Periodic Reporting for period 1 - PDT-NEO-immunother (Generation of tumor neoantigens with photodynamic therapy: a new strategy for anticancer vaccines to fight head and neck cancer)
Periodo di rendicontazione: 2023-12-01 al 2025-11-30
Head and neck squamous cell carcinomas (HNSCC) represent a major global health challenge, ranking among the most common cancers in men, with nearly 900,000 new cases and 450,000 deaths annually worldwide. Despite advances in treatment, including surgery combined with radiotherapy or chemotherapy, outcomes for patients with advanced disease remain poor, with treatment failure occurring in up to 40% of cases. This highlights a critical unmet need for more effective and durable therapeutic strategies capable of overcoming tumor resistance and preventing relapse.
In this context, there is growing interest in the development of more selective and personalized cancer therapies that specifically target tumor cells while minimizing toxicity to healthy tissues. One promising approach is immunotherapy, which works by helping the body’s own immune system recognize and attack cancer. This approach can be even more effective when combined with treatments that actively stimulate immune responses against tumors.
An important concept in this field is immunogenic cell death (ICD). This is a special type of cancer cell death that not only destroys tumor cells but also activates the immune system. ICD works through two key components. First, it provides adjuvanticity, meaning that dying cancer cells release “danger signals” that alert and stimulate the immune system. Second, it provides antigenicity, meaning that cancer cells carry specific markers (antigens) that the immune system can recognize and target. Both components are essential: the danger signals activate the immune response, while the antigens guide the immune system to specifically recognize and attack cancer cells. While the role of these danger signals is well established, it is still unclear whether ICD also enhances antigenicity, for example, by increasing the amount or diversity of tumor-specific antigens, including newly formed ones during treatment.
The overall objective of this project is to investigate whether ICD induction promotes increased antigenicity of tumor cells and to exploit this mechanism for the development of personalized cancer vaccines. To this end, photodynamic therapy (PDT) has been selected as a clinically relevant and controllable method to induce ICD. PDT is a minimally invasive treatment that involves administering a light-sensitive drug (called photosensitizer) that accumulates in tumor cells and is then activated by a specific wavelength of light. This activation produces reactive oxygen species that damage and kill the cancer cells.
PDT has demonstrated strong immunogenic potential and offers a unique opportunity to locally trigger tumor cell death while also influencing the immune system. By using PDT as a tool to induce ICD, the project aims to determine how this process reshapes the neoantigen repertoire of cancer cells and whether it can enhance their capacity to stimulate effective anti-tumor immune responses.
The project follows a structured pathway to impact. First, it aims to characterize the effects of PDT-induced ICD on both adjuvanticity and antigenicity of HNSCC cells, with a particular focus on understanding how therapy influences the tumor antigen landscape. Importantly, the project explores how different types or combinations of antigens could contribute to more effective immune activation, providing insights that can guide future vaccine design strategies. Finally, the project lays the groundwork for the development of personalized vaccination approaches, including dendritic cell-based and mRNA-based platforms, by identifying promising antigenic targets and defining strategies for their potential use.
Beyond HNSCC, this project is expected to generate knowledge and methodologies that are transferable to other tumor types. By establishing a strategy to enhance antigenicity through ICD induction and to identify actionable neoantigens, the project contributes to a broader framework for precision immunotherapy. Its outcomes could therefore have significant implications not only for improving treatment responses and preventing relapse in HNSCC patients, but also for advancing personalized cancer vaccination strategies across oncology.
Overall, this project aims to bridge a critical gap in current cancer immunotherapy by linking ICD induction to enhanced antigenicity and vaccine design, setting the foundation for more effective and widely applicable anti-cancer treatments.
In this context, there is growing interest in the development of more selective and personalized cancer therapies that specifically target tumor cells while minimizing toxicity to healthy tissues. One promising approach is immunotherapy, which works by helping the body’s own immune system recognize and attack cancer. This approach can be even more effective when combined with treatments that actively stimulate immune responses against tumors.
