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Introduction

Developed by a team of researchers at Stanford University, CIBERSORT uses a deconvolutional algorithm to estimate the composition and abundance of immune cells in a mixture of cells based on transcriptomic data, and has been cited nearly a thousand times. The first version of CIBERSORT was published in Nature Methods in 2015, and the current upgraded version of CIBERSORTx was published in Nature Biotechnonogy in 2019.

CIBERSORT is a web-based tool for deconvolution of expression matrices of human leukocyte subtypes based on linear support vector regression. It is mostly used for microarray expression matrices and is superior to other methods (LLSR,LLSR,PERT,RLR,MMAD,DSA) for deconvolution analysis of expression matrices of unknown mixtures and expression matrices containing similar cell types. The method is still based on a known reference set that provides a gene expression signature set for 22 leukocyte subtypes - LM22. Website link: http://cibersort.stanford.edu/

CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression, which can be used to estimate immune cell infiltration with RNA-Seq data. Users only need to register an account and get 500M space for storing data and results. To operate, simply upload a standard expression matrix file to analyze immune infiltration; if you want to analyze the percentage of infiltration that includes other cell types, you need to upload the appropriate file according to the format prompted by the official website.

Tool Features

Single-cell RNA sequencing has become a powerful technology for modern medical research, allowing scientists to study the expression and behavior of individual cells for diseases such as cancer. However, the technology is not yet available for preserved tissue samples and is too expensive to be used on a large scale for routine clinical testing.

To address these shortcomings, researchers at the Stanford University School of Medicine have invented a computational software called CIBERSORTx that can analyze individual cell gene expression directly from whole tissue samples or data sets.

Define cell type and status

CIBERSORTx is a big leap forward from the team's previously developed software, CIBERSORT, Alizadeh says, "In the original version of CIBERSORT, it was possible to analyze the frequency of specific molecules in a group of cells, and without physically separating the cells, tell us which cells were in this group of cells " "We can make an analogy, it's like analyzing a fruit milkshake", says Newman, "you can't see what fruit is in the milkshake, but you can taste it and know that there's a lot of apples, a little bit of bananas, and you see the red color of some strawberries in there". CIBERSORTx takes this principle one step further, as the researchers first perform single-cell RNA analysis on a small amount of tissue, such as tumor tissue, where they separate the tumor cells and take a closer look at the RNA (as well as the proteins) produced by each cell. This process yields an RNA expression pattern for the cell type: a "bar code" that identifies not only the cell type, but also the subtype or mode of operation in which it operates. For example, the same immune cells infiltrating a tumor will produce different RNAs and proteins, and therefore a different RNA barcode than that of the peripheral blood. "What CIBERSORTx does is let us know not only how many apples are in the milkshake, but also how many are small green apples, how many are red apples, how many are green, and how many are purple," says Alizadeh, "Similarly, starting with a mix of RNA in the tumor barcodes to begin with, can give us insights into the status of cell types and affected cells in these tumors, and how we can address these deficiencies for cancer therapy". The scientists say that such a tool would be able to identify not only cell types, but also the state or behavior of cells in a particular environment, which could lead to the discovery of new mechanisms of action and improved treatments.

The team used the technique to analyze more than 1,000 tumor samples and found not only that the expected cancer cells were different from normal cells, but also that the immune cells infiltrating the tumors acted differently from the circulating immune cells, and that even the cells of the normal structures surrounding the cancer cells were different from the same types of cells in other parts of the organ.

"The cancer cells are changing all the other cells in the tumor", Newman said. The researchers even found large differences after the same immune cells infiltrated different types of lung cancer.

The main advantage of CIBERSORTx is that it can be applied to FFPE tissue samples (most tumor sample types). Most FFPE samples cannot be analyzed by single-cell RNA sequencing because the cell membranes are disrupted or the cells cannot be separated from each other, making single-cell RNA analysis impractical or impossible for most large studies and clinical trials.

Predicting treatment responseThe researchers also tested the tool's diagnostic capabilities by analyzing melanomas. Blocking the PD-1 and CTLA4 proteins that infiltrate T cells with drugs is one of the most effective treatments for metastatic melanoma or some other cancers, but these "checkpoint inhibitor" drugs work well in only a small number of patients and there is no easy way to tell which patients will respond.

