The Role of Melanoma Phenotype on Immune-Cell Surveillance and Lymph Node Metastasis
The Role of Melanoma Phenotype on Immune-Cell Surveillance and Lymph Node Metastasis
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DissertationDate of Exam
10.09.2026Date of Publication
29.09.2026Advisor
Hölzel, MichaelCo-Referee
Gebhardt, ThomasMetadata
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Abstract
Melanoma is the most aggressive form of skin cancer. MITF is a transcription factor involved in melanocytic differentiation and melanoma progression. In recent years, different melanoma phenotypes have been described, which can be classified as cells with high MITF or low MITF expression. Different therapies are currently used to treat melanoma, which include targeted therapies and immunotherapies. However, resistance develops and melanoma often relapses.
One such resistance mechanism that develops is the result of phenotype switching. In the tumor microenvironment, melanoma cells can progress from a differentiated proliferative phenotype with high MITF expression to a de-differentiated invasive phenotype with low MITF expression and this switch between the different states leads to melanoma progression. Better understanding the molecular regulation of these melanoma phenotypes could therefore inform on resistance mechanisms and immune-cell interactions promoting immune escape.
It has been difficult to generate melanoma cells with stable phenotypes due to the dynamic shift in melanoma cell states as a result of transcriptional reprogramming in response to cues within the tumor microenvironment. However, our laboratory has recently used a CRISRP-Cas9 based prime editing technique to insert clinically relevant oncogenic point mutations in TRP53 and NRAS genes leading to amino acid substitutions at sites Trp53 Y217C and Nras Q61K in the B16F1 murine cell line. This yielded stable and distinct melanoma cells of differentiated and de-differentiated phenotypes with high MITF and low MITF expression, respectively. Thus, the aim of this project was to use a suite of clones representing different phenotypes in combination with an in vivo epicutaneous melanoma model to understand how melanoma phenotypes impact on immune surveillance and tumor growth kinetics.
We first aimed at understanding the behavior of melanoma cells with different phenotypes in vitro. Our results indicate that melanoma clones with de-differentiated phenotypes were less sensitive to IFNγ treatment compared to differentiated clones that underwent more pronounced cell death upon IFNγ exposure. As we observed melanoma phenotype-specific differences in response to inflammatory cytokines, we performed transcriptomic analysis using 3'mRNA-sequencing on the clones and showed a higher inflammatory pathway activity in cells with de-differentiated phenotypes. Furthermore, we analyzed the expression of MHC-I and PD-L1 molecules involved in antigen presentation and immune regulation. Our results showed that the de-differentiated clones displayed higher expression of MHC-I upon IFNγ stimulation. Next, we generated melanoma clones expressing the gB antigen from herpes simplex virus as a model antigen and exposed these to gB-specific, transgenic gBT-I CD8+ T cells in co-culture assays. This revealed that all gB-expressing melanoma clones, regardless of their phenotypes, were equally targeted and killed by CD8+ T cells. However, killing of differentiated melanoma clones was dominated by perforin-mediated cytotoxicity, whereas elaboration of this killing pathway alone was not sufficient to eliminate melanoma clones with de-differentiated phenotypes.
Given these phenotype-specific differences in responding to immune attack in vitro, we analyzed tumor kinetics of differentiated and de-differentiated melanoma clones in vivo using a mouse model involving epicutaneous challenge with melanoma lines. To allow for longitudinal melanoma tracking via bioluminescence imaging, we transduced the cell lines with different constructs to express firefly luciferase. We then chose a matched pair of differentiated and de-differentiated clones and inoculated the cells to assess tumor growth and lymph node metastasis. This revealed that regardless of their phenotype, both clones were immunogenic as few tumors developed in WT C57Bl/6 mice and the majority of mice also remained free of metastatic spread. In contrast, severely immunodeficient Rag2–/–;Il2rg–/– mice readily developed cutaneous melanoma after challenge with differentiated and de-differentiated melanoma lines.
Overall, we demonstrated that melanoma phenotypes impact inflammatory response and immune surveillance in vitro with de-differentiated cells showing an increased inflammatory pathway activity and a different response to immune attack. This could potentially alter immune-cell dynamics in vivo, as we found delayed tumor growth with inoculated cells of de-differentiated phenotype, although we observed few tumors from inoculated cells of both phenotypes. The presented work serves as a first step towards understanding melanoma plasticity using a model of cells with stable phenotypes to study tumor growth kinetics via epicutaneous challenge by inoculating cells at the uppermost layer of the epidermis. This serves as an analogy to melanoma progression in patients and therefore the model could provide deeper insights into reducing resistance mechanisms to drug exposure potentially speculating disease progression and improving drug timing, dosage, and treatment options to improve patient care.
