GSM7388875: D886T219_tumor_Tex_patient1; Homo sapiens; RNA-Seq
Tumor tissue and  normal adjacent (juxta) tissue  samples were obtained from early stage, untreated patients with NSCLC. Tumor tissue samples were obtained from surgical specimens, after macroscopical examination of the tissue by a pathologist. Tissue samples were stored in C02-independent medium (Invitrogen) with 5% human serum and transferred within 1-hour post-surgery to the research institute. For each specimen, a fragment was formalin-fixed, and paraffin embedded for histology and immunohistochemistry. Tumor tissue samples were gently cut in approximately 1mm3 pieces. Tissues were digested enzymatically, by an incubation of 20-40 minutes, based on the size of the tissue, at 37oC in agitation, in C02-independent medium (Invitrogen) with Collagenase I (2mg/ml, Sigma-Aldrich), Hyaluronidase (2mg/ml, Sigma-Aldrich) and DNase (25µg/ml, Sigma-Aldrich). Tissue fragments were gently triturated with a 20 ml syringe plunger on a 40 µm cell strainer (BD) in 1X PBS (Invitrogen) with 1% fetal bovine serum (FBS) and 2mM EDTA (Gibco), until uniform cell suspensions were obtained. The suspended cells were subsequently centrifuged for 10 minutes at 400g.  Tumor-infiltrating lymphocytes were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich). After tissue digestion, cells are resuspended in C02-independent medium and layered onto Ficoll-Paque PLUS solution. Subsequently cells were centrifuged for 20 minutes at room temperature at 800g without breaks. After centrifugation tumor-infiltrating lymphocytes were carefully transferred to a new tube and washed with 1X PBS with 1% FBS and 2mM EDTA (Gibco). CD3+ T cells were isolated using the Pan T cell Isolation Kit (Miltenyi), that leads to approximately 80% purity.  CD3+ T cells were resuspended 1X PBS with 1% FBS and 2mM EDTA (Gibco). Cell numbers and viability were measured using a Countess II Automated Cell Counter (Thermo Fisher Scientific) and hemocytometer/ trypan blue.   For isolation of the distinct CD8+ T cell subsets, upon TILs isolation, cells were washed and resuspended in 100ul staining buffer containing antibodies. After 20 minutes incubation at 4oC, cells were washed twice and resuspended in 200ul of 1X Phosphate Buffered Saline (PBS) buffer with 1mM EDTA and 1% fetal bovine serum. Cell sorting was performed using MoFlo ASTRIOS cell sorter from Beckman Coulter Life Sciences. Cells were stained with a combination of surface markers, using a set of monoclonal antibodies from the following:  CD3-AF532 (1:50, clone UCHT1, #2067979, Thermofischer Scientific), CD8-BV510 (1:50, clone 2ST8SH7, #9259590, BD Biosciences), PD1-BV421 (1:40, clone EH12.2H7, #B268454, Biolegend), KLRG1-PE-Cy7 (1:40, clone SA231A2 ,#B256810, Biolegend), TIM3-BV786 (1:40, clone 7D3, #9259393, BD Biosciences), CD39-APC (1:50, clone eBioA1, 2#2071264, Thermofischer Scientific). Cells were gated on single cells based on the FSC-A/FSC-H and SSC-A/ SSC-H scatters, live cells (Zombie NIR negative), CD3+, CD8+ cells and afterwards on the population of interest, on the basis of the expression of the protein markers: KLRG1, CD103, PD1, TIM3 and CD39.   Upon sorting of the CD8+ subsets, cells were centrifuged at 300g for 3 minutes, resuspended in 350ul of TCL buffer (QIAGEN) with 1% b-mercaptoethanol inside a 1.5ml eppendorf tube and rapidly stored at -80oC. Within a period of a maximum 7 days, cells were thawed and prepared for RNA isolation. Norgen single cell RNA purification kit was used for RNA extraction. Residual genomic DNA was removed using the RNase free DNase set (Qiagen). RNA quantity and quality was measured by Agilent RNA 6000 Pico kit (Agilent Technologies). Samples with high quality RNA (RIN>7) were further selected. On average the samples had between 500pg-1ng of total RNA. To synthesize and amplify cDNA from those samples, the SMART-Seq v4 Ultra Low Input RNA kit (TaKaRa) was used, according to manufacturer's instructions. cDNA quantity and quality were measured by High Sensitivity DNA kit (Agilent Technologies). 9ng of amplified cDNA was used for library synthesis, using the KAPA HyperPlus library preparation kit (Roche) according to manufacturer's instructions. Sequencing libraries were quantified with the LabChip® GX Touch™ nucleic acid analyzer and libraries were sequenced on a HiSeq4000 sequencer (Illumina).    Tumor tissue samples were obtained from surgical specimens, after macroscopical examination of the tissue by a pathologist. Tissue samples were stored in C02-independent medium (Invitrogen) with 5% human serum and transferred within 1-hour post-surgery to the research institute. For each specimen, a fragment was formalin-fixed, and paraffin embedded for histology and immunohistochemistry.    