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
Materials & Equipment Checklist
7 items
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Equipment3
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Protocol Steps
View Abstract
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]
1
Gene expression profiling of melanoma biopsies
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
Not specifiedNot specified
Note: Analysis revealed segregation of samples correlating lymphocyte presence with chemokine gene expression
View evidence from paper
“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”
2
Identify and confirm chemokine expression
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
Not specifiedNot specified
Note: Six specific chemokines were identified as preferentially expressed in T cell-containing tumors
View evidence from paper
“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”
3
Assess chemokine receptor expression on CD8+ T cells
Determine that corresponding chemokine receptors are up-regulated on human CD8+ effector T cells
Not specifiedNot specified
Note: Receptor expression correlates with chemokine presence in tumors
View evidence from paper
“Corresponding chemokine receptors were found to be up-regulated on human CD8+ effector T cells”
4
Perform in vitro transwell migration assays
Conduct transwell migration assays to confirm the ability of each of the six identified chemokines to promote migration of CD8+ effector cells in vitro
Not specifiedNot specified
Note: All six chemokines demonstrated ability to promote CD8+ T cell migration
View evidence from paper
“transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8+ effector cells in vitro”
5
Screen melanoma cell lines for chemokine production
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
Not specifiedNot specified
Note: Selected cell lines produce chemokine profiles matching those in T cell-infiltrated tumors
View evidence from paper
“Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines”
6
Implant melanoma xenografts in immunodeficient mice
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
Not specifiedNot specified
Note: Immunodeficient mice allow human cell xenograft establishment and human T cell recruitment assessment
View evidence from paper
“These melanoma cells more effectively recruited human CD8+ effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo”
7
Perform chemokine blockade experiments
Administer specific chemokine-blocking antibodies to inhibit chemokine signaling and assess the effect on CD8+ T cell migration to xenograft tumors
Not specifiedNot specified
Note: Antibody blockade inhibits CD8+ T cell migration, confirming chemokine role in recruitment
View evidence from paper
“Chemokine blockade with specific antibodies inhibited migration of CD8+ T cells”