Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers

Abstract

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.

Why responses to immune checkpoint inhibitors are heterogeneous in head and neck cancers: Contributions from tumor-intrinsic and host-intrinsic factors

“…We employed conventional flow cytometry and single-cell RNAsequencing to identify the difference in TILs. We found that TAb2 and TCh3 KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their unresponsiveness or sensitivity to anti-PD-L1 (18). Others have established murine HNSCC cell lines from primary 4NQO-induced tumors in the tongue of C57BL/6 (B6) mice, which were designated 4MOSC, short for 4NQO-induced murine oral squamous cells (65).…”

“…We recently established a genetically engineered mouse model by deleting p53 and constitutively activating PIK3CA in mouse keratin 15-expressing (K15 + ) stem cells, which leads to the spontaneous development of multi-lineage tumors including SCCs, termed keratin-15-p53-PIK3CA (KPPA) tumors ( 14 ). Furthermore, we derived transplantable daughter cell lines from KPPA tumors, which may provide a platform for testing new therapeutic strategies in HNSCCs ( 18 ).…”

Recent Advances and Challenges in Cancer Immunotherapy

“…TAMs’ role as major carriers of checkpoint inhibitor ligands (e.g., PDL-1/2, B7-H4, VISTA) and as mediators of T-cell exhaustion has been well described [ 181 ]. Studies have also revealed how an increased concentration of M2-polarized TAMs mediate anti-PD-L1 ICI resistance in HNSCC and in prostate cancer [ 182 , 183 ].…”

Perspectives for 3D-Bioprinting in Modeling of Tumor Immune Evasion

“…Therefore, immune evasion was added to the list of hallmarks of cancer in 2010 [5]. Although some of the mechanisms of immune evasion can be related to direct interactions between malignant cells and the immune effectors, there are numerous instances when TME serves for cancer as a protector against the immune system and/or immunotherapies [6][7][8].…”