Tissue dissociation  Tumor tissue samples were gently cut in approximately 1mm3 pieces. Tissues were digested enzymatically, by an incubation of 20-40 minutes, based on the size of the tissue, at 37oC in agitation, in C02-independent medium (Invitrogen) with Collagenase I (2mg/ml, Sigma-Aldrich), Hyaluronidase (2mg/ml, Sigma-Aldrich) and DNase (25µg/ml, Sigma-Aldrich).    The tissue pieces were gently triturated with a 20 ml syringe plunger on a 40 µm cell strainer (BD) in 1X PBS (Invitrogen) with 1% fetal bovine serum (FBS) and 2mM EDTA (Gibco), until uniform cell suspensions were obtained. The suspended cells were subsequently centrifuged for 10 minutes at 400g.      Tumor- infiltrating lymphocytes isolation  Tumor-infiltrating lymphocytes were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich). After tissue digestion, cells are resuspended in C02-independent medium and layered onto Ficoll-Paque PLUS solution. Subsequently cells were centrifuged for 20 minutes at room temperature at 800g without breaks. After centrifugation tumor-infiltrating lymphocytes were carefully transferred to a new tube and washed with 1X PBS with 1% FBS and 2mM EDTA (Gibco).    CD3+ T cell isolation and purification  CD3+ T cells were isolated using the Pan T cell Isolation Kit (Miltenyi), that leads to approximately 80% purity.  CD3+ T cells were resuspended 1X PBS with 1% FBS and 2mM EDTA (Gibco). Cell numbers and viability were measured using a Countess II Automated Cell Counter (Thermo Fisher Scientific) and hemocytometer/ trypan blue.     CD8+ T cell sorting  For isolation of the distinct CD8+ T cell subsets, upon TILs isolation, cells were washed and resuspended in 100ul staining buffer containing antibodies. After 20 minutes incubation at 4oC, cells were washed twice and resuspended in 200ul of 1X Phosphate Buffered Saline (PBS) buffer with 1mM EDTA and 1% fetal bovine serum. Cell sorting was performed using MoFlo ASTRIOS cell sorter from Beckman Coulter Life Sciences. Cells were stained with a combination of surface markers, using a set of monoclonal antibodies from the following:  CD3-AF532 (1:50, clone UCHT1, #2067979, Thermofischer Scientific), CD8-BV510 (1:50, clone 2ST8SH7, #9259590, BD Biosciences), PD1-BV421 (1:40, clone EH12.2H7, #B268454, Biolegend), KLRG1-PE-Cy7 (1:40, clone SA231A2 ,#B256810, Biolegend), TIM3-BV786 (1:40, clone 7D3, #9259393, BD Biosciences), CD39-APC (1:50, clone eBioA1, 2#2071264, Thermofischer Scientific). Cells were gated on single cells based on the FSC-A/FSC-H and SSC-A/ SSC-H scatters, live cells (Zombie NIR negative), CD3+, CD8+ cells and afterwards on the population of interest, on the basis of the expression of the protein markers: KLRG1, CD103, PD1, TIM3 and CD39.     Samples preparation for RNAseq  Upon sorting of the CD8+ subsets, cells were centrifuged at 300g for 3 minutes, resuspended in 350ul of TCL buffer (QIAGEN) with 1% b-mercaptoethanol inside a 1.5ml eppendorf tube and rapidly stored at -80oC.    Within a period of a maximum 7 days, cells were thawed and prepared for RNA isolation. Norgen single cell RNA purification kit was used for RNA extraction. Residual genomic DNA was removed using the RNase free DNase set (Qiagen). RNA quantity and quality was measured by Agilent RNA 6000 Pico kit (Agilent Technologies). Samples with high quality RNA (RIN>7) were further selected. On average the samples had between 500pg-1ng of total RNA. To synthesize and amplify cDNA from those samples, the SMART-Seq v4 Ultra Low Input RNA kit (TaKaRa) was used, according to manufacturer's instructions. cDNA quantity and quality were measured by High Sensitivity DNA kit (Agilent Technologies). 9ng of amplified cDNA was used for library synthesis, using the KAPA HyperPlus library preparation kit (Roche) according to manufacturer's instructions. Sequencing libraries were quantified with the LabChip® GX Touch™ nucleic acid analyzer and libraries were sequenced on a HiSeq4000 sequencer (Illumina).  Study design  The overall objective of this study was to characterize the diversity and ontogeny of CD8+ T cells in untreated lung tumors. This was accomplished by performing bulk RNAseq and flow cytometry analysis on CD8+ T cell subsets isolated from tumor tissue, normal adjacent to tumor tissue, and blood samples from patients undergoing surgical resection for early-stage lung cancer. All samples were collected from the Institute Mutualist Montsouris, under a dedicated protocol for lung cancer specimens approved by the French Ethics and Informatics Commission (EUdract 2017-A03081-52). All patients in this study provided written informed consent for sample collection and data analysis. Cohort size was selected to assess interpatient variability in subpopulation levels.    