Development of a Circulating Tumour Cell Culture Model from Head and Neck Squamous Cell Carcinoma

Development of a Circulating Tumour Cell Culture Model from Head and Neck Squamous Cell Carcinoma | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL View This is a preprint and has not been peer reviewed. Data may be preliminary. 3 June 2025 V1 Latest version Share on Development of a Circulating Tumour Cell Culture Model from Head and Neck Squamous Cell Carcinoma Authors : Xiaomin Huang , Xi Zhang 0000-0001-6148-8837 , Chameera Ekanayake Weeramange , Paul Leo , Brett Hughes , Riccardo Dolcetti , Bijun Zeng , … Show All … , Roberta Mazzieri , Gunter Hartel , Rahul Ladwa , Rouraj Taheri , Omar Briek , Lizbeth Kenny , Sarju Vasani , and Chamindie Punyadeera 0000-0001-9039-8259 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.174893062.20203367/v1 Published VIEW Version of record Peer review timeline 419 views 189 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract not-yet-known not-yet-known not-yet-known unknown Despite the advancements made in the diagnosis and treatment of head and neck squamous cell carcinoma (HNSCC), metastasis remains the primary cause of cancer-related deaths. Circulating tumour cells (CTCs) may offer a valuable platform for personalised treatment selection. Towards this goal, the establishment of short-term CTC cultures would allow ex vivo characterisation and drug sensitivity testing. A negative enrichment technique (RosetteSepTM, Human CD45 depletion cocktail) was used to capture CTCs for downstream culturing. Whole exome sequencing (WES) was applied to compare the genomic features in primary tumour and CTC cultures. In vitro functional assays and in vivo mouse models were used to confirm the origin and biological properties of cultured CTCs. Cytotoxicity assays were used to measure CTCs’ responsiveness to chemotherapeutic drugs. We successfully derived 16 CTC cell lines from HNSCC patients’ blood samples (16/107). Tumoural p16-positivity notably increased the successful culture rate. CTC cultures displayed similar migratory and invasive capabilities compared to The American Type Culture Collection (ATCC) HNSCC cell lines. In addition, CTC cultures generated a subcutaneous tumour in immunocompromised NSG mice, which was confirmed by a pathologist to be of HNSCC. CTCs derived from blood samples at post-treatment or HNSCC patients were less sensitive to Cisplatin and 5-FU (Mann-Whitney test, p>0.05). HNSCC-derived CTC cultures closely recapitulate the features of primary tumours, providing a model system for in vitro drug testing and may pave the way towards a personalized medicine approach for HNSCC. Introduction Cancer metastasis significantly influences the prognosis and mortality in cancer patients. It is estimated that over 90% of cancer-related deaths are due to metastasis 1 . The spread of cancer cells to distant organs through the bloodstream and the formation of metastatic cancer leads to disease progression and reduces treatment options. Key mechanisms underlying cancer metastasis include epithelial-to-mesenchymal transition (EMT), angiogenesis, immune evasion and microenvironment interactions. Circulating tumour cells (CTCs) arise when rare epithelial cells detach from primary or metastatic tumour sites and enter the bloodstream, and they are thought to play a crucial role in initiating cancer metastasis 2 . CTCs gain invasive properties through the activation of EMT and survive in the bloodstream by interacting with the blood microenvironment 3 . CTCs have been detected in patients with many solid tumours, and their numbers have been associated with cancer progression 4, 5 . CTCs can provide a real-time view of the tumour using only peripheral blood samples, avoiding repeated invasive tissue biopsies. The generation of CTCs is a rare event and, as such, requires sensitive detection methods. Head and neck cancer (HNC) encompasses various cancers that affect the head and neck region, with over 90% originating from mucosal squamous cells (HNSCC). In 2020, an estimated 890,000 new cases of HNC were reported worldwide, ranking it as the seventh most common cancer globally 6 . Treatment strategies for HNSCC have not evolved over the past decade, and this may partly be due to the lack of an in vitro model system to characterise response to drugs accurately. CTC culture models may provide a personalized approach to treatment selection. The development of culture models enables researchers to understand the characteristics of primary tumours 7 and explore therapeutic targets. The techniques for isolating CTCs are primarily categorised into two groups: those based on physical properties and those based on immunoaffinity. CTCs can be isolated and enriched using cellular physical properties such as cell density, size, or electrical properties. For example, in the size-based