Ncounter pancancer immune profiling panel

Manufactured by NanoString

Sourced in United States

The NCounter PanCancer Immune Profiling Panel is a laboratory equipment product from NanoString that enables comprehensive analysis of the immune response in tumor samples. It provides a direct, multiplexed measurement of gene expression for over 700 genes related to the tumor microenvironment and immune system.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (15 mentions) Ncounter digital analyzer, by NanoString (7 mentions) Nsolver software, by NanoString (6 mentions) Nsolver analysis software, by NanoString (5 mentions) Ncounter max analysis system, by NanoString (5 mentions) Ncounter flex analysis system, by NanoString (4 mentions) Nsolver analysis software v4, by NanoString (4 mentions) Rneasy ffpe kit, by Qiagen (3 mentions) Pancancer immune profiling advanced analysis module, by NanoString (3 mentions) Ncounter prep station, by NanoString (3 mentions) Rneasy plus mini kit, by Qiagen (3 mentions) Agilent bioanalyzer, by Agilent Technologies (2 mentions) Rna 6000 nano kit, by Agilent Technologies (2 mentions) High pure ffpet rna isolation kit, by Roche (2 mentions) Rnalater, by Qiagen (2 mentions) Allprep dna rna ffpe kit, by Qiagen (2 mentions) Ncounter sample prep station, by NanoString (2 mentions) Ncounter max, by NanoString (2 mentions) Rneasy extraction kit, by Qiagen (2 mentions) Nsolver 4.0 analysis software, by NanoString (2 mentions)

91 protocols using ncounter pancancer immune profiling panel

Comprehensive Immune Profiling with NanoString

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The NanoString nCounter PanCancer Immune Profiling Panel (NanoString Technologies) is a commercialized multiplexed gene expression panel, which can simultaneously quantitate 770 immune-related genes. This was used for further mRNA quantification of two 150-ng ribonucleic acid extracted from patient’s both tissue samples. All procedures, including preparation, hybridization, detection, scanning, and normalization, were performed according to the manufacturer’s instructions. Detailed information and the gene list are available on the official website at https://www.nanostring.com/products/gene-expression-panels/gene-expression-panels-overview/hallmarks-cancer-gene-expression-panel-collection/pancancer-immune-profiling-panel.

Liang Y.H., Tsai J.H., Cheng Y.M., Chan K.Y., Hsu W.L., Lee C.C., Chen K.H, & Yeh K.H. (2021). Chemotherapy agents stimulate dendritic cells against human colon cancer cells through upregulation of the transporter associated with antigen processing. Scientific Reports, 11, 9080.

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Immune Gene Expression Profiling of PBMCs

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RNA was extracted from PBMCs. The NanoString nCounter PanCancer Immune Profiling panel (Nanostring Technologies, Seattle, WA, USA) was used to characterize immune gene expression. Analysis was performed in nCounter Software 3. The software first identifies and checks standard QC controls such as imaging, binding density, positive controls to ensure the samples were read and reported properly. The normalization was performed with positive controls as well as housekeeping gene normalization. The positive control normalization normalizes all platform-associated sources of variation. Reference (housekeeping) gene normalization was also performed to adjust counts of all probes relative to a probe set that is not expected to vary between samples.

Mahalingam D., Wilkinson G.A., Eng K.H., Fields P., Raber P., Moseley J.L., Cheetham K., Coffey M., Nuovo G., Kalinski P., Zhang B., Arora S.P, & Fountzilas C. (2019). Pembrolizumab in combination with the oncolytic virus pelareorep and chemotherapy in patients with advanced pancreatic adenocarcinoma: a Phase 1b study. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(1), 71-81.

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Nanostring Immune Profiling Protocol

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In cohort 1, RNA was purified using AllPrep^®DNA/RNA/miRNA Universal Kit according to manufacturer’s instructions (Qiagen). For multiplex gene expression analysis, the Nanostring nCounter^® platform was used. 100 ng total RNA in 7-20 µl (5-14ng/µl) was used as input. Samples were hybridized to target specific barcodes consisting of a capture probe and a reporter probe using the Nanostring nCounter^® PanCancer Immune Profiling panel (NanoString Technologies Inc, Seattle, WA, USA). After hybridization, target RNA was isolated by magnetic bead sorting and immobilized by applying an electric current as described in the hybridization protocol in the nCounter XT Assay user manual (NanoString Technologies Inc). Gene expression data were normalized with CD8A and CD8B that are stably expressed in CD8⁺ T cells (34 (link)). Two outliers were excluded after nSolver gene expression analysis, why only 38 genotype-selected HS were analysed in cohort 1.