An important concept in this field is immunogenic cell death (ICD). This is a special type of cancer cell death that not only destroys tumor cells but also activates the immune system. ICD works through two key components. First, it provides adjuvanticity, meaning that dying cancer cells release “danger signals” that alert and stimulate the immune system. Second, it provides antigenicity, meaning that cancer cells carry specific markers (antigens) that the immune system can recognize and target. Both components are essential: the danger signals activate the immune response, while the antigens guide the immune system to specifically recognize and attack cancer cells. While the role of these danger signals is well established, it is still unclear whether ICD also enhances antigenicity, for example, by increasing the amount or diversity of tumor-specific antigens, including newly formed ones during treatment.
The overall objective of this project is to investigate whether ICD induction promotes increased antigenicity of tumor cells and to exploit this mechanism for the development of personalized cancer vaccines. To this end, photodynamic therapy (PDT) has been selected as a clinically relevant and controllable method to induce ICD. PDT is a minimally invasive treatment that involves administering a light-sensitive drug (called photosensitizer) that accumulates in tumor cells and is then activated by a specific wavelength of light. This activation produces reactive oxygen species that damage and kill the cancer cells.
PDT has demonstrated strong immunogenic potential and offers a unique opportunity to locally trigger tumor cell death while also influencing the immune system. By using PDT as a tool to induce ICD, the project aims to determine how this process reshapes the neoantigen repertoire of cancer cells and whether it can enhance their capacity to stimulate effective anti-tumor immune responses.
The project follows a structured pathway to impact. First, it aims to characterize the effects of PDT-induced ICD on both adjuvanticity and antigenicity of HNSCC cells, with a particular focus on understanding how therapy influences the tumor antigen landscape. Importantly, the project explores how different types or combinations of antigens could contribute to more effective immune activation, providing insights that can guide future vaccine design strategies. Finally, the project lays the groundwork for the development of personalized vaccination approaches, including dendritic cell-based and mRNA-based platforms, by identifying promising antigenic targets and defining strategies for their potential use.
Beyond HNSCC, this project is expected to generate knowledge and methodologies that are transferable to other tumor types. By establishing a strategy to enhance antigenicity through ICD induction and to identify actionable neoantigens, the project contributes to a broader framework for precision immunotherapy. Its outcomes could therefore have significant implications not only for improving treatment responses and preventing relapse in HNSCC patients, but also for advancing personalized cancer vaccination strategies across oncology.
Overall, this project aims to bridge a critical gap in current cancer immunotherapy by linking ICD induction to enhanced antigenicity and vaccine design, setting the foundation for more effective and widely applicable anti-cancer treatments.
The work carried out in this project focused on investigating the immunogenic potential of photodynamic therapy (PDT) and other immunogenic cell death (ICD) inducers, with the aim of understanding their ability to enhance tumor antigenicity and support the development of personalized cancer vaccines.
A major part of the project was dedicated to studying the clinically approved photosensitizer temoporfin, which is currently used mainly in palliative settings or for resistant tumors. Given its established clinical use and promising therapeutic outcomes, we aimed to evaluate whether temoporfin-based PDT can effectively induce ICD and modulate tumor antigenicity, potentially supporting its repositioning as a first-line or adjuvant therapy.
To address this, we first optimized the conditions required to induce ICD in head and neck cancer cell lines using temoporfin. Specifically, we systematically evaluated key parameters including drug concentration, incubation time prior to irradiation, and light exposure conditions (fluence and total light dose). This optimization allowed us to define conditions under which temoporfin-PDT efficiently induces ICD and to assess how these parameters influence immunogenic responses.
Before investigating the generation of new tumor-specific antigens, we first evaluated whether temoporfin-based PDT could cause mutations in cancer cells. This step was important because some types of antigens, called neoantigens, are created as a result of genetic mutations. For these neoantigens to be meaningful targets for the immune system, the mutations need to be stable and passed on as cancer cells divide. To assess this, we used a standard test called the micronucleus assay, which detects signs of DNA damage and mutation in cells. Under our experimental conditions, we found no evidence that temoporfin-PDT induces mutations. This result indicated that the treatment is unlikely to generate neoantigens through genetic changes.
Based on these findings, we shifted our focus to alternative ways in which antigens can be modified. In particular, we decided to investigate post-translational modifications (PTMs), which are chemical changes that occur to proteins after they are produced by the cell. One example is protein oxidation, a process that is known to occur during PDT due to the production of reactive oxygen species. These modifications can alter antigen structure and potentially make them more visible or recognizable to the immune system, thereby increasing their immunogenicity.
