A Collective Route to Head and Neck Cancer Metastasis

Distant metastasis (DM) from head and neck cancers (HNC) portends a poor patient prognosis. Despite its important biological role, little is known about the cells which seed these DM. Circulating tumour cells (CTCs) represent a transient cancer cell population, which circulate in HNC patients’ peripheral blood and seed at distant sites. Capture and analysis of CTCs offers insights into tumour metastasis and can facilitate treatment strategies. Whilst the data on singular CTCs have shown clinical significance, the role of CTC clusters in metastasis remains limited. In this pilot study, we assessed 60 treatment naïve HNC patients for CTCs with disease ranging from early to advanced stages, for CTC clusters utilizing spiral CTC enrichment technology. Single CTCs were isolated in 18/60–30% (Ranging from Stage I-IV), CTC clusters in 15/60–25% (exclusively Stage IV) with 3/15–20% of CTC clusters also containing leukocytes. The presence of CTC clusters associated with the development of distant metastatic disease(P = 0.0313). This study demonstrates that CTC clusters are found in locally advanced patients, and this may be an important prognostic marker. In vivo and in vitro studies are warranted to determine the role of these CTC clusters, in particular, whether leukocyte involvement in CTC clusters has clinical relevance.

are better suited to survive the journey through the circulation by cooperation of heterogeneous cell types within the cluster, which can include immune evading cells 2,17,19 . The prevalence and the number of CTC clusters can be underestimated due to their short detection window and lack of appropriate detection methods 20 . Studies have also documented that CTC clusters have a shorter circulation half-life, with faster entrapment within distant organs where metastatic growth may initiate 10,19,21,22 . CTC clusters may provide clues to their evolution during the course of cancer treatment and the mechanisms of cluster mediated treatment resistance 23 . CTC clusters have been associated with decreased metastasis-free survival and a greater metastatic capacity than single CTCs 16,17,21 . Notably, it has been shown that CTC clusters, held by plakoglobin-dependent adhesions, have arisen from oligoclonal expansion of tumour cell groupings, rather than aggregation or proliferation of single CTCs 17 . Recent studies suggest that CTC clusters have the ability to traverse narrow capillaries in a 'single-file' and retain the ability to re-form the intact cluster upon exiting, highlighting the metastatic seeding capacity of these large cellular aggregates that were previously thought to extravasate upon reaching narrow capillaries 16,24 .
To date, the role of CTC clusters, including CTM, has not been well established. Despite their biological importance, in comparison to single CTCs, the data on CTC clusters remains unclear. In this pilot study, we investigated whether CTC clusters and CTM were found in the circulation of HNC patients. This study evaluated 60 treatment-naive HNC patients, with disease ranging from early to advanced stages, for CTC clusters using spiral microfluidic CTC enrichment technology. This technology enriches for CTCs by a function of cell size and deformability and provides a robust methodology for CTC enrichment 15,25,26 .

Results
Patients' HNC disease staging ranged from early to advanced stages of HNC (Stage I-IV). CTCs and CTC clusters were successfully isolated using the spiral microfluidic chip. All patients had no evidence of distant metastatic disease upon presentation, however, at later time points (3-6 months follow up rescan), 7 stage IV patients developed lung and/or liver lesions (Fig. 1). CTC clusters were present in the blood of 6/7 patients that progressed to have distant disease. The patient demographics and clinicopathological features are presented in Table 1 and Table 2 respectively. Single CTCs. Single CTCs were detected in 20/60 patients (33.3%) ranging from 1-10 CTCs/5 ml (   (Fig. 4).
In 10/20 patients positive for single CTCs, CTC clusters were not present. Whereas, in 5/15 patients positive for CTC clusters, single CTCs were not found. Patients presented with both single and CTC clusters in 10/60 patient samples. No CTC-like (single/clusters) events were observed in the 10 normal healthy volunteer (NHV) cohort.