approach, CTCs are separated from leukocytes by size, as CTCs are large in diameter. Using the size-based method, we recently demonstrated that the number of CTCs directly correlates with treatment response in a 119-HNSCC patients’ cohort 8, 9 . On the other hand, the immunoaffinity-based method stands out as highly effective in isolating CTCs. This approach involves either positive enrichment, which directly targets CTCs, or negative enrichment, which targets and eliminates other cells. CD45 antigen is a protein tyrosine phosphatase exclusively expressed on the surface of nucleated hematopoietic lineage cells. With negative enrichment, CD45 antibodies can deplete leucocytes by binding to CD45 antigens 10 . We have previously reported that CTCs from HNSCC patients, isolated by CD45 negative enrichment can be expanded ex vivo 11 . However, extensive characterisation was not performed. We hypothesise that CTCs derived from HNSCC patients can be used for drug sensitivity testing, enabling personalised drug models. To achieve these aims, DNA from CTC cultures was analysed by WES to reveal their genomic features. Functional assays and in vivo mouse models were used to explore the proliferation, migration and tumorigenicity abilities of CTC cultures. Cytotoxicity assays were used to provide the proof-of-principle supporting the role of CTCs as an in vitro drug testing model. Study participants, ethics and patient cohorts The study complies with the 2013 Declaration of Helsinki 12 and the Australian Code for Responsible Conduct of Research 13 . We obtained ethics approvals from Metro South Health District’s human research ethics committee (approval number: HREC/12/QPAH/381). The study was also approved by the Queensland University of Technology (approval number: 1400000617) and Griffith University (approval number: 2022/009). Inclusion criteria for patients were (a) aged >18 years; (b) primary HNSCC originating from either the oral cavity, oropharynx, larynx, or hypopharynx, regardless of the treatment status. (c) either locoregionally-advanced or metastatic. Exclusion criteria included (a) patients with secondary head and neck cancer, (b) cancers that originated from the nasopharynx, or non-mucosal Head & Neck sites. All study participants provided informed written consent before recruitment to this study. A total of 10 mL of blood was collected in Heparin vacutainers. Isolation of CTCs for ex vivo culture Whole blood (10ml) collected in heparin blood tubes (BD-Plymouth, UK) was incubated with 500ul of RosetteSep ® Human CD45 depletion cocktail (Stemcell Technologies, Vancouver, Canada) for 20 minutes at room temperature as previously published 7, 11 . The cellular separation was performed using a density gradient media (Lymphoprep™, Stemcell Technologies, Vancouver, Canada) in SepMate™-50 mL tubes (Stemcell Technologies, Vancouver, Canada). After centrifugation at room temperature at 1,200×g for 10 minutes, the supernatant was poured into a new 15ml falcon tube, followed by another centrifugation at 300×g for 10 minutes. After removing the supernatant, the remaining 4 mL of residual sample was mixed with MSK media, and the mixture was then ready for culture. Ex vivo culture of CTCs Isolated CTCs were cultured in 96 well-standard microplates (Thermo Scientific, USA) in MSK culture medium. MSK Culture media contain Advanced DMEM/F12 (Cat# 2537022, ThermoFisher, Massachusetts, USA) and conditioned media with the following additives: 50ng/ml EGF (Sigma-Aldrich, Massachusetts, USA), 5% v/v R-spondin 1, 10% v/v Noggin, 10ng/ml FGF10 (PeproTech, USA), 1ng/ml FGF2 (PeproTech, New Jersey, USA), 10nM Nicotinamide (Acros Organics, Antwerpen, Belgium), 0.5µM A83-01 (Tocris, UK), 10 µM SB202190 (Sigma Aldrich, USA), 10 µM Y-27632 (Selleck Chemical, Texas, USA), 1X B27 Additive (Invitrogen, California, USA), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich, Massachusetts, USA), 2nM Glutamax (Invitrogen, California, USA), 10mM HEPES (Sigma-Aldrich, Massachusetts, USA), 1:100 v/v Primocin (Invivogen, California, USA). Cultures were incubated under hypoxic conditions (2% O 2 , 5% CO 2 ) and monitored for up to 60 days. Samples with substantial cell growth were dissociated with TrypLE Express Enzyme (ThermoFisher, Massachusetts, USA) and sub-cultured in a T25 culture flask (Greiner, Bio-one, Kremsmünster, Austria) to allow further expansion of the cells. Comparison of cultural conditions To optimise the culture condition, one of the CTC cultures (QUT1368) was cultured under normoxia (5% O 2 ) and hypoxia (2% O 2 ) with 10% of Foetal Bovine Serum (Cat# A4766801, ThermoFisher, Massachusetts, USA) in either MSK, RPMI-1640 (Cat# 2472414, ThermoFisher, Massachusetts, USA) or DEME (Cat# 2561473, ThermoFisher, Massachusetts, USA). The morphology of cells was examined under a microscope (ECLIPSE Ti2, Nikon, Japan), and photos