and 2) an aneuploid stage, where mATCs gained aneuploid genomes and mesenchymal phenotypes producing excessive collagens and collagen-interacting receptors. In parallel, cancer-associatedfibroblasts showed strong interactions among mesenchymal cell-types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for ATC.

Single-cell nanopore sequencing of full-length mRNAs transforms single-cell multi-omics studies. However, challenges include high sequencing errors and dependence on short-reads and/or barcode whitelists. To address these, we develop scNanoGPS to calculate same-cell genotypes (mutations) and phenotypes (gene/isoform expressions) without short-read nor whitelist guidance. We apply scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell-lines. Standalone, scNanoGPS deconvolutes error-prone long-reads into single-cells and single-molecules, and simultaneously accesses both phenotypes and genotypes of individual cells. Our analyses reveal that tumor and stroma/immune cells express distinct combination of isoforms (DCIs). In a kidney tumor, we identify 924 DCI genes involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Transcriptome-wide mutation analyses identify many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting the critical roles of different mutant populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing technologies.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC), however the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alterations data of patients with different subtypes of thyroid cancer. The resulting model started from stress-responsive DTC cells to inflammatory ATC cells, to mitotic defective ATC cells and extended all the way to mesenchymal ATC cells. In parallel with tumor cell evolution, macrophages shifted from anti-tumor to tumor-promoting states and T cells reprogrammed from cytotoxic to exhausted states. Further, our analysis identified two important milestones: 1) diploid stage, where ATC cells were commonly diploids with non-RAS mutations and inflammatory phenotypes. 2) aneuploid stage, where ATC cells gained aneuploidy with frequent RAS mutations and mesenchymal phenotypes leading to the extreme lethal stage of ATC progression. Citation Format: Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Yuanqing Yan, Tuan M Tran, Jianzhuo Li, Cheng-Kai Shiau, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Lamki Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao. Anaplastic transformation model in thyroid cancer revealed by single cell lineage and fate analysis [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 3131.

Single-cell nanopore sequencing of full-length mRNAs (scNanoRNAseq) is transforming single-cell multi-omics studies. However, challenges include computational complexity and dependence on short-read curation. To address this, we developed a comprehensive toolkit, scNanoGPS to calculate same-cell genotypes-phenotypes without short-read guidance. We applied scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell lines. Standalone, scNanoGPS accurately deconvoluted error-prone long-reads into single-cells and single-molecules. Further, scNanoGPS simultaneously accessed both phenotypes (expressions/isoforms) and genotypes (mutations) of individual cells. Our analyses revealed that tumor and stroma/immune cells often expressed significantly distinct combinations of isoforms (DCIs). In a kidney tumor, we identified 924 genes with DCIs involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Moreover, transcriptome-wide mutation analyses identified many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting critical roles of different populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing.

Single-cell Nanopore sequencing of full-length mRNAs is transforming single-cell multi-omics studies. However, challenges include computational complexity and dependence on short-read curation. To address this, we developed a comprehensive toolkit, scNanoGPS to compute same-cell genotype and phenotype without short-read guidance. We applied scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell lines. Standalone, scNanoGPS accurately deconvoluted error-prone long reads into single cells and single molecules. Further, scNanoGPS simultaneously accessed both phenotypes (expressions/isoforms) and genotypes (single nucleotide variants) of individual cells. Our analyses revealed that tumor and stroma/immune cells often expressed significantly distinct combinations of isoforms. In a kidney tumor, we identified 924 genes with distinct combinations of isoforms involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Moreover, transcriptome-wide mutation analyses identified many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting critical roles of different populations in tumors. In summary, scNanoGPS facilitates applications of single-cell long-read sequencing. Citation Format: Cheng-Kai Shiau, Lina Lu, Rachel Kieser, Kazutaka Fukumura, Timothy Pan, Hsiao-Yun Lin, Jie Yang, Eric L. Tong, GaHyun Lee, Yuanqing Yan, Jason T. Huse, Ruli Gao. ScNanoGPS: A robust tool for simultaneous calculation of same-cell genotypes and phenotypes from single cell long read transcriptomes [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 2069.