Source Paper
Chemokine Expression in Melanoma Metastases Associated with CD8+ T-Cell Recruitment
Helena Harlin, Yuru Meng, Amy C. Peterson, Yuanyuan Zha, Maria Tretiakova et al.
Cancer Research • 2009
Source Paper
Helena Harlin, Yuru Meng, Amy C. Peterson, Yuanyuan Zha, Maria Tretiakova et al.
Cancer Research • 2009
Abstract Despite the frequent detection of circulating tumor antigen–specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients. One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites. Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell–associated transcripts. The presence of lymphocytes correlated with the expression of defined chemokine genes. A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells. Corresponding chemokine receptors were found to be up-regulated on human CD8+ effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8+ effector cells in vitro. Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines. These melanoma cells more effectively recruited human CD8+ effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo. Chemokine blockade with specific antibodies inhibited migration of CD8+ T cells. Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumor immunity. [Cancer Res 2009;69(7):3077–85]
Objective: Assess recruitment of human CD8+ effector T cells to melanoma xenografts in vivo and evaluate the role of chemokine expression in T cell migration to tumor sites
This is a Melanoma Xenograft Model protocol using mouse as the model organism. The procedure involves 7 procedural steps, 3 equipment items, 3 materials. Extracted from a 2009 paper published in Cancer Research.
Model and subjects
mouse • nonobese diabetic/severe combined immunodeficient (NOD/SCID) • unknown • Not specified • Not specified
Study window
Estimated timing pending
Core workflow
Gene expression profiling of melanoma biopsies • Identify and confirm chemokine expression • Assess chemokine receptor expression on CD8+ T cells
Primary readouts
Key equipment and reagents
Verified items
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Perform Affymetrix gene expression profiling on a series of metastatic melanoma biopsies to identify samples based on presence or absence of T-cell-associated transcripts
Note: Analysis revealed segregation of samples correlating lymphocyte presence with chemokine gene expression
“Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts”
Use chemokine protein array and/or quantitative reverse transcription-PCR to confirm expression of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) in tumors containing T cells
Note: Six specific chemokines were identified as preferentially expressed in T cell-containing tumors
“A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells”
Determine that corresponding chemokine receptors are up-regulated on human CD8+ effector T cells
Note: Receptor expression correlates with chemokine presence in tumors
“Corresponding chemokine receptors were found to be up-regulated on human CD8+ effector T cells”
Conduct transwell migration assays to confirm the ability of each of the six identified chemokines to promote migration of CD8+ effector cells in vitro
Note: All six chemokines demonstrated ability to promote CD8+ T cell migration
“transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8+ effector cells in vitro”
Use chemokine protein array to screen melanoma cell lines and identify a subset that produces a broad array of chemokines similar to those found in T cell-containing tumors
Note: Selected cell lines produce chemokine profiles matching those in T cell-infiltrated tumors
“Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines”
Implant chemokine-producing melanoma cells as xenografts in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to assess in vivo recruitment of human CD8+ effector T cells
Note: Immunodeficient mice allow human cell xenograft establishment and human T cell recruitment assessment
“These melanoma cells more effectively recruited human CD8+ effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo”
Administer specific chemokine-blocking antibodies to inhibit chemokine signaling and assess the effect on CD8+ T cell migration to xenograft tumors
Note: Antibody blockade inhibits CD8+ T cell migration, confirming chemokine role in recruitment
“Chemokine blockade with specific antibodies inhibited migration of CD8+ T cells”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Assess recruitment of human CD8+ effector T cells to melanoma xenografts in vivo and evaluate the role of chemokine expression in T cell migration to tumor sites
Objective
Assess recruitment of human CD8+ effector T cells to melanoma xenografts in vivo and evaluate the role of chemokine expression in T cell migration to tumor sites
Subjects
From papermouse • nonobese diabetic/severe combined immunodeficient (NOD/SCID) • unknown • Not specified • Not specified
Cohort notes
From paperImmunodeficient mice used to allow human cell xenograft implantation
Gene expression profiling of melanoma biopsies (Not specified)
Identify and confirm chemokine expression (Not specified)
Assess chemokine receptor expression on CD8+ T cells (Not specified)
Perform in vitro transwell migration assays (Not specified)
Presence or absence of T-cell-associated transcripts in melanoma biopsies
From paperGene expression profiling data analyzed to identify segregation of samples based on T-cell-associated transcripts; chemokine expression confirmed by protein array and quantitative reverse transcription-PCR; transwell migration assays quantified T cell migration responses
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Expression levels of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10) in tumors
From paperGene expression profiling data analyzed to identify segregation of samples based on T-cell-associated transcripts; chemokine expression confirmed by protein array and quantitative reverse transcription-PCR; transwell migration assays quantified T cell migration responses
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Chemokine receptor expression on human CD8+ effector T cells
From paperGene expression profiling data analyzed to identify segregation of samples based on T-cell-associated transcripts; chemokine expression confirmed by protein array and quantitative reverse transcription-PCR; transwell migration assays quantified T cell migration responses
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
In vitro CD8+ T cell migration in response to individual chemokines
From paperGene expression profiling data analyzed to identify segregation of samples based on T-cell-associated transcripts; chemokine expression confirmed by protein array and quantitative reverse transcription-PCR; transwell migration assays quantified T cell migration responses
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Presence or absence of T-cell-associated transcripts in melanoma biopsies
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Expression levels of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10) in tumors
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Chemokine receptor expression on human CD8+ effector T cells
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
In vitro CD8+ T cell migration in response to individual chemokines
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Gene expression profiling data analyzed to identify segregation of samples based on T-cell-associated transcripts; chemokine expression confirmed by protein array and quantitative reverse transcription-PCR; transwell migration assays quantified T cell migration responses
Scoring or quantification
Quantify the primary readouts for this experiment: Presence or absence of T-cell-associated transcripts in melanoma biopsies; Expression levels of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10) in tumors; Chemokine receptor expression on human CD8+ effector T cells; In vitro CD8+ T cell migration in response to individual chemokines.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Presence or absence of T-cell-associated transcripts in melanoma biopsies, Expression levels of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10) in tumors, Chemokine receptor expression on human CD8+ effector T cells, In vitro CD8+ T cell migration in response to individual chemokines.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Helena Harlin et al. (2009). Chemokine Expression in Melanoma Metastases Associated with CD8+ T-Cell Recruitment. Cancer Research
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