were taken on days 1, 3, 5 and 7 for comparison purposes. Whole exome sequencing and variant selection DNA from the primary tumour was extracted from formalin-fixed paraffin-embedded (FFPE) tissue using the QIAamp DNA FFPE Tissue Kit (Cat# 56404, Qiagen, Hilden, Germany), following the manufacturer’s instructions. Germline DNA from peripheral blood leucocytes and DNA from CTC cultures were also isolated using the QIAamp DNA Blood Mini kit (Cat# 51104, Qiagen, Hilden, Germany). WES was performed on tumour tissue DNA from FFPE and the corresponding germline DNA, with 100ng input, and was randomly broken into small fragments by Covaris (Chicago, USA). Adapters were ligated to the DNA fragments with the unique molecule identifier (UMI) after end repair and cleanup. Then the fragments with UMIs were amplified with PCR. DNA libraries were constructed according to the manufacturer’s protocol, and their concentrations and sizes were measured with Qubit (Thermofisher, USA) and TapeStation (Agilent, USA) to ensure the library quality. The library was then blocked with the xGen universal blockers, followed by annealing to the WES panel (Twist Bioscience, USA). Isolated DNA was sent to the NovaSeq platform (Illumina, USA) at 2×150bp, with a reading depth of more than 200× coverage. UDI indices with UMIs were used to reduce sample cross-contamination and reduce sequencing errors. Base-pair-specific error correction was applied using the estimates of the background error rate under a binomial model. All the raw data was stored in FASTA format. Somatic mutations from each patient were sorted out by bioinformatics using version hg37 and were used to design probes (IDT, USA). Tumorigenicity of the CTC cultures in Mouse Xenograft Models and Immunohistochemical Staining CTCs were cultured in multiple T-75 flasks under hypoxia conditions until 70% of confluency. Cells were detached from the flasks using Trypsin-EDTA (Gibco™, cat# 25200056) and resuspended in phosphate-buffered saline (PBS). Afterwards, a total number of 10 7 cells were injected subcutaneously into the right flank of immunocompromised NSG mice. Tumour growth was measured twice weekly using a digital calliper. To calculate the tumour volume, the following formula was used: tumour volume = [LxW 2 ]/2, where W = width of the tumour and L = length of the tumour. After 5 months, the mice were sacrificed for further tumour analysis. not-yet-known not-yet-known not-yet-known unknown Immunohistochemistry staining on the mice tumour Harvested tumours were immediately fixed in 10% neutral-buffered formalin for 24 hours at room temperature. After fixation, tissues were sectioned to fit into plastic cassettes. Using the automated Sakura Tissue Tech VIP processor, the tissues were dehydrated through a graded series of formalin, ethanol, and paraffin and then cleared with xylene. The process tissue is then embedded in paraffin. The paraffin-embedded tissues were then sectioned at 4 µm thickness on a Microtome. Sides were deparaffinised for H&E and immunohistochemistry (IHC) staining. H&E stain was performed by automation using Sakura tissue tech prisma plus Stainer. Staining was performed automatically using the Ventana Benchmark Ultrastainer using antibodies CKAE1/AE3 (clone AE1/3, dilution 1:100, Dako, California, USA) and P63 (clone EC4A4, dilution 1:150, Biocare Medical, California, USA), visualised with DAB OptiView detection kit (760-700). The IHC slides were automatically counterstained with hematoxylin, dehydrated, and mounted. Stained slides from H&E and IHC were analysed via light microscopy for histopathological and biomarkers assessment. Cell proliferation CTC cultures (N=11) and HNSCC cell lines (N=2, SCC9 and SCC2) were seeded at a density of 4,000 cells per well in triplicate in a 96-well flat-bottom plate (Nunclon TC, USA Scientific, USA) to achieve 20%-30% cell confluence. The plate was placed into a real-time cell imaging system (IncuCyte TM live-cell ESSEN BbioScience Inc, Michigan, USA), and cells were monitored for 7 days. Data were analysed using the logistic growth model in GraphPad Prism 10.0, which was defined by a linear decrease in the relative growth rate. Cell proliferation was ranked by comparing the rate constant (k). Cell migration CTC cultures (N=6) and ATCC HNSCC cell lines (N=2, SCC2 and SCC9) were plated at a density of 35,000 cells per well in a 96-well flat-bottom plate (Nunclon TC, USA Scientific, Ocala, USA) to achieve 100% confluency. Cells were cultured overnight, and on the next day, 10µg/100µl of mitomycin C was added to each well for 2 hours to inhibit cell proliferation without affecting viability. After replacing the media, wounds were created on the cell surface using the IncuCyte TM WoundMaker (ESSEN BbioScience Inc, Michigan, USA). The 96-well plate was placed into a real-time cell imaging system (IncuCyte TM live-cell ESSEN BbioScience Inc, Michigan, USA), and migration was measured according to the manufacturer’s algorithm. Data were analysed using the logistic growth model in GraphPad Prism 10.0, which was defined by a linear decrease of the relative growth rate. Cell migration rate was ranked by comparing the rate constant (k). Cytotoxic assay CTC cultures (N=8) were plated at a density of 5,000-8,000 cells/per well in 96-well plates and were cultured in a 2% O 2 hypoxia incubator. After the attachment of cells, different concentrations of Cisplatin (1mg/ml, ACCORD, Melbourne, Australia) and 5-Fluorouracil (Cat# F6627, Sigma-Aldrich, Missouri, USA) were applied to cells with 24h and 48h incubation. 