One such resistance mechanism that develops is the result of phenotype switching. In the tumor microenvironment, melanoma cells can progress from a differentiated proliferative phenotype with high MITF expression to a de-differentiated invasive phenotype with low MITF expression and this switch between the different states leads to melanoma progression. Better understanding the molecular regulation of these melanoma phenotypes could therefore inform on resistance mechanisms and immune-cell interactions promoting immune escape.
It has been difficult to generate melanoma cells with stable phenotypes due to the dynamic shift in melanoma cell states as a result of transcriptional reprogramming in response to cues within the tumor microenvironment. However, our laboratory has recently used a CRISRP-Cas9 based prime editing technique to insert clinically relevant oncogenic point mutations in TRP53 and NRAS genes leading to amino acid substitutions at sites Trp53 Y217C and Nras Q61K in the B16F1 murine cell line. This yielded stable and distinct melanoma cells of differentiated and de-differentiated phenotypes with high MITF and low MITF expression, respectively. Thus, the aim of this project was to use a suite of clones representing different phenotypes in combination with an in vivo epicutaneous melanoma model to understand how melanoma phenotypes impact on immune surveillance and tumor growth kinetics.
We first aimed at understanding the behavior of melanoma cells with different phenotypes in vitro. Our results indicate that melanoma clones with de-differentiated phenotypes were less sensitive to IFNγ treatment compared to differentiated clones that underwent more pronounced cell death upon IFNγ exposure. As we observed melanoma phenotype-specific differences in response to inflammatory cytokines, we performed transcriptomic analysis using 3'mRNA-sequencing on the clones and showed a higher inflammatory pathway activity in cells with de-differentiated phenotypes. Furthermore, we analyzed the expression of MHC-I and PD-L1 molecules involved in antigen presentation and immune regulation. Our results showed that the de-differentiated clones displayed higher expression of MHC-I upon IFNγ stimulation. Next, we generated melanoma clones expressing the gB antigen from herpes simplex virus as a model antigen and exposed these to gB-specific, transgenic gBT-I CD8+ T cells in co-culture assays. This revealed that all gB-expressing melanoma clones, regardless of their phenotypes, were equally targeted and killed by CD8+ T cells. However, killing of differentiated melanoma clones was dominated by perforin-mediated cytotoxicity, whereas elaboration of this killing pathway alone was not sufficient to eliminate melanoma clones with de-differentiated phenotypes.
Given these phenotype-specific differences in responding to immune attack in vitro, we analyzed tumor kinetics of differentiated and de-differentiated melanoma clones in vivo using a mouse model involving epicutaneous challenge with melanoma lines. To allow for longitudinal melanoma tracking via bioluminescence imaging, we transduced the cell lines with different constructs to express firefly luciferase. We then chose a matched pair of differentiated and de-differentiated clones and inoculated the cells to assess tumor growth and lymph node metastasis. This revealed that regardless of their phenotype, both clones were immunogenic as few tumors developed in WT C57Bl/6 mice and the majority of mice also remained free of metastatic spread. In contrast, severely immunodeficient Rag2–/–;Il2rg–/– mice readily developed cutaneous melanoma after challenge with differentiated and de-differentiated melanoma lines.
Overall, we demonstrated that melanoma phenotypes impact inflammatory response and immune surveillance in vitro with de-differentiated cells showing an increased inflammatory pathway activity and a different response to immune attack. This could potentially alter immune-cell dynamics in vivo, as we found delayed tumor growth with inoculated cells of de-differentiated phenotype, although we observed few tumors from inoculated cells of both phenotypes. The presented work serves as a first step towards understanding melanoma plasticity using a model of cells with stable phenotypes to study tumor growth kinetics via epicutaneous challenge by inoculating cells at the uppermost layer of the epidermis. This serves as an analogy to melanoma progression in patients and therefore the model could provide deeper insights into reducing resistance mechanisms to drug exposure potentially speculating disease progression and improving drug timing, dosage, and treatment options to improve patient care.
Subjects
Oncology, Immunology, Melanoma, Melanoma Plasticity, Melanoma Phenotypes
Classification (DDC)
570 Biowissenschaften, BiologieAbdelAziz, Farah: The Role of Melanoma Phenotype on Immune-Cell Surveillance and Lymph Node Metastasis. - Bonn, 2026. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn, University of Melbourne.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-93058
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-93058
@phdthesis{handle:20.500.11811/14554,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-93058,
author = {{Farah AbdelAziz}},
title = {The Role of Melanoma Phenotype on Immune-Cell Surveillance and Lymph Node Metastasis},
school = {{Rheinische Friedrich-Wilhelms-Universität Bonn} and {University of Melbourne}},
year = 2026,
month = sep,
note = {Melanoma is the most aggressive form of skin cancer. MITF is a transcription factor involved in melanocytic differentiation and melanoma progression. In recent years, different melanoma phenotypes have been described, which can be classified as cells with high MITF or low MITF expression. Different therapies are currently used to treat melanoma, which include targeted therapies and immunotherapies. However, resistance develops and melanoma often relapses.
