Abstract
Background
Circulating tumour cells (CTCs) are a source of prognostic and predictive biomarkers across multiple cancers, including head and neck squamous cell carcinoma (HNSCC). Characterisation of CTCs has historically focussed on RNA expression. Although a powerful approach, important cellular changes can occur in the absence of transcriptional modification through processes such as alterations in protein stability, subcellular localisation or post-translational modification such as phosphorylation. To increase the number of protein markers that can be measured per cell we developed and validated a protocol to apply mass cytometry to CTCs enriched from blood using Parsortix technology.
Methods
Blood samples collected from a pilot cohort of advanced HNSCC patients and processed using the microfluidic-based Parsortix CTC enrichment device were used for panel validation. A 42-marker mass cytometry panel was developed and optimised using a HNSCC cell line model. The mass cytometry panel comprised of lineage markers and druggable targets such as PD-L1, EGFR and CTLA4. In addition, the activation status (phosphorylation) of key signalling pathways, including STAT, ERK, AKT and PARP, was assessed.
Results
The combination of Parsortix-enrichment and mass cytometric analysis allowed successful identification of HNSCC cell lines in spiked donor samples. In patient samples, CTCs were identified and grouped into phenotypic sub-populations. Phospho-markers allowed identification of ‘activated cells’.
Conclusions
We present novel data demonstrating the utility of mass cytometry to characterise CTCs in HNSCC; allowing a previously unseen view of functional marker expression at the single cell level. Our protocol was not only able to identify CTCs in patient samples, but also phenotypic sub-groups within the CTC population. Furthermore, we demonstrate how multiple druggable targets can be detected in these sub-groups. This approach has high clinical translational potential given the cost and complexity of alternative sequencing approaches. Our data serves as a foundation for further research and clinical trials.
Circulating tumour cells (CTCs) are a source of prognostic and predictive biomarkers across multiple cancers, including head and neck squamous cell carcinoma (HNSCC). Characterisation of CTCs has historically focussed on RNA expression. Although a powerful approach, important cellular changes can occur in the absence of transcriptional modification through processes such as alterations in protein stability, subcellular localisation or post-translational modification such as phosphorylation. To increase the number of protein markers that can be measured per cell we developed and validated a protocol to apply mass cytometry to CTCs enriched from blood using Parsortix technology.
Methods
Blood samples collected from a pilot cohort of advanced HNSCC patients and processed using the microfluidic-based Parsortix CTC enrichment device were used for panel validation. A 42-marker mass cytometry panel was developed and optimised using a HNSCC cell line model. The mass cytometry panel comprised of lineage markers and druggable targets such as PD-L1, EGFR and CTLA4. In addition, the activation status (phosphorylation) of key signalling pathways, including STAT, ERK, AKT and PARP, was assessed.
Results
The combination of Parsortix-enrichment and mass cytometric analysis allowed successful identification of HNSCC cell lines in spiked donor samples. In patient samples, CTCs were identified and grouped into phenotypic sub-populations. Phospho-markers allowed identification of ‘activated cells’.
Conclusions
We present novel data demonstrating the utility of mass cytometry to characterise CTCs in HNSCC; allowing a previously unseen view of functional marker expression at the single cell level. Our protocol was not only able to identify CTCs in patient samples, but also phenotypic sub-groups within the CTC population. Furthermore, we demonstrate how multiple druggable targets can be detected in these sub-groups. This approach has high clinical translational potential given the cost and complexity of alternative sequencing approaches. Our data serves as a foundation for further research and clinical trials.
| Original language | English |
|---|---|
| Pages (from-to) | S802 |
| Journal | Annals of Oncology |
| Volume | 32 |
| Issue number | supplement 5 |
| DOIs | |
| Publication status | Published - 21 Sept 2021 |
| Event | ESMO Congress 2021 - Paris, France Duration: 16 Sept 2021 → 21 Sept 2021 https://www.esmo.org/meeting-calendar/past-meetings/esmo-congress-2021 |