20µl of reagent from CellTiter 96 AQueous One Solution Cell Proliferation Assay (Cat# G3582, Promega, Wisconsin, USA) dissolved in 100µl of culture media were added to each well. OD490 was taken by BioTek Synergy 2SL Luminescence reader (BioTek, Vermont, USA) to analyse the half maximal inhibitory concentration (IC50). IC50 is defined as the concentration inhibiting 50% of the cell’s growth, and it was analysed using a logarithmic sigmoidal dose-response model (GraphPad Prism 9.0). Statistical analysis All statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software Inc., La Jolla, CA, USA) and R (R Development Core Team. Vienna, Austria). GraphPad was used to compare the clinical characteristics (continuous variables) of the HNSCC patients using the Shapiro–Wilk normality test to test for normal distribution. Logarithmic transformation was performed to normalise the data. The Kruskal–Wallis’s test (one-way ANOVA) and Dunn’s multiple comparisons tests were performed on unpaired data with non-normal distribution to compare values between multiple groups. Fisher’s exact test was performed on qualitative data. Chi-square was used to compare the cultural rate. Results Establishment of CTC culture CTCs were derived using samples from both metastatic (N=18) and locoregionally advanced HNSCC patients (N=58). Among the 107 blood samples collected, 50 were from treatment-naïve patients, and 57 were from post-treatment samples (either 13 weeks, 6 months, 12 months, or 24 months after treatments). An illustration of the study is shown in Figure 1A. Out of 107 blood samples, 16 successfully established CTC cultures, resulting in an overall culturable rate of 14.95%. The successful CTC culture rate in treatment-naïve blood samples was 20.00% (10/50), nearly twice as the post-treatment blood samples at 10.53% (6/57) (Figure ID). The clinical information of the patients whose cultures were successful is listed in Table 1. As illustrated in the pie charts in Figure 1B, of the 16 CTC cell cultures, 2/16 (12.50%) originated from metastatic patients, while 14/16 (87.50%) came from locoregionally advanced patients. Regarding treatment status (Figure 1C), 10 samples (62.50%, 10/16) were treatment-naïve, and 6 samples (37.50%, 6/16) were post-treatment. We then investigated factors such as alcohol intake and smoking status, gender, tumoural p16 positivity in the primary tumour tissue, and tumour anatomical location in relation with the successful cultural rate. Figure 1D-H demonstrates that, although no statistically significant differences were observed, alcohol drinkers (27.59% vs. 13.79%), smokers (26.67% vs. 13.33%), males (13.21% vs. 1.89%), and tumours in the oropharynx (compared to those in the oral cavity, 13.83% vs. 3.19%) exhibited higher success culture rates. Notably, patients with p16-positive tumours had a significantly higher culture rate than those with p16-negative (15.66% vs. 2.41%, p=0.0386) (Figure 1I). not-yet-known not-yet-known not-yet-known unknown CTC cultivability and comparison of cultural conditions Different percentages of oxygen (5% in normoxia, 2% in hypoxia) and three types of cell culture media (MSK, DMEM, RPMI-1640) were further investigated to optimize cultural conditions (Figure 2). CTCs (QUT1368) were plated at a density of 20,000 cells in each well and cultured in 10% FBS with MSK, RPMI-1640 or DMEM under normoxia and hypoxia conditions. As illustrated in Figure 3, the morphology of CTCs in MSK remained intact, whereas CTCs in DMEM showed signs of cell death (cells detached from plates) and increasing debris over time. CTCs in RPMI-1640 exhibited slow growth under normoxic conditions from Day 1 to Day 5; however, by Day 7, the cells became rounded and surrounded by debris, indicating cell death. Additionally, in MSK media, a greater number of cells were observed growing on Day 7 under hypoxic conditions compared to normoxic conditions. MSK media was also compared with media used in other studies (details of the media were shown in Supplementary Table 1 and Supplementary Table 2), and it significantly stimulated CTC expansion (Supplementary Figure 1). Thus, it can be concluded that MSK is the optimal media