Buhelt S., Laigaard H.M., von Essen M.R., Ullum H., Oturai A., Sellebjerg F, & Søndergaard H.B. (2021). IL2RA Methylation and Gene Expression in Relation to the Multiple Sclerosis-Associated Gene Variant rs2104286 and Soluble IL-2Rα in CD8+ T Cells. Frontiers in Immunology, 12, 676141.

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Comprehensive Immune Gene Expression Analysis

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Immune gene expression analysis of nasal cell lysate and extracted RNA from blood was performed using a commercially available NanoString nCounter PanCancer Immune Profiling panel (NanoString Technologies). This panel contained 40 references (housekeeping) genes and 730 immune genes and was used in combination with the nCounter panel plus probe set which contained an additional 30 immune genes relating to the allergic response and mechanism of action of steroids and antihistamines (760 immune genes in total). Gene expression data underwent imaging quality control and normalization checks before analysis and interpretation of data. Genes that were expressed at counts below 20 in 80% or more samples were excluded from further analysis. Reference (housekeeping) normalization was performed using the GeNorm Algorithm where 20 out of 40 housekeeping genes were used for the nasal lysate samples and 33/40 housekeeping genes were used for the peripheral blood samples.

Watts A.M., West N.P., Smith P.K., Zhang P., Cripps A.W, & Cox A.J. (2021). Nasal immune gene expression in response to azelastine and fluticasone propionate combination or monotherapy. Immunity, Inflammation and Disease, 10(3), e571.

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NanoString-based Immune Profiling

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The NanoString nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) was used to assess the expression of 770 genes. Each biotinylated capture probe in the panel was manufactured with specificity for a 100-base region of the target mRNA. A reporter probe tagged with a fluorescent barcode was also included, thus resulting in two sequence-specific probes for each target transcript. Probes were hybridized to 60 ng of total RNA for 20 h at 65 °C and applied to the nCounter preparation station for automated removal of excess probe and immobilization of probe-transcript complexes on a streptavidin-coated cartridge. Data were collected with the nCounter digital analyzer by counting individual barcodes and analyzed using the software nSolver version 4.0.

Andric F., Al-Fairouzi A., Wettergren Y., Szeponik L., Bexe-Lindskog E., Cusack JC J.r., Tumusiime G., Quiding-Järbrink M, & Ljungman D. (2023). Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer. Cancers, 15(5), 1457.

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FFPE Tumor RNA Extraction and Profiling

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RNA extraction was performed using QIAGEN RNeasy FFPE Kit from two slides of 5 µm-thick unstained formalin fixed paraffin embedded (FFPE) tumor specimens. In 66 cases of CCC whose serum cytokines were analyzed in this study, no left over or only a little remaining FFPE blocks were found in 10 of 66 cases. We excluded 5 out of 56 samples due to the poor quality, and performed the final analysis in 51 cases (Supplementary Fig. 2). The samples were subjected to gene expression profiling using the NanoString nCounter PanCancer Immune Profiling Panel codesets and IO360 codesets (NanoString Technologies Inc; Seattle, WA) according to the manufacturer’s instructions. The gene expression data normalization was performed using nSolver analysis software version 3.0 (NanoString Technologies Inc; Seattle, WA). The raw count from NanoString was subjected to background subtraction, positive control normalization and housekeeping genes normalization as defined by the PanCancer Immune Profiling Panel. Local cytokines gene expression was extracted from the normalized data.

Yabuno A., Matsushita H., Hamano T., Tan T.Z., Shintani D., Fujieda N., Tan D.S., Huang R.Y., Fujiwara K., Kakimi K, & Hasegawa K. (2020). Identification of serum cytokine clusters associated with outcomes in ovarian clear cell carcinoma. Scientific Reports, 10, 18503.