At the same time, we also explored whether other treatments that induce ICD could help generate new tumor-specific antigens. We tested two different compounds, mitoxantrone and RSL3, which kill cancer cells in ways that can also activate the immune system. Our goal was to see whether these treatments could change the types of antigens presented by cancer cells.
Using advanced techniques to analyze gene activity, we identified potential new antigens that might be recognized by the immune system. To understand whether these antigens could actually trigger an immune response, we carried out functional tests using dendritic cells (DCs), which are key immune cells responsible for activating other immune cells.
In these experiments, DCs were exposed to cancer cells that had been treated to undergo ICD, allowing them to “pick up” tumor antigens and use them to prevent and cure glioblastoma in mice. We then assessed whether this process could stimulate an immune response. This approach allowed us to test the potential of these treatments to activate the immune system before moving on to more complex and time-consuming steps, such as producing and validating individual antigens.
Overall, these activities have established robust experimental platforms to study ICD-induced antigenicity and have provided key insights into both mutation-dependent and mutation-independent mechanisms of tumor antigen modulation, as well as a proof of concept for ICD-based personalized cancer vaccination.
A major part of the project was dedicated to studying the clinically approved photosensitizer temoporfin, which is currently used mainly in palliative settings or for resistant tumors. Given its established clinical use and promising therapeutic outcomes, we aimed to evaluate whether temoporfin-based PDT can effectively induce ICD and modulate tumor antigenicity, potentially supporting its repositioning as a first-line or adjuvant therapy.
To address this, we first optimized the conditions required to induce ICD in head and neck cancer cell lines using temoporfin. Specifically, we systematically evaluated key parameters including drug concentration, incubation time prior to irradiation, and light exposure conditions (fluence and total light dose). This optimization allowed us to define conditions under which temoporfin-PDT efficiently induces ICD and to assess how these parameters influence immunogenic responses.
Before investigating the generation of new tumor-specific antigens, we first evaluated whether temoporfin-based PDT could cause mutations in cancer cells. This step was important because some types of antigens, called neoantigens, are created as a result of genetic mutations. For these neoantigens to be meaningful targets for the immune system, the mutations need to be stable and passed on as cancer cells divide. To assess this, we used a standard test called the micronucleus assay, which detects signs of DNA damage and mutation in cells. Under our experimental conditions, we found no evidence that temoporfin-PDT induces mutations. This result indicated that the treatment is unlikely to generate neoantigens through genetic changes.
Based on these findings, we shifted our focus to alternative ways in which antigens can be modified. In particular, we decided to investigate post-translational modifications (PTMs), which are chemical changes that occur to proteins after they are produced by the cell. One example is protein oxidation, a process that is known to occur during PDT due to the production of reactive oxygen species. These modifications can alter antigen structure and potentially make them more visible or recognizable to the immune system, thereby increasing their immunogenicity.
At the same time, we also explored whether other treatments that induce ICD could help generate new tumor-specific antigens. We tested two different compounds, mitoxantrone and RSL3, which kill cancer cells in ways that can also activate the immune system. Our goal was to see whether these treatments could change the types of antigens presented by cancer cells.
Using advanced techniques to analyze gene activity, we identified potential new antigens that might be recognized by the immune system. To understand whether these antigens could actually trigger an immune response, we carried out functional tests using dendritic cells (DCs), which are key immune cells responsible for activating other immune cells.
In these experiments, DCs were exposed to cancer cells that had been treated to undergo ICD, allowing them to “pick up” tumor antigens and use them to prevent and cure glioblastoma in mice. We then assessed whether this process could stimulate an immune response. This approach allowed us to test the potential of these treatments to activate the immune system before moving on to more complex and time-consuming steps, such as producing and validating individual antigens.
Overall, these activities have established robust experimental platforms to study ICD-induced antigenicity and have provided key insights into both mutation-dependent and mutation-independent mechanisms of tumor antigen modulation, as well as a proof of concept for ICD-based personalized cancer vaccination.
