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Updated: Sep 28, 2025
Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells
Published on: March 7, 2025
Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in
Samantha M Y Chen1,2, Vince Popolizio1, Rachel A Woolaver1
1Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, School of Medicine, Aurora, CO, 80045, USA.
Background:
While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood.
Method:
To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs).
Results:
We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors.
Conclusions:
We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.
Insights
Tumor-intrinsic differences, not just genetic mutations, dictate head and neck cancer response to immune checkpoint inhibitors (ICI). Targeting tumor-specific or TAM-specific players may improve ICI efficacy.
Area of Science:
- Oncology
- Immunology
- Cancer Biology
Background:
- Immune checkpoint inhibitors (ICI) show limited efficacy in head and neck squamous cell carcinomas (HNSCCs).
- Mechanisms of ICI resistance versus sensitivity in HNSCCs are not fully understood.
Purpose of the Study:
- To investigate tumor-intrinsic factors influencing differential responses to ICI in HNSCC.
- To establish and utilize novel mouse squamous cell carcinoma (SCC) models with frequently mutated HNSCC genes (TP53 deletion and PIK3CA hyperactivation).
Main Methods:
- Generated two distinct KPPA SCC mouse tumor lines (TAb2 and TCh3) by deleting p53 and activating PIK3CA.
- Transplanted tumors into C57BL/6 mice and analyzed the tumor immune microenvironment (TME) via flow cytometry and single-cell RNA sequencing.
- Assessed tumor infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) in response to anti-PD-L1 treatment.
Main Results:
- Pre-existing heterogeneous immune profiles in KPPA tumors determined sensitivity or unresponsiveness to anti-PD-L1.
- Unresponsive TAb2 tumors showed enrichment of M2-TAMs, while sensitive TCh3 tumors had more CD8 TILs with effector functions.
- TAb2 tumors expanded TAMs requiring CSF1 and VEGF; high VEGF-C/CSF1 expression correlated with worse survival in HNSCC patients. Anti-PD-L1 did not alter TAb2 TME but increased CD8 TILs in TCh3 tumors.
Conclusions:
- Tumor-intrinsic differences, beyond genetic alterations, significantly impact ICI response in HNSCC.
- Evaluating tumor-intrinsic cues and TME immune profiles may improve prediction of ICI efficacy.
- Developed experimental models offer a platform for testing combination immunotherapies targeting tumor-specific or TAM-specific pathways to overcome ICI resistance.
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