Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma
BackgroundImmune checkpoint inhibitors (ICIs) have become an integral part of cancer therapy, but only a minority of patients experience durable responsiveness. Response rates vary greatly and are often unpredictable, highlighting the urgent need for predictive biomarkers to guide treatment decision...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Oncology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fonc.2025.1622008/full |
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| author | Verena Schweihofer Verena Schweihofer Daniela Schulz Daniela Schulz Raquel Blazquez Gero Brockhoff Gero Brockhoff Tobias Ettl Tobias Ettl Mathias Fiedler Mathias Fiedler Sina Heimer Sina Heimer Juliane Schikora Richard J. Bauer Richard J. Bauer Anja Kathrin Wege Anja Kathrin Wege |
| author_facet | Verena Schweihofer Verena Schweihofer Daniela Schulz Daniela Schulz Raquel Blazquez Gero Brockhoff Gero Brockhoff Tobias Ettl Tobias Ettl Mathias Fiedler Mathias Fiedler Sina Heimer Sina Heimer Juliane Schikora Richard J. Bauer Richard J. Bauer Anja Kathrin Wege Anja Kathrin Wege |
| author_sort | Verena Schweihofer |
| collection | DOAJ |
| description | BackgroundImmune checkpoint inhibitors (ICIs) have become an integral part of cancer therapy, but only a minority of patients experience durable responsiveness. Response rates vary greatly and are often unpredictable, highlighting the urgent need for predictive biomarkers to guide treatment decisions.MethodsWe investigated immune- and tumor-specific expression and secretion profiles in peripheral blood and tumor samples derived from patients with head and neck squamous cell carcinoma (HNSCC). We combined flow cytometry, LEGENDplex™ immune profiling, and preoperative/postoperative serum cytokine analyses to determine checkpoint molecules (e.g., PD-1, TIM-3, LAG-3), immune cell profiles, as well as key markers on tumor cells (CD44, PD-L1, MHC class I/II). In addition, a 3D co-culture model using tumor slices and autologous mononuclear cells from selected HNSCC patients were analyzed upon atezolizumab and pembrolizumab treatment.ResultsCo-expression of PD-1 and TIM-3 on a subset of CD8+ tumor-infiltrating T cells was frequently observed, alongside a pronounced infiltration of myeloid cells in the tumor microenvironment. In the peripheral blood, we detected elevated levels of soluble CD27 in patients compared to controls and distinct preoperative cytokine profiles (e.g., reduced IFN-γ, CCL3, CCL20; elevated IL-15/IL-16). Postoperatively, most cytokines showed lower levels compared to healthy controls but significantly higher CCL2 levels. Furthermore, tumor–immune co-cultures from selected patients showed a stronger apoptotic response and phenotypic differences (e.g., increased PD-1 and CD137 expression) upon atezolizumab treatment. Individual changes in soluble factor release (e.g., Gal-9, sPD-L1, sCD25, and sTIM-3) was noticeable upon co-culture under immune checkpoint therapy.ConclusionsThis study provides proof-of-principle data suggesting that a combined multiplexed marker profiling and a functional 3D co-culture assay may help to explore predictive ICI response for HNSCC patients in the future. However, extensive studies with larger cohorts are warranted to validate and refine this approach. |
| format | Article |
| id | doaj-art-fb128e4297c04b18a0c38729cd5e51ea |
| institution | Kabale University |
| issn | 2234-943X |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Oncology |
| spelling | doaj-art-fb128e4297c04b18a0c38729cd5e51ea2025-08-20T03:41:08ZengFrontiers Media S.A.Frontiers in Oncology2234-943X2025-08-011510.3389/fonc.2025.16220081622008Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinomaVerena Schweihofer0Verena Schweihofer1Daniela Schulz2Daniela Schulz3Raquel Blazquez4Gero Brockhoff5Gero Brockhoff6Tobias Ettl7Tobias Ettl8Mathias Fiedler9Mathias Fiedler10Sina Heimer11Sina Heimer12Juliane Schikora13Richard J. Bauer14Richard J. Bauer15Anja Kathrin Wege16Anja Kathrin Wege17Department of Gynecology and Obstetrics, University Medical Center Regensburg, Regensburg, GermanyBavarian Cancer Research Center (BZKF), Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, Experimental Oral and Maxillofacial Surgery, Center for Medical Biotechnology, Regensburg, GermanyDepartment of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, GermanyDepartment of Gynecology and Obstetrics, University Medical Center Regensburg, Regensburg, GermanyBavarian Cancer Research Center (BZKF), Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, Experimental Oral and Maxillofacial