Limits...
Effects of epithelial to mesenchymal transition on T cell targeting of melanoma cells.

Woods K, Pasam A, Jayachandran A, Andrews MC, Cebon J - Front Oncol (2014)

Bottom Line: In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells.We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing.However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunobiology Laboratory, Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Olivia Newton John Cancer and Wellness Centre , Melbourne, VIC , Australia ; School of Cancer Medicine, La Trobe University , Melbourne, VIC , Australia.

ABSTRACT
Melanoma cells can switch phenotype in a manner similar to epithelial to mesenchymal transition (EMT). In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells. During the EMT-like switch in phenotype, a concomitant change in expression of multiple tumor antigens occurs. Melanoma cells undergoing EMT escape from killing by T cells specific for antigens whose expression is downregulated by this process. We discuss melanoma antigens whose expression is influenced by EMT. We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing. In addition to escape from T cell immunity via changes in antigen expression, mesenchymal-like melanoma cells are generally more resistant to classical chemotherapy and radiotherapy. However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype. When considering immune therapies such as cancer vaccination, these data suggest escape from T cell killing due to phenotype switching in melanoma could potentially be avoided by careful selection of target antigen.

No MeSH data available.


Related in: MedlinePlus

Antigen expression and T cell-mediated lysis of melanoma cell lines following TGFβ induced EMT. Epithelial-like melanoma cells lines, LM Mel 1a, LM Mel 28, SK Mel 8, and LM Mel 34, and mesenchymal-like cell line LM Mel 53, were incubated with 5 ng/ml TGFβ for 72 h to induce EMT. Cells were lysed, and cDNA was generated (as in Section “Materials and Methods”). (A) Expression levels of N-cadherin in treated and untreated cell lines were determined by qPCR, in order to assess switching to the mesenchymal-like phenotype, and expressed relative to 10,000 copies of β-actin. (B) Immunofluorescence confirmed increase in N-cadherin protein expression (Red) and concomitant reduction in E-cadherin (Green) after 72 h of TGF-β1 treatment, shown here for a representative cell line (LM-Mel 28). Scale bar = 100 μM. Expression levels of (C) Melan-A and (D) NY-ESO-1 were compared between TGFβ treated/untreated melanoma cell lines expressing these antigens. Melanoma cell lines expressing Melan-A, and/or NY-ESO-1 were treated with/without 5 ng/ml TGFβ for 72 h. Specific T cell clones, which recognized the HLA-A*0201 epitopes (E) Melan-A 26–35 or (F) NY-ESO-1 157–165 were incubated with 2 × 104 treated/untreated melanoma cells at a 1:1 effector to target ratio. Cells were incubated for 24 h at 37°C. T cells were washed off, followed by addition of MTS reagent (Promega) and 1 h incubation at 37°C. Absorbance was read at 490 nm, and normalized to untreated control cells for each treatment condition. Each measurement was in triplicate, and treated/untreated samples were compared using a paired T-test. ns = not significant. **P ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4269118&req=5

Figure 2: Antigen expression and T cell-mediated lysis of melanoma cell lines following TGFβ induced EMT. Epithelial-like melanoma cells lines, LM Mel 1a, LM Mel 28, SK Mel 8, and LM Mel 34, and mesenchymal-like cell line LM Mel 53, were incubated with 5 ng/ml TGFβ for 72 h to induce EMT. Cells were lysed, and cDNA was generated (as in Section “Materials and Methods”). (A) Expression levels of N-cadherin in treated and untreated cell lines were determined by qPCR, in order to assess switching to the mesenchymal-like phenotype, and expressed relative to 10,000 copies of β-actin. (B) Immunofluorescence confirmed increase in N-cadherin protein expression (Red) and concomitant reduction in E-cadherin (Green) after 72 h of TGF-β1 treatment, shown here for a representative cell line (LM-Mel 28). Scale bar = 100 μM. Expression levels of (C) Melan-A and (D) NY-ESO-1 were compared between TGFβ treated/untreated melanoma cell lines expressing these antigens. Melanoma cell lines expressing Melan-A, and/or NY-ESO-1 were treated with/without 5 ng/ml TGFβ for 72 h. Specific T cell clones, which recognized the HLA-A*0201 epitopes (E) Melan-A 26–35 or (F) NY-ESO-1 157–165 were incubated with 2 × 104 treated/untreated melanoma cells at a 1:1 effector to target ratio. Cells were incubated for 24 h at 37°C. T cells were washed off, followed by addition of MTS reagent (Promega) and 1 h incubation at 37°C. Absorbance was read at 490 nm, and normalized to untreated control cells for each treatment condition. Each measurement was in triplicate, and treated/untreated samples were compared using a paired T-test. ns = not significant. **P ≤ 0.01.

Mentions: Based on our previous studies (7, 8), we selected four E-like melanoma cell lines, which were HLA-A*0201+, and which were also positive for expression of either Melan-A, or NY-ESO-1, or both. As a control, we also obtained an M-like cell line (LM-Mel 53). The selected lines were treated with TGFβ for 72 h to induce EMT, and successful switch to an M-like phenotype was confirmed by demonstrating upregulation of N-cadherin gene expression by qPCR, or protein expression by immunofluorescence (Figures 2A,B). In all four E-like cell lines, upregulation of N-cadherin expression was observed following TGFβ incubation, whereas there was no significant change in N-cadherin expression by the M-like cell line.


