CAL-27

Molecular correlates for HPV-negative head and neck cancer engraftment prognosticate patient outcomes

Nature Communications | DOI | Paper 2024 | By Matthew Waas; Christina Karamboulas; Benson Z. Wu; Shahbaz Khan; Stephanie Poon; Jalna Meens; Meinusha Govindarajan; Amanda Khoo; Salvador Mejia-Guerrero; Annie Ha; Lydia Y. Liu; Kevin C. J. Nixon; Joseph Walton; Scott V. Bratman; Shao Hui Huang; David Goldstein; Federico Gaiti; Laurie Ailles; Thomas Kislinger

Background

HPV-negative head and neck squamous cell carcinoma (HNSCC) needs better risk stratification due to treatment toxicity. Lymph node involvement is a key prognostic factor, and patient-derived xenograft engraftment predicts poor outcomes but is impractical clinically. The authors performed transcriptomic and proteomic profiling of 88 HPV-negative HNSCC tumors and found that the relationship between engraftment and clinical outcomes is recapitulated by molecular phenotype. They identified LAMC2 and TGM3 as candidate prognostic biomarkers and validated them in an independent cohort of 404 HPV-negative HNSCC patients, showing improved outcome prediction beyond nodal status alone.

Key Findings

Engraftment of HPV-negative HNSCC tumors in mice was significantly associated with worse disease-specific survival and provided risk stratification beyond N category. Molecular profiling linked engraftment to partial epithelial-mesenchymal transition (p-EMT), extracellular matrix reorganization, diminished immune signaling, and fibroblast-epithelial cell communication. LAMC2 and TGM3 were identified as top candidate biomarkers: high LAMC2 intensity correlated with worse outcomes, while normal-like TGM3 staining correlated with better outcomes. In an independent cohort of 404 HNSCC patients, immunohistochemical scoring of LAMC2 and TGM3 validated these associations and significantly stratified outcomes beyond nodal status. Combining LAMC2 and TGM3 improved prognostication within N0 patients, identifying subsets with markedly different failure rates and survival, and suggested potential utility for elective neck dissection decisions and risk-tailored treatment planning.

Contribution

Creative Bioarray provided GFP-labeled Cal33 and Cal27 head and neck squamous cell carcinoma cell lines used in co-culture spheroid experiments with patient-derived cancer-associated fibroblasts. These reagents enabled the authors to assess fibroblast-epithelial crosstalk and p-EMT induction, supporting mechanistic links between CAF signaling and engraftment-associated molecular phenotypes.

Effect of Honokiol-Loaded Titanium Dioxide Nanotube Drug Delivery System on CAL-27 Cells

Tongue cancer is the most common type of oral cancer with a low postoperative prognosis. Honokiol (HNK), a compound extracted from Houpu, has been shown to have anti-tumor effects. Titanium dioxide nanotubes (TNTs) can be used as drug carriers. The purpose of this experiment was to study the anti-tumor effect of HNK-loaded TNTs (HNK-TNTs) on CAL-27 cells.

The results of CCK-8 experiment showed that compared with the control group, the proliferation rate of CAL-27 cells in group HNK and HNK-TNTs was negatively correlated with the time of drug action, and the proliferation rate in group HNK-TNTs was lower than that in group HNK (Fig. 1), suggesting that HNK-TNTs has a better effect on CAL-27 cells proliferation inhibition than HNK. As shown in this scratching experiment results, after 12 and 24 h of cell culture, the migration ability of CAL-27 cells in group HNK-TNTs was significantly lower than that in group control and group HNK (Fig. 2A), and the difference was statistically significant (Fig. 2B). It was indicated that HNK-TNTs has a more significant inhibitory effect on the cell migration.

Bacterial Antigens Reduced the Inhibition Effect of Capsaicin on Cal 27 Oral Cancer Cell Proliferation

Oral cancer is a significant global health issue with high incidence and low survival rates. The oral cavity contains biofilms, including dental plaques, which harbor bacterial antigens like LPS and LTA from Gram-negative and Gram-positive bacteria. These antigens can stimulate cancer cell growth, while capsaicin has been reported to counteract this effect. Chakraborty et al. aim to evaluate the efficacy of capsaicin in treating oral cancer, particularly in the presence of LPS, LTA, or both.

Titration of capsaicin found that the maximum clinically relevant concentration that could be used without adversely affecting normal cells was 150 µM. Capsaicin at 150 µM produced maximum inhibition of oral cancer cell Cal 27 (Fig. 3a), but a small effect on cell viability of normal oral cell OKF6 (Fig. 4b). Therefore, they could not increase the concentration of capsaicin in the study. Within one hour of application, capsaicin at 150 µM reduced 60% of Cal 27 metabolism (Fig. 3b). The level of inhibition remained steady for up to 24 h (Fig. 3b). There was no significant change in normal oral cell OKF6 metabolism (Fig. 4a) and a small effect on cell viability (Fig. 4b) when treated with 150 µM capsaicin or stimulated with bacterial antigens.