Discussion
Single CTCs have been previously reported in other solid tumour types including HNC 5,6,25,[27][28][29] . CTC clusters are defined as ≥3 tumour cells, held in close proximity by strong cell-cell adhesions, detected in the blood of cancer patients 2,30,31 . Studies have reported that CTC clusters have a shorter half-life in blood, an increased metastatic capacity compared to single CTCs 21,24,30 , and that disrupting the interactions within clusters may provide a strategy to reduce CTC cluster mediated metastasis 16 . An example of this would be the knockdown of plakoglobin, a protein which is highly expressed in CTC clusters that may facilitate in the reduction of cluster generation 21,32 . The observation of CTC clusters or CTMs has been associated with adverse outcomes 33 . To date, the clinical utility of CTCs in HNC remains limited, and few studies have reported on the presence of these subpopulations of CTC clusters 2,13,14,34-37 . In our cross sectional pilot study, single CTCs were found in 33.3% of patients (Stage I-IV) and CTC clusters in 25% of patients (Stage IV). Whilst the number of single CTCs is comparable to previous HNC studies 2,14,26,34,[38][39][40][41] , the presence of CTC clusters in 25% of the cohort is of importance 42,43 . Furthermore, from 7 stage IV HNC patients in the study, that progressed to developing lung/liver metastasis (3-6 months later), 6 patients presented with CTC clusters (P = 0.0313). In patients with the absence of CTC clusters, the predicitive value of not developing distant disease within 6 months was found to be 95%. Chalmers et al., 2012 has shown that HNC CTC clusters can co-express Vimentin and CD44, epithelial-mesenchymal transition (EMT) and stemness traits which may represent an aggressive phenotype 34 . It is not fully understood whether the expression of mesenchymal traits on CTC clusters is due to single proliferating CTCs which had undergone EMT or an EMT transformed CTC cluster 44 . Single CTCs, CTC clusters, or both cell types were found in 25/60 of the sampled HNC patient bloods. Importantly, the presence of CTC clusters did not depend on single CTCs being present and could be potentially used as an independent prognostic marker 44 .
Notably, in 3/21 of the CTC clusters from stage IV HNC patients, white blood cells were found within the cluster. This feature in a number of CTC clusters is of importance as the incorporation of white blood cells (WBCs) in the CTC cluster may provide a mechanism by which these CTC clusters evade the immune system 31,[45][46][47] . To this end, recent studies have highlighted that PD-L1 is frequently expressed on CTCs and may be involved in immune evasion 45,46,48 . Studies have shown that the presence of non tumour cells (e.g. platelets and leukocytes) within CTC clusters promotes metastasis, by protecting the clusters from the shear stressors and immune attacks 44,49 .

Conclusion
This pilot study challenges the notion of only reporting on individual CTCs in HNC studies. Whilst single CTCs were found in the screened population, a comparable population presented with CTC clusters. Whilst the role of CTC clusters, including clusters containing WBCs is not fully understood, studies into this area are warranted to understand cluster mediated immune escape and their role in metastasis.

Materials and Methods
Study design. This prospective study was conducted across three major academic hospitals in Brisbane,  Committee (HREC/12/QPAH/381 and HREC/11/QPAH/331) in accordance with the National Health and Medical Research Council's (NHMRC) guidelines to collect blood from the Royal Brisbane and Women's Hospital (RBWH), Logan Hospital and Princess Alexandra Hospital (PAH). This study also has QUT ethics approval (1400000617 and 1100001420). All participants gave written informed consent and 10 ml blood samples were collected in BD Vacutainer K2E tubes (EDTA) from 60 HNC patients before treatment and 10 normal healthy volunteers (NHV), with CTC assessment made as described below.
Enrichment of CTCs using spiral technology. An initial red blood cell (RBC) lysis (Astral Scientifix) was performed to the 10 ml blood sample to reduce the cellular components passing through the spiral chip. Thereafter, cells were centrifuged and the pellet resuspended in 10 ml of sheath buffer (1xPBS, 2 mM EDTA, 0.5% BSA). The spiral device was setup as previously described 15,26 . In brief, after the spiral chip had an initial priming run, the sample was loaded onto a syringe and pumped through the spiral chip at 1.7 ml/min. The CTC output were collected and spun down at 300 × g for 5 mins.     CTC and CTM parameters. CTCs were visualized using immunofluorescence post enrichment. Cells were classified as CTCs after meeting the following criteria (i) high nucleus to cytoplasmic ratio (ii) morphologically larger than the background cells with intact nuclei (iii) cytokeratin-8,18,19 positive (iv) EGFR positive (v) CD45 negative. CTC clusters were reported as 3 or more CTCs in close proximity and CTM when CTC clusters included leukocytes (CD45 positive cells). The results were reported as the number of CTCs, CTC clusters, and CTM per 5 ml whole blood.
Statistical Analysis. The development of distant metastatic disease (confirmed by imaging and biopsy where possible) were compared to CTC groups using Fisher's exact test. All statistical analysis were performed using Graphpad Prism 7.0 software, were two-sided, and P-values <0.05 considered statistically significant.
Data Availability. All data generated or analysed during this study are included in this published article.