Previous hypotheses have suggested that these drugs are more likely to work if a patient's infiltrating T cells have high levels of PD-1 and CTLA4, but researchers have had difficulty determining whether this is true. cibersortx can explore this question. After training the algorithm on single-cell RNA data from a small number of melanoma samples, the researchers analyzed previously published datasets of melanoma tumor tissue and test fixed samples. They confirmed the hypothesis, finding that high expression of PD-1 and CTLA4 in certain T cells was associated with reduced mortality in patients treated with PD-1 blocking drugs. CIBERSORTx may also help discover new cellular markers that could provide avenues for cancer treatment, the researchers said. Using the tool to analyze preserved tissue samples and correlate cell types with clinical outcomes may reveal genes and proteins important for cancer growth. "Discovering PD-1 and CTLA4 as important target proteins took 30 years, but when using CIBERSORTx to correlate tumor cell gene expression with therapeutic outcomes, these markers jumped right out at us," Alizadeh said. "We are seeing so many new molecules that could prove interesting", says Newman, "it's a treasure".

Registration Process

CIBERSORTThe database requires a nonprofit organization edu education email address to register, we open the registration address https://cibersort.stanford.edu/register.php

As shown in the figure below:

For Non-Commercial use only. Non-academic and commercial users should go here. If you are a member of an academic or non-commercial organization, please use your organization's email. Personal (e.g. Gmail, Yahoo, Hotmail, etc.) and commercial emails ending in .com will be automatically rejected. Personal (e.g. Gmail, Yahoo, Hotmail, etc.) and commercial emails ending in .com will be automatically rejected.

For non-commercial use only. Non-academic and commercial users should go tohere are. If you are a member of an academic or non-commercial organization, please use your organization's email. Personal (e.g., Gmail, Yahoo, Hotmail, etc.) and commercial e-mail ending in .com will be automatically rejected.

When we fill out the information:

Name section: First Name* Last Name*

Mailbox section: Email* School name: Organization* Your place of work or the research organization you belong to.

City of organization: City* Street of organization: State or Province* Country of organization: Country*

Account Verification

Thank you for registering! Confirmation email has been sent to ljaime4698@xxxx.edu
Please click on the activation link to activate your account. Please note that the link will expire after 1 hour. If your link has expired, you will receive another activation link when you click on the old one.

After we fill out the information and click submit, our edu mailbox will receive a verification email from cibersortx with the subject line: "CIBERSORTx Registration Confirmation".

To activate your account, please click on this link:Activation Link嚗If clicking on this link does not work, please copy the link below and paste it into If clicking on this link does not work, please copy the link below and paste it into your browser.

Audit Success

After we verified, the account is not available for immediate use, it needs to be approved by cibersortx's official manual review and approval, and you can log in and use it only after the manual review and approval is approved, and you will receive a notification email at the same time.We are straight seconds past cibersortx if we use US edu mailboxes.

Approved: CIBERSORTx Account Activation Request

Your request for CIBERSORTx account activation has been approved. You may now log in to the CIBERSORTx website.

Simple to use

The first step is to upload the data, as shown below, by clicking on theMenu-Upload files-Add filesUpload txt data in the format detailed in the example data.

second stepto configure the parameters and prepare to run, clickMenu-Run CIBERSORTx-2.Impute Cell FractionsThe specific configurations are as follows:

To speed up the run, Permutations for significance analysis is selected here 50 times:

third step, and after running it for a while, you can see the results thatMenu-Job ResultsIf you click on the CSV or XLSX to get the predicted result, it will beModule 1of the output data.

E-mail access

This tutorial uses the edumail.vip official purchase platform of the U.S. series of educational mailboxes, lifelong use, free FQ, domestic can log in to use, the platform also help to register cibersortx account in passing, so it is very convenient and fast.

edu education network mailboxes on behalf of the registration on behalf of the application to buy price

CIBERSORTx immune cell infiltration analysis tool edu education email registration application original tutorials

Introduction

Tool Features

Registration Process

Account Verification

Audit Success

Simple to use

E-mail access

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