Surgery, Center for Medical Biotechnology, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, Experimental Oral and Maxillofacial Surgery, Center for Medical Biotechnology, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, GermanyDepartment of Dermatology, University Hospital Regensburg, Regensburg, GermanyExperimental Ophthalmology, University Marburg, Marburg, GermanyDepartment of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, GermanyDepartment of Oral and Maxillofacial Surgery, Experimental Oral and Maxillofacial Surgery, Center for Medical Biotechnology, Regensburg, GermanyDepartment of Gynecology and Obstetrics, University Medical Center Regensburg, Regensburg, GermanyBavarian Cancer Research Center (BZKF), Regensburg, GermanyBackgroundImmune checkpoint inhibitors (ICIs) have become an integral part of cancer therapy, but only a minority of patients experience durable responsiveness. Response rates vary greatly and are often unpredictable, highlighting the urgent need for predictive biomarkers to guide treatment decisions.MethodsWe investigated immune- and tumor-specific expression and secretion profiles in peripheral blood and tumor samples derived from patients with head and neck squamous cell carcinoma (HNSCC). We combined flow cytometry, LEGENDplex™ immune profiling, and preoperative/postoperative serum cytokine analyses to determine checkpoint molecules (e.g., PD-1, TIM-3, LAG-3), immune cell profiles, as well as key markers on tumor cells (CD44, PD-L1, MHC class I/II). In addition, a 3D co-culture model using tumor slices and autologous mononuclear cells from selected HNSCC patients were analyzed upon atezolizumab and pembrolizumab treatment.ResultsCo-expression of PD-1 and TIM-3 on a subset of CD8+ tumor-infiltrating T cells was frequently observed, alongside a pronounced infiltration of myeloid cells in the tumor microenvironment. In the peripheral blood, we detected elevated levels of soluble CD27 in patients compared to controls and distinct preoperative cytokine profiles (e.g., reduced IFN-γ, CCL3, CCL20; elevated IL-15/IL-16). Postoperatively, most cytokines showed lower levels compared to healthy controls but significantly higher CCL2 levels. Furthermore, tumor–immune co-cultures from selected patients showed a stronger apoptotic response and phenotypic differences (e.g., increased PD-1 and CD137 expression) upon atezolizumab treatment. Individual changes in soluble factor release (e.g., Gal-9, sPD-L1, sCD25, and sTIM-3) was noticeable upon co-culture under immune checkpoint therapy.ConclusionsThis study provides proof-of-principle data suggesting that a combined multiplexed marker profiling and a functional 3D co-culture assay may help to explore predictive ICI response for HNSCC patients in the future. However, extensive studies with larger cohorts are warranted to validate and refine this approach.https://www.frontiersin.org/articles/10.3389/fonc.2025.1622008/fullhead and neck squamous cell carcinoma (HNSCC)immune checkpoint inhibitors (ICI)tumor microenvironment (TME)multiplexed immune profiling(patient-derived) organotypic slice co-culturepredictive biomarkers |
| spellingShingle | Verena Schweihofer Verena Schweihofer Daniela Schulz Daniela Schulz Raquel Blazquez Gero Brockhoff Gero Brockhoff Tobias Ettl Tobias Ettl Mathias Fiedler Mathias Fiedler Sina Heimer Sina Heimer Juliane Schikora Richard J. Bauer Richard J. Bauer Anja Kathrin Wege Anja Kathrin Wege Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma Frontiers in Oncology head and neck squamous cell carcinoma (HNSCC) immune checkpoint inhibitors (ICI) tumor microenvironment (TME) multiplexed immune profiling (patient-derived) organotypic slice co-culture predictive biomarkers |
| title | Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| title_full | Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| title_fullStr | Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| title_full_unstemmed | Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| title_short | Multiplexed immune profiling and 3D co-culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| title_sort | multiplexed immune profiling and 3d co culture assays to assess the individual checkpoint therapy response in head and neck squamous cell carcinoma |
| topic | head and neck squamous cell carcinoma (HNSCC) immune checkpoint inhibitors (ICI) tumor microenvironment (TME) multiplexed immune profiling (patient-derived) organotypic slice co-culture predictive biomarkers |
| url | https://www.frontiersin.org/articles/10.3389/fonc.2025.1622008/full |
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