This project goes significantly beyond the current state of the art by redefining how tumor antigenicity can be generated and exploited for cancer immunotherapy. In particular, it lays the groundwork for a new class of personalized cancer vaccines based on ICD-induced antigen modulation, with potential applicability across multiple cancer types and significant implications for the future of precision oncology.
First, we demonstrate that temoporfin-based PDT induces immunogenic responses in head and neck cancer models. Importantly, our data suggest that its immunogenicity may not be driven solely by mutagenesis but is also likely associated with post-translational modifications of tumor antigens, such as oxidative changes induced during PDT. This represents a conceptual advance, as it highlights an alternative and previously underexplored mechanism to enhance antigenicity without relying on genetic alterations.
In parallel, we show that different ICD inducers, including non-PDT-based approaches, can reshape the antigenic landscape of tumor cells. These findings support the broader hypothesis that ICD induction, regardless of the modality, can be strategically exploited to enhance tumor antigenicity and improve immune recognition. Importantly, we have developed and tested a prototype anti-tumor vaccine based on ICD-treated cells, demonstrating the translational potential of this approach.
Looking forward, several key steps are required to ensure further uptake and impact of these results. From a research perspective, further work is needed to characterize the immunogenic epitopes generated through post-translational modifications fully and to validate their relevance across different tumor types.
From an innovation and impact perspective, the project opens a new avenue for the development of personalized cancer vaccines based not only on genetically derived neoantigens but also on treatment-induced antigenic modifications. This significantly expands the pool of targetable antigens and enables more flexible and patient-specific therapeutic strategies, including the use of single antigens or antigen combinations to maximize immune responses.
To ensure further uptake and exploitation, several key actions are underway. We are actively exploring the creation of a spin-off company to advance the development, validation, and commercialization of ICD-based cancer vaccines. The use of clinically approved compounds such as temoporfin or mitoxantrone provides a strong advantage for regulatory translation and market entry, potentially shortening the pathway to clinical implementation. Future work will focus on preclinical validation across tumor types, scalability of vaccine production, and alignment with regulatory and standardization frameworks.
Overall, the project delivers a high-impact contribution to the field by demonstrating the translational feasibility of ICD-based vaccination strategies and laying the foundation for a new generation of broadly applicable, personalized cancer immunotherapies.
First, we demonstrate that temoporfin-based PDT induces immunogenic responses in head and neck cancer models. Importantly, our data suggest that its immunogenicity may not be driven solely by mutagenesis but is also likely associated with post-translational modifications of tumor antigens, such as oxidative changes induced during PDT. This represents a conceptual advance, as it highlights an alternative and previously underexplored mechanism to enhance antigenicity without relying on genetic alterations.
In parallel, we show that different ICD inducers, including non-PDT-based approaches, can reshape the antigenic landscape of tumor cells. These findings support the broader hypothesis that ICD induction, regardless of the modality, can be strategically exploited to enhance tumor antigenicity and improve immune recognition. Importantly, we have developed and tested a prototype anti-tumor vaccine based on ICD-treated cells, demonstrating the translational potential of this approach.
Looking forward, several key steps are required to ensure further uptake and impact of these results. From a research perspective, further work is needed to characterize the immunogenic epitopes generated through post-translational modifications fully and to validate their relevance across different tumor types.
From an innovation and impact perspective, the project opens a new avenue for the development of personalized cancer vaccines based not only on genetically derived neoantigens but also on treatment-induced antigenic modifications. This significantly expands the pool of targetable antigens and enables more flexible and patient-specific therapeutic strategies, including the use of single antigens or antigen combinations to maximize immune responses.
To ensure further uptake and exploitation, several key actions are underway. We are actively exploring the creation of a spin-off company to advance the development, validation, and commercialization of ICD-based cancer vaccines. The use of clinically approved compounds such as temoporfin or mitoxantrone provides a strong advantage for regulatory translation and market entry, potentially shortening the pathway to clinical implementation. Future work will focus on preclinical validation across tumor types, scalability of vaccine production, and alignment with regulatory and standardization frameworks.
Overall, the project delivers a high-impact contribution to the field by demonstrating the translational feasibility of ICD-based vaccination strategies and laying the foundation for a new generation of broadly applicable, personalized cancer immunotherapies.