run_metadata: 61559

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61559	SRR12851621	SRX9318588	SRS7543398	SRP287858	PRJNA670235	Spatially resolved transcriptomics reveals the architecture of the tumor microenvironment interface	GSE159709	Transcriptome Analysis	Cancer cells interact with a wide variety of other cell types but our understanding of microenvironmental heterogeneity and how it influences tumor phenotypes is limited. While single cell RNA seq scRNA seq has helped define these TME cell types it provides limited information on the mechanisms that define how individual tumor cells interact with TME. Here we integrate spatial transcriptomics with scRNA seq to define the architecture and nature of nascent tumor and surrounding microenvironment cells as they come into contact through the process of invasion. Using a well defined transgenic zebrafish model of BRAFV600E driven melanoma we identify a transcriptionally unique “interface” cluster localized at the boundary between tumor cells and surrounding tissues. Using an unbiased data driven approach we identify spatially patterned gene modules specific to the interface and show that the interface is a distinct transcriptional entity that histologically resembles the microenvironment but transcriptionally resembles the tumor. By complementing ST with scRNA seq we demonstrate that the interface is composed of specialized tumor and microenvironment cells. Both cell types in the interface upregulate a common set of cilia genes and we find enrichment of cilia proteins only where the tumor meets the TME. Cilia gene expression is regulated by ETS family transcription factors which normally act to suppress their expression outside of this region. This unique ETS driven interface transcriptional state is conserved across ten different human patient samples suggesting this is a conserved feature of human melanoma. Taken together our results demonstrate the power of spatial and single cell transcriptomics techniques in uncovering novel biological mechanisms that drive tumor invasion into new tissues. Overall design: Spatial transcriptomics Visium platform of zebrafish bearing BRAF V600E driven melanomas. Single cell RNA seq 10X Genomics v3 of two such zebrafish. Single nucleus RNA seq 10X Genomics v3 of three such zebrafish pooled into one reaction.		pubmed:34725363		SingleCell E	GSM4838134		tissue:Zebrafish melanomas BRAF V600E\|sample type:Single cell suspension	SingleCell E	Illumina RTA v2 software was used for basecalling and quality determination. The Visium raw FASTQ data was processed using 10X Genomics proprietary SpaceRanger v1.0.0 pipeline. The single cell RNA seq raw FASTQ data was processed using the 10X Genomics proprietary CellRanger v3.1.0 pipeline Genome build: GRCz10.88 Supplementary files format and content: For 10x Genomics spatial transcriptomic data: output of SpaceRanger pipeline including gene expression and spatial contents. For 10x Genomics single cell RNA Seq: output of CellRanger pipeline including gene expression features and barcodes.	Zebrafish melanomas BRAF V600E		For spatial transcriptomics using the Visium platform from 10X genomics: Adult tumor bearing fish were euthanized on ice and washed in 1X PBS. post dissection of the head and tail the remaining tissue was equilibrated in cold OCT for 2 minutes before transfer to a tissue mold filled with fresh OCT for snap freezing in liquid nitrogen chilled isopentane. Tissue blocks were stored at 80°C. For cryosectioning both the tissue block and the ST slide were equilibrated inside the cryostat for 15 30 minutes at 16°C before sectioning. Transverse sections through the entire fish were cut at a thickness of 10 µm and immediately placed on the ST slide Visium Spatial Gene Expression Slides 10X Genomics. ST slides containing sections were stored at 80°C for a maximum of one week before use. For single cell RNA seq using the 10X genomics 3’ GEX v3 platform: Adult tumor bearing fish were dissected to obtain only the tumor and surrounding tissues i.e head and tail were removed. Tissue was minced with a fresh scalpel and incubated in 0.16 mg/mL liberase Sigma Aldrich #5401020001 in 0.9X PBS for 15 minutes at RT. Tissue was then further dissociated by repeated pipetting with a wide bore P1000 followed by incubation for an additional 15 minutes at RT. post adding 500 uL FBS to stop the dissociation reaction samples were filtered through a 70 µm filter and centrifuged at 500g for 5 minutes at RT. The resulting pellet was resuspended in DMEM supplemented with 2% FBS and cells were sorted at room temperature to remove debris and doublets using a BD FACSAria III cell sorter BD Biosciences. Equal numbers of GFP+ tumor and GFP macroenvironment cells were collected. For spatial transcriptomics using the Visium platform from 10X genomics: Samples were processed according to the Visium Spatial Gene Expression User Guide 10X Genomics and all reagents were from the Visium Spatial Gene Expression Kit 10X Genomics. Briefly sections were fixed in chilled methanol for 30 min at 20°C stained with hematoxylin and eosin and mounted in 85% glycerol for imaging. Imaging was performed on a Leica SCN400 F whole slide scanner at 40X magnification. post imaging sections were permeabilized at 37°C for 45 minutes. post