Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets.

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV⁺ patients contained increased proportions of CD39⁻CD73⁻CD4⁺ T cells and reduced proportions of CD39^−/+CD73⁺CD4⁺ T cells compared to the equivalent cells from HPV⁻ patients. Notably, the pretreatment CD4⁺:CD8⁺ T cell ratios and CD39⁺CD73⁺CD19⁺ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.

Peripheral blood mononuclear cells (PBMCs) are commonly cryopreserved for immune cell profiling analyses, like flow cytometry. Cryopreservation of PBMCs may affect the recovery of immune cell subsets or the presence of cell-surface molecules, potentially skewing results compared to freshly isolated PBMCs. The ectonucleotidase CD39 promotes immunosuppression through the hydrolysis of extracellular nucleotides such as adenosine 5’-triphosphate (ATP) to limit P2X7 activation and downstream immune responses. Because of this, CD39 and P2X7 are emerging as potential biomarkers in cancer and other diseases. Using flow cytometry, this study demonstrated that an anti-human CD39 monoclonal antibody (mAb) (clone TU66) bound human embryonic kidney (HEK) 293 cells transfected with human CD39, but not mock-transfected HEK293 cells. In contrast, an anti-human P2X7 mAb (clone 1058613) bound similarly to HEK293 cells transfected with human P2X7 and non-transfected HEK293 cells. Using flow cytometry, this study then assessed the impact of cryopreservation on the population frequencies of immune cell subsets including CD39 ⁺ cells in human PBMC samples. These analyses revealed that cryopreservation had a minor, yet statistically significant impact on the population frequencies of CD4 ⁺ , CD8 ⁺ and CD4 ⁻ CD8 ⁻ T cells. Cryopreservation also had a significant impact on the frequencies of CD39 ⁺ CD19 ⁺ B cells and CD39 ⁺ non-classical monocytes. Overall, this study informs researchers and biobank workers about the need to consider the impact of cryopreservation when interpreting data from stored samples.