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Immune Profiling of Tumor Samples

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An additional 50-μm section of each tumor was used for mRNA extraction (Absolutely RNA miniprep kit, Cat. No. 400800, Agilent, Santa Clara, CA) and gene expression analysis with Nanostring nCounter PanCancer Immune Profiling Panel (Nanostring Inc, Seattle, WA), which contains 770 genes involved in cancer immune response and include 19 housekeeping genes for data normalization. Read counts from the raw data output were assessed for differential gene expression after normalization using NanoString nSolver (version 4.0). Immune cell type scoring in nSolver was used to assess relative abundance of tumor infiltrating leukocytes. This method of characterizing immune cell populations was previously validated against IHC and flow cytometry [28] . The results were plotted using Prism GraphPad (San Diego, CA).

Khokhlova T.D., Wang Y.N., Son H., Totten S., Whang S, & Ha Hwang J. (2023). Chronic effects of pulsed high intensity focused ultrasound aided delivery of gemcitabine in a mouse model of pancreatic cancer. Ultrasonics, 132.

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Immune Gene Expression Profiling

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Analysis of the expression profiles of >700 immune-related genes was performed by the nanoString nCounter Pan-Cancer Immune Profiling Panel (NanoString Technologies, Seattle, WA).
In detail, 150 ng of RNA from each sample was hybridized with the nCounter PanCancer Immune Profiling Panel (GX Assay) CodeSet. All the procedures related to mRNA quantification, including sample preparation, hybridization, detection, and scanning, were performed following the manufacturer's instructions. The counts were normalized according to the standard protocol. Raw nano-String counts for each mRNA within each experiment were subjected to technical normalization with the counts obtained for positive-control probe sets before biological normalization using the 40 reference genes included in the CodeSet. The normalized data were log2 transformed and then used as input for differential expression analysis. The data were filtered to exclude relatively invariant features and features below the detection threshold (defined for each sample by a cutoff value corresponding to twice the SD of the negative control probes plus the means).

Giannini R., Zucchelli G., Giordano M., Ugolini C., Moretto R., Ambryszewska K., Leonardi M., Sensi E., Morano F., Pietrantonio F., Cremolini C., Falcone A, & Fontanini G. (2020). Immune Profiling of Deficient Mismatch Repair Colorectal Cancer Tumor Microenvironment Reveals Different Levels of Immune System Activation. The Journal of molecular diagnostics : JMD, 22(5).

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Molecular Profiling of Treatment-Naive Cancer Samples

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A total of 24 treatment‐naive samples (sLDH Elevated, n = 12; sLDH Not Elevated, n = 12) balanced for gender and accession site were selected from the TMA samples to undergo additional molecular profiling (Figure 1). RNA was isolated as previously described.¹⁷ Gene expression data were collected for analysis via the NanoString nCounter Vantage 3D Cancer Metabolism Panel and NanoString nCounter PanCancer Immune Profiling Panel and analyzed via nSolver™ Analysis Software. Details of analyses are provided in Data S1.

Fischer G.M., Carapeto F.C., Joon A.Y., Haydu L.E., Chen H., Wang F., Van Arnam J.S., McQuade J.L., Wani K., Kirkwood J.M., Thompson J.F., Tetzlaff M.T., Lazar A.J., Tawbi H.A., Gershenwald J.E., Scolyer R.A., Long G.V, & Davies M.A. (2020). Molecular and immunological associations of elevated serum lactate dehydrogenase in metastatic melanoma patients: A fresh look at an old biomarker. Cancer Medicine, 9(22), 8650-8661.

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Immune Profiling of Tumor Samples

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Total RNA was extracted from whole tumor lysates using TRIzol (Invitrogen) and purified with ethanol. RNA quality was confirmed with a fragment analyzer (Advanced Analytical Technologies, Iowa, USA). Immune profiling was performed with a digital multiplexed NanoString nCounter PanCancer Immune Profiling panel (NanoString Technologies), using 100 ng of total RNA isolated from tumor tissues. Data analysis was performed with nSolver software (NanoString Technologies). The mRNA profiling data was normalized by housekeeping genes and analyzed with R software (www.r-project.org), as previously described.28 29 (link)

Park H.S., Kim Y.M., Kim S., Lee W.S., Kong S.J., Yang H., Kang B., Cheon J., Shin S.J., Kim C, & Chon H.J. (2021). High endothelial venule is a surrogate biomarker for T-cell inflamed tumor microenvironment and prognosis in gastric cancer. Journal for Immunotherapy of Cancer, 9(10), e003353.

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