Effects of epithelial to mesenchymal transition on T cell targeting of melanoma cells.

Woods K, Pasam A, Jayachandran A, Andrews MC, Cebon J - Front Oncol (2014)

Antigen expression and T cell-mediated lysis of melanoma cell lines following TGFβ induced EMT. Epithelial-like melanoma cells lines, LM Mel 1a, LM Mel 28, SK Mel 8, and LM Mel 34, and mesenchymal-like cell line LM Mel 53, were incubated with 5 ng/ml TGFβ for 72 h to induce EMT. Cells were lysed, and cDNA was generated (as in Section “Materials and Methods”). (A) Expression levels of N-cadherin in treated and untreated cell lines were determined by qPCR, in order to assess switching to the mesenchymal-like phenotype, and expressed relative to 10,000 copies of β-actin. (B) Immunofluorescence confirmed increase in N-cadherin protein expression (Red) and concomitant reduction in E-cadherin (Green) after 72 h of TGF-β1 treatment, shown here for a representative cell line (LM-Mel 28). Scale bar = 100 μM. Expression levels of (C) Melan-A and (D) NY-ESO-1 were compared between TGFβ treated/untreated melanoma cell lines expressing these antigens. Melanoma cell lines expressing Melan-A, and/or NY-ESO-1 were treated with/without 5 ng/ml TGFβ for 72 h. Specific T cell clones, which recognized the HLA-A*0201 epitopes (E) Melan-A 26–35 or (F) NY-ESO-1 157–165 were incubated with 2 × 104 treated/untreated melanoma cells at a 1:1 effector to target ratio. Cells were incubated for 24 h at 37°C. T cells were washed off, followed by addition of MTS reagent (Promega) and 1 h incubation at 37°C. Absorbance was read at 490 nm, and normalized to untreated control cells for each treatment condition. Each measurement was in triplicate, and treated/untreated samples were compared using a paired T-test. ns = not significant. **P ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269118&req=5

Figure 2: Antigen expression and T cell-mediated lysis of melanoma cell lines following TGFβ induced EMT. Epithelial-like melanoma cells lines, LM Mel 1a, LM Mel 28, SK Mel 8, and LM Mel 34, and mesenchymal-like cell line LM Mel 53, were incubated with 5 ng/ml TGFβ for 72 h to induce EMT. Cells were lysed, and cDNA was generated (as in Section “Materials and Methods”). (A) Expression levels of N-cadherin in treated and untreated cell lines were determined by qPCR, in order to assess switching to the mesenchymal-like phenotype, and expressed relative to 10,000 copies of β-actin. (B) Immunofluorescence confirmed increase in N-cadherin protein expression (Red) and concomitant reduction in E-cadherin (Green) after 72 h of TGF-β1 treatment, shown here for a representative cell line (LM-Mel 28). Scale bar = 100 μM. Expression levels of (C) Melan-A and (D) NY-ESO-1 were compared between TGFβ treated/untreated melanoma cell lines expressing these antigens. Melanoma cell lines expressing Melan-A, and/or NY-ESO-1 were treated with/without 5 ng/ml TGFβ for 72 h. Specific T cell clones, which recognized the HLA-A*0201 epitopes (E) Melan-A 26–35 or (F) NY-ESO-1 157–165 were incubated with 2 × 104 treated/untreated melanoma cells at a 1:1 effector to target ratio. Cells were incubated for 24 h at 37°C. T cells were washed off, followed by addition of MTS reagent (Promega) and 1 h incubation at 37°C. Absorbance was read at 490 nm, and normalized to untreated control cells for each treatment condition. Each measurement was in triplicate, and treated/untreated samples were compared using a paired T-test. ns = not significant. **P ≤ 0.01.
Mentions: Based on our previous studies (7, 8), we selected four E-like melanoma cell lines, which were HLA-A*0201+, and which were also positive for expression of either Melan-A, or NY-ESO-1, or both. As a control, we also obtained an M-like cell line (LM-Mel 53). The selected lines were treated with TGFβ for 72 h to induce EMT, and successful switch to an M-like phenotype was confirmed by demonstrating upregulation of N-cadherin gene expression by qPCR, or protein expression by immunofluorescence (Figures 2A,B). In all four E-like cell lines, upregulation of N-cadherin expression was observed following TGFβ incubation, whereas there was no significant change in N-cadherin expression by the M-like cell line.

Bottom Line: In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells.We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing.However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunobiology Laboratory, Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Olivia Newton John Cancer and Wellness Centre , Melbourne, VIC , Australia ; School of Cancer Medicine, La Trobe University , Melbourne, VIC , Australia.

ABSTRACT
Melanoma cells can switch phenotype in a manner similar to epithelial to mesenchymal transition (EMT). In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells. During the EMT-like switch in phenotype, a concomitant change in expression of multiple tumor antigens occurs. Melanoma cells undergoing EMT escape from killing by T cells specific for antigens whose expression is downregulated by this process. We discuss melanoma antigens whose expression is influenced by EMT. We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing. In addition to escape from T cell immunity via changes in antigen expression, mesenchymal-like melanoma cells are generally more resistant to classical chemotherapy and radiotherapy. However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype. When considering immune therapies such as cancer vaccination, these data suggest escape from T cell killing due to phenotype switching in melanoma could potentially be avoided by careful selection of target antigen.

No MeSH data available.


Related in: MedlinePlus