permeabilization the on slide reverse transcription RT reaction was performed at 53°C for 2 hours. Permeabilization time and RT reaction length were determined using the Visium Spatial Tissue Optimization Kit 10X Genomics. Second strand synthesis was subsequently performed on slide for 15 minutes at 65°C. All on slide reactions were performed in a thermocycler with a metal slide adaptor plate. Following second strand synthesis samples were transferred to tubes for cDNA amplification and cleanup. Library quality was assayed using a Bioanalyzer High Sensitivity chip Agilent. For single cell RNA seq using the 10X genomics 3’ GEX v3 platform: Equal numbers of sorted GFP+ tumor and GFP macroenvironment cells were centrifuged at 300g for 5 minutes at RT and resuspended in DMEM + 10% FBS. Droplet based scRNA seq was performed using the Chromium Single Cell 3’ Library & Gel Bead Kit v3 10X Genomics and Chromium Single Cell 3’ Chip G 10X Genomics. Approximately 10 000 cells from two fish were split encapsulated in a single v3 reaction. GEM generation and library preparation were performed according to manufacturer’s instructions.		sample type:Single cell suspension	GSM4838134	GSM4838134: SingleCell E; Danio rerio; RNA Seq	GSM4838134		1	For spatial transcriptomics using the Visium platform from 10X genomics: Adult tumor bearing fish were euthanized on ice and washed in 1X PBS. post dissection of the head and tail the remaining tissue was equilibrated in cold OCT for 2 minutes before transfer to a tissue mold filled with fresh OCT for snap freezing in liquid nitrogen chilled isopentane. Tissue blocks were stored at 80°C. For cryosectioning both the tissue block and the ST slide were equilibrated inside the cryostat for 15 30 minutes at 16°C before sectioning. Transverse sections through the entire fish were cut at a thickness of 10 µm and immediately placed on the ST slide Visium Spatial Gene Expression Slides 10X Genomics. ST slides containing sections were stored at 80°C for a maximum of one week before use. For single cell RNA seq using the 10X genomics three prime GEX v3 platform: Adult tumor bearing fish were dissected to obtain only the tumor and surrounding tissues i.e head and tail were removed. Tissue was minced with a fresh scalpel and incubated in 0.16 mg/mL liberase Sigma Aldrich #5401020001 in 0.9X PBS for 15 minutes at RT. Tissue was then further dissociated by repeated pipetting with a wide bore P1000 followed by incubation for an additional 15 minutes at RT. post adding 500 uL FBS to stop the dissociation reaction samples were filtered through a 70 µm filter and centrifuged at 500g for 5 minutes at RT. The resulting pellet was resuspended in DMEM supplemented with 2% FBS and cells were sorted at room temperature to remove debris and doublets using a BD FACSAria III cell sorter BD Biosciences. Equal numbers of GFP+ tumor and GFP macroenvironment cells were collected. For spatial transcriptomics using the Visium platform from 10X genomics: Samples were processed according to the Visium Spatial Gene Expression User Guide 10X Genomics and all reagents were from the Visium Spatial Gene Expression Kit 10X Genomics. Briefly sections were fixed in chilled methanol for 30 min at 20°C stained with hematoxylin and eosin and mounted in 85% glycerol for imaging. Imaging was performed on a Leica SCN400 F whole slide scanner at 40X magnification. post imaging sections were permeabilized at 37°C for 45 minutes. post permeabilization the on slide reverse transcription RT reaction was performed at 53°C for 2 hours. Permeabilization time and RT reaction length were determined using the Visium Spatial Tissue Optimization Kit 10X Genomics. Second strand synthesis was subsequently performed on slide for 15 minutes at 65°C. All on slide reactions were performed in a thermocycler with a metal slide adaptor plate. Following second strand synthesis samples were transferred to tubes for cDNA amplification and cleanup. Library quality was assayed using a Bioanalyzer High Sensitivity chip Agilent. For single cell RNA seq using the 10X genomics three prime GEX v3 platform: Equal numbers of sorted GFP+ tumor and GFP macroenvironment cells were centrifuged at 300g for 5 minutes at RT and resuspended in DMEM + 10% FBS. Droplet based scRNA seq was performed using the Chromium Single Cell three prime Library & Gel Bead Kit v3 10X Genomics and Chromium Single Cell three prime Chip G 10X Genomics. Approximately 10 000 cells from two fish were split encapsulated in a single v3 reaction. GEM generation and library preparation were performed according to manufacturer's instructions.	GEO Accession:GSM4838134	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP287858			10X_SingleCell_Sample_E_S1_L000_R1_001.fastq.gz 10X_SingleCell_Sample_E_S1_L000_R2_001.fastq.gz	fastq fastq	44430354080.0	277689713.0	GSM4838134 r1	0:28 1:132	A:12355044923;C:9923404810;G:10323616312;T:11822807950;N:5480085	28	132			12355044923	9923404810	10323616312	11822807950	5480085	SRX9318588	SRS7543398	SRA1145723	GEO	Memorial Sloan Kettering Cancer Center	2	0.00204	0.9315	0.00092	0.09404	0.9964	0.83027	0.40865	0.54937	28	132	T	B	sc-like readlen	illumina	nextseq	unknown	random_priming	unknown	sc	single_cell_droplet	10x		United States	2020-10-20	Adult	Adult	Cancer or Tumor	Cancer or Tumor