Human samples  Tumor tissue, normal adjacent (juxta) tissue and blood samples were obtained from early stage, untreated patients with NSCLC.  All patients enrolled in this study patients in this study provided written informed consent for sample collection and data analyses.    Tumor tissue samples were obtained from surgical specimens, after macroscopical examination of the tissue by a pathologist. Tissue samples were stored in C02-independent medium (Invitrogen) with 5% human serum and transferred within 1-hour post-surgery to the research institute. For each specimen, a fragment was formalin-fixed, and paraffin embedded for histology and immunohistochemistry.    Tissue dissociation  Tumor tissue samples were gently cut in approximately 1mm3 pieces. Tissues were digested enzymatically, by an incubation of 20-40 minutes, based on the size of the tissue, at 37oC in agitation, in C02-independent medium (Invitrogen) with Collagenase I (2mg/ml, Sigma-Aldrich), Hyaluronidase (2mg/ml, Sigma-Aldrich) and DNase (25µg/ml, Sigma-Aldrich).    The tissue pieces were gently triturated with a 20 ml syringe plunger on a 40 µm cell strainer (BD) in 1X PBS (Invitrogen) with 1% fetal bovine serum (FBS) and 2mM EDTA (Gibco), until uniform cell suspensions were obtained. The suspended cells were subsequently centrifuged for 10 minutes at 400g.      Tumor- infiltrating lymphocytes isolation  Tumor-infiltrating lymphocytes were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich). After tissue digestion, cells are resuspended in C02-independent medium and layered onto Ficoll-Paque PLUS solution. Subsequently cells were centrifuged for 20 minutes at room temperature at 800g without breaks. After centrifugation tumor-infiltrating lymphocytes were carefully transferred to a new tube and washed with 1X PBS with 1% FBS and 2mM EDTA (Gibco).    CD3+ T cell isolation and purification  CD3+ T cells were isolated using the Pan T cell Isolation Kit (Miltenyi), that leads to approximately 80% purity.  CD3+ T cells were resuspended 1X PBS with 1% FBS and 2mM EDTA (Gibco). Cell numbers and viability were measured using a Countess II Automated Cell Counter (Thermo Fisher Scientific) and hemocytometer/ trypan blue.     CD8+ T cell sorting  For isolation of the distinct CD8+ T cell subsets, upon TILs isolation, cells were washed and resuspended in 100ul staining buffer containing antibodies. After 20 minutes incubation at 4oC, cells were washed twice and resuspended in 200ul of 1X Phosphate Buffered Saline (PBS) buffer with 1mM EDTA and 1% fetal bovine serum. Cell sorting was performed using MoFlo ASTRIOS cell sorter from Beckman Coulter Life Sciences. Cells were stained with a combination of surface markers, using a set of monoclonal antibodies from the following:  CD3-AF532 (1:50, clone UCHT1, #2067979, Thermofischer Scientific), CD8-BV510 (1:50, clone 2ST8SH7, #9259590, BD Biosciences), PD1-BV421 (1:40, clone EH12.2H7, #B268454, Biolegend), KLRG1-PE-Cy7 (1:40, clone SA231A2 ,#B256810, Biolegend), TIM3-BV786 (1:40, clone 7D3, #9259393, BD Biosciences), CD39-APC (1:50, clone eBioA1, 2#2071264, Thermofischer Scientific). Cells were gated on single cells based on the FSC-A/FSC-H and SSC-A/ SSC-H scatters, live cells (Zombie NIR negative), CD3+, CD8+ cells and afterwards on the population of interest, on the basis of the expression of the protein markers: KLRG1, CD103, PD1, TIM3 and CD39.     Samples preparation for RNAseq  Upon sorting of the CD8+ subsets, cells were centrifuged at 300g for 3 minutes, resuspended in 350ul of TCL buffer (QIAGEN) with 1% b-mercaptoethanol inside a 1.5ml eppendorf tube and rapidly stored at -80oC.    Within a period of a maximum 7 days, cells were thawed and prepared for RNA isolation. Norgen single cell RNA purification kit was used for RNA extraction. Residual genomic DNA was removed using the RNase free DNase set (Qiagen). RNA quantity and quality was measured by Agilent RNA 6000 Pico kit (Agilent Technologies). Samples with high quality RNA (RIN>7) were further selected. On average the samples had between 500pg-1ng of total RNA. To synthesize and amplify cDNA from those samples, the SMART-Seq v4 Ultra Low Input RNA kit (TaKaRa) was used, according to manufacturer's instructions. cDNA quantity and quality were measured by High Sensitivity DNA kit (Agilent Technologies). 9ng of amplified cDNA was used for library synthesis, using the KAPA HyperPlus library preparation kit (Roche) according to manufacturer's instructions. Sequencing libraries were quantified with the LabChip® GX Touch™ nucleic acid analyzer and libraries were sequenced on a HiSeq4000 sequencer (Illumina). RNA libraries were prepared for sequencing using standard Illumina protocols (NovaSeq 6000)