One such resistance mechanism that develops is the result of phenotype switching. In the tumor microenvironment, melanoma cells can progress from a differentiated proliferative phenotype with high MITF expression to a de-differentiated invasive phenotype with low MITF expression and this switch between the different states leads to melanoma progression. Better understanding the molecular regulation of these melanoma phenotypes could therefore inform on resistance mechanisms and immune-cell interactions promoting immune escape.
It has been difficult to generate melanoma cells with stable phenotypes due to the dynamic shift in melanoma cell states as a result of transcriptional reprogramming in response to cues within the tumor microenvironment. However, our laboratory has recently used a CRISRP-Cas9 based prime editing technique to insert clinically relevant oncogenic point mutations in TRP53 and NRAS genes leading to amino acid substitutions at sites Trp53 Y217C and Nras Q61K in the B16F1 murine cell line. This yielded stable and distinct melanoma cells of differentiated and de-differentiated phenotypes with high MITF and low MITF expression, respectively. Thus, the aim of this project was to use a suite of clones representing different phenotypes in combination with an in vivo epicutaneous melanoma model to understand how melanoma phenotypes impact on immune surveillance and tumor growth kinetics.
We first aimed at understanding the behavior of melanoma cells with different phenotypes in vitro. Our results indicate that melanoma clones with de-differentiated phenotypes were less sensitive to IFNγ treatment compared to differentiated clones that underwent more pronounced cell death upon IFNγ exposure. As we observed melanoma phenotype-specific differences in response to inflammatory cytokines, we performed transcriptomic analysis using 3'mRNA-sequencing on the clones and showed a higher inflammatory pathway activity in cells with de-differentiated phenotypes. Furthermore, we analyzed the expression of MHC-I and PD-L1 molecules involved in antigen presentation and immune regulation. Our results showed that the de-differentiated clones displayed higher expression of MHC-I upon IFNγ stimulation. Next, we generated melanoma clones expressing the gB antigen from herpes simplex virus as a model antigen and exposed these to gB-specific, transgenic gBT-I CD8+ T cells in co-culture assays. This revealed that all gB-expressing melanoma clones, regardless of their phenotypes, were equally targeted and killed by CD8+ T cells. However, killing of differentiated melanoma clones was dominated by perforin-mediated cytotoxicity, whereas elaboration of this killing pathway alone was not sufficient to eliminate melanoma clones with de-differentiated phenotypes.
Given these phenotype-specific differences in responding to immune attack in vitro, we analyzed tumor kinetics of differentiated and de-differentiated melanoma clones in vivo using a mouse model involving epicutaneous challenge with melanoma lines. To allow for longitudinal melanoma tracking via bioluminescence imaging, we transduced the cell lines with different constructs to express firefly luciferase. We then chose a matched pair of differentiated and de-differentiated clones and inoculated the cells to assess tumor growth and lymph node metastasis. This revealed that regardless of their phenotype, both clones were immunogenic as few tumors developed in WT C57Bl/6 mice and the majority of mice also remained free of metastatic spread. In contrast, severely immunodeficient Rag2–/–;Il2rg–/– mice readily developed cutaneous melanoma after challenge with differentiated and de-differentiated melanoma lines.
Overall, we demonstrated that melanoma phenotypes impact inflammatory response and immune surveillance in vitro with de-differentiated cells showing an increased inflammatory pathway activity and a different response to immune attack. This could potentially alter immune-cell dynamics in vivo, as we found delayed tumor growth with inoculated cells of de-differentiated phenotype, although we observed few tumors from inoculated cells of both phenotypes. The presented work serves as a first step towards understanding melanoma plasticity using a model of cells with stable phenotypes to study tumor growth kinetics via epicutaneous challenge by inoculating cells at the uppermost layer of the epidermis. This serves as an analogy to melanoma progression in patients and therefore the model could provide deeper insights into reducing resistance mechanisms to drug exposure potentially speculating disease progression and improving drug timing, dosage, and treatment options to improve patient care.},
url = {https://hdl.handle.net/20.500.11811/14554}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-93058,
author = {{Farah AbdelAziz}},
title = {The Role of Melanoma Phenotype on Immune-Cell Surveillance and Lymph Node Metastasis},
school = {{Rheinische Friedrich-Wilhelms-Universität Bonn} and {University of Melbourne}},
year = 2026,
month = sep,
note = {Melanoma is the most aggressive form of skin cancer. MITF is a transcription factor involved in melanocytic differentiation and melanoma progression. In recent years, different melanoma phenotypes have been described, which can be classified as cells with high MITF or low MITF expression. Different therapies are currently used to treat melanoma, which include targeted therapies and immunotherapies. However, resistance develops and melanoma often relapses.
One such resistance mechanism that develops is the result of phenotype switching. In the tumor microenvironment, melanoma cells can progress from a differentiated proliferative phenotype with high MITF expression to a de-differentiated invasive phenotype with low MITF expression and this switch between the different states leads to melanoma progression. Better understanding the molecular regulation of these melanoma phenotypes could therefore inform on resistance mechanisms and immune-cell interactions promoting immune escape.
It has been difficult to generate melanoma cells with stable phenotypes due to the dynamic shift in melanoma cell states as a result of transcriptional reprogramming in response to cues within the tumor microenvironment. However, our laboratory has recently used a CRISRP-Cas9 based prime editing technique to insert clinically relevant oncogenic point mutations in TRP53 and NRAS genes leading to amino acid substitutions at sites Trp53 Y217C and Nras Q61K in the B16F1 murine cell line. This yielded stable and distinct melanoma cells of differentiated and de-differentiated phenotypes with high MITF and low MITF expression, respectively. Thus, the aim of this project was to use a suite of clones representing different phenotypes in combination with an in vivo epicutaneous melanoma model to understand how melanoma phenotypes impact on immune surveillance and tumor growth kinetics.
We first aimed at understanding the behavior of melanoma cells with different phenotypes in vitro. Our results indicate that melanoma clones with de-differentiated phenotypes were less sensitive to IFNγ treatment compared to differentiated clones that underwent more pronounced cell death upon IFNγ exposure. As we observed melanoma phenotype-specific differences in response to inflammatory cytokines, we performed transcriptomic analysis using 3'mRNA-sequencing on the clones and showed a higher inflammatory pathway activity in cells with de-differentiated phenotypes. Furthermore, we analyzed the expression of MHC-I and PD-L1 molecules involved in antigen presentation and immune regulation. Our results showed that the de-differentiated clones displayed higher expression of MHC-I upon IFNγ stimulation. Next, we generated melanoma clones expressing the gB antigen from herpes simplex virus as a model antigen and exposed these to gB-specific, transgenic gBT-I CD8+ T cells in co-culture assays. This revealed that all gB-expressing melanoma clones, regardless of their phenotypes, were equally targeted and killed by CD8+ T cells. However, killing of differentiated melanoma clones was dominated by perforin-mediated cytotoxicity, whereas elaboration of this killing pathway alone was not sufficient to eliminate melanoma clones with de-differentiated phenotypes.
Given these phenotype-specific differences in responding to immune attack in vitro, we analyzed tumor kinetics of differentiated and de-differentiated melanoma clones in vivo using a mouse model involving epicutaneous challenge with melanoma lines. To allow for longitudinal melanoma tracking via bioluminescence imaging, we transduced the cell lines with different constructs to express firefly luciferase. We then chose a matched pair of differentiated and de-differentiated clones and inoculated the cells to assess tumor growth and lymph node metastasis. This revealed that regardless of their phenotype, both clones were immunogenic as few tumors developed in WT C57Bl/6 mice and the majority of mice also remained free of metastatic spread. In contrast, severely immunodeficient Rag2–/–;Il2rg–/– mice readily developed cutaneous melanoma after challenge with differentiated and de-differentiated melanoma lines.
Overall, we demonstrated that melanoma phenotypes impact inflammatory response and immune surveillance in vitro with de-differentiated cells showing an increased inflammatory pathway activity and a different response to immune attack. This could potentially alter immune-cell dynamics in vivo, as we found delayed tumor growth with inoculated cells of de-differentiated phenotype, although we observed few tumors from inoculated cells of both phenotypes. The presented work serves as a first step towards understanding melanoma plasticity using a model of cells with stable phenotypes to study tumor growth kinetics via epicutaneous challenge by inoculating cells at the uppermost layer of the epidermis. This serves as an analogy to melanoma progression in patients and therefore the model could provide deeper insights into reducing resistance mechanisms to drug exposure potentially speculating disease progression and improving drug timing, dosage, and treatment options to improve patient care.},
url = {https://hdl.handle.net/20.500.11811/14554}
}
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