Multimodal Spatial Biology Approach Reveals a Novel Cancer-Associated Fibroblast (CAF) Subset in HNSCC
Head and neck squamous cell carcinomas (HNSCC) require treatment with radiotherapy in two-thirds of cases.1 However, the location of such tumors means they can be particularly challenging to treat due to the close proximity of a number of delicate organs. Therefore, any radiotherapy treatment needs to be highly targeted to avoid unwanted damage to nearby healthy tissues.
Surgical removal of tumors is one treatment approach that may be suitable for smaller cancers, though there are sometimes concerns about the challenge of removing all cancerous tissues. One way of improving surgical outcomes and suppressing tumor recurrence is neoadjuvant immunotherapy.2 Neoadjuvant immunotherapy attempts to boost the immune system’s response to the cancerous tumor before secondary interventions such as surgery take place to improve treatment outcomes.
While immunotherapy has revolutionized cancer treatment and survival rates, and neoadjuvant immunotherapy also shows further improved outcomes, neoadjuvant immunotherapy does not appear to be equally successful for all patients.3 As a result, there is a huge research effort into identifying and profiling biomarkers and potential therapeutic targets in the tumor microenvironment to help make the technique more universal and suitable for the treatment of early-stage cancers.
CD8+ T Cells
Recent research has focused on trying to understand how CD8+ T-cell infiltration occurs.4 CD8+ T cells are cytotoxic, so they play an important role in helping the immune system combat tumors. In cases of HNSCC, the infiltration mechanism of CD8+ T cells often becomes restricted and dysfunctional. In this study, the team successfully identified a relationship between cancer-associated fibroblasts (CAF) and T cells that demonstrate this impairment.
To understand the mechanisms behind CD8+ T-cell regulation, the team needed to perform spatial transcription analysis of the cancerous specimens. Spatial transcriptomics is widely used to understand cell dynamics in the native issue environments.
However, while spatial transcriptomics is a very useful tool for the visualization of gene expressions and their distributions, it is based on mRNA sequencing and expression. The disadvantage of spatial transcriptomics is that the mRNA expression does not necessarily translate directly to protein expression and distribution, meaning a second method needs to be used to investigate novel targets uncovered by spatial transcriptomics that operate on the protein level.
Multiplex Immunofluorescence
One approach that is sensitive to protein expression and distribution is multiplex immunofluorescence. Immunofluorescence is a well-established method for profiling tumor microenvironments and immune changes.6
Multiplexing of fluorescence microscopy measurements means the introduction of multiple markers with different emission wavelengths into a single sample. By having multiple fluorescent tags for several environments of interest in the cell or tissue section, a more comprehensive understanding of the cell environment can be obtained, and it will enable the possibility for more complex, multiparameter imaging studies and analysis.7
Applications
For the CD8+ T-cell studies, the team performed multiplex immunofluorescence staining of three immune types of the carcinoma samples, which they had also studied using spatial transcriptome sequencing. With the combined results, the team could evaluate the spatial relationships between the Gal9+ expression in the CAF and the TCF1+CD8+ memory-like T-cells.
Gal9+ is known to regulate the immune system and affect the prognosis of many kinds of cancer. The team also used their methodology to analyze the percentage of Gal9+ fibroblasts amongst the total fibroblasts and what percentage of the T cells were TCF1+ as well the distance between the Gal9+ fibroblasts to malignant cells.
From Supplementary Figure 8. Li C, Guo H, Zhai P, Yan M, Liu C, Wang X, Shi C, Li J, Tong T, Zhang Z, Ma H, Zhang J. Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells. Cancer Res., 2024
TissueFAXS Spectra
While the information-rich nature of multiplex immunofluorescence offers clear advantages, the challenge is capturing the image efficiently and performing the subsequent image analysis.
TissueGnostics TissueFAXS Spectra, in combination with the StrataQuest analysis package, is designed to address exactly these challenges.
TissueFAXS Spectra is a multispectral imaging platform that allows for the recording of up to 8 different markers. The default size is an 8-slide stage, but this can be upgraded to handle up to 120 slides as TissueFAXS Spectra SL configuration for the highest throughput measurements.
One of the biggest advantages of the TissueFAXS Spectra platform is the unique liquid crystal tuneable filter, which allows for rapid tuning over a broad spectral range to detect emissions. This eliminates the time-consuming step of needing to swap filters in and out to capture different wavelengths, and as long as there is a suitable staining protocol, making immunofluorescence imaging easier.
The team took advantage of these high throughput capabilities of the TissueFAXS Spectra SL platform in combination with StrataQuest to calculate the proximity distance between cell populations as well as for StrataQuest’s automated contextual image analysis to identify cells, structures and tissues. The same frozen slides could be used for both the spatial transcriptome and the multiplex immunofluorescence measurements to ensure the results were truly comparable and there was no compromise on accuracy.
Contact TissueGnostics today to find out how the flexibility and high throughput nature of the TissueFAXS platform could provide a new level of whole-slide imaging capabilities coupled with powerful, automated data analysis. TissueGnostics platforms ensure smooth workflows and incorporation of multiplex immunofluorescence into the pipeline.
References
- Beddok, A., Vela, A., Calugaru, V., Tessonnier, T., Kubes, J., Dutheil, P., Gerard, A., Vidal, M., Goudjil, F., Florescu, C., Kammerer, E., Benezery, K., Herault, J., Poortmans, P., Bourhis, J., & Thariat, J. (2020). Proton therapy for head and neck squamous cell carcinomas : A review of the physical and clinical challenges. Radiotherapy and Oncology, 147, 30–39. https://doi.org/10.1016/j.radonc.2020.03.006
- Boydell, E., Sandoval, J. L., Michielin, O., Obeid, M., Addeo, A., & Friedlaender, A. (2023). Neoadjuvant Immunotherapy : A Promising New Standard of Care. International Journal of Molecular Sciences, 24, 11849. https://doi.org/10.3390/ijms241411849
- Mittendorf, E. A., Burgers, F., Haanen, J., & Cascone, T. (2022). Neoadjuvant Immunotherapy : Leveraging the Immune System to Treat Early-Stage Disease. In ASCO Educational Book. https://ascopubs.org/doi/pdfdirect/10.1200/EDBK_349411
- Li, C., Guo, H., Zhai, P., Yan, M., Liu, C., Wang, X., Shi, C., Li, J., Tong, T., Zhang, Z., & Zhang, J. (2024). Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts In fi ltration and Antitumor Activity of CD8 þ T Cells. Cancer Research, 84(2), 258–275. https://doi.org/10.1158/0008-5472.CAN-23-1448
- Vickovic, S., Magnusson, J., Giacomello, S., Asp, M., Westholm, J. O., Huss, M., Mollbrink, A., Linnarsson, S., Codeluppi, S., Costea, P. I., Mulder, J., Bergmann, O., & Lundeberg, J. (2016). Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science, 353(6294), 78–82. https://doi.org/10.1126/science.aaf2403
- Lee, C. W., Ren, Y. J., Marella, M., Wang, M., Hartke, J., & Couto, S. S. (2020). Multiplex immunofluorescence staining and image analysis assay for diffuse large B cell lymphoma. Journal of Immunological Methods, 478, 112714. https://doi.org/10.1016/j.jim.2019.112714
- Rojas, F., Hernandez, S., Lazcano, R., Laberiano-Fernandez, C., & Parra, E. R. (2022). Multiplex Immunofluorescence and the Digital Image Analysis Workflow for Evaluation of the Tumor Immune Environment in Translational Research. Frontiers in Oncology, 12, 1–15. https://doi.org/10.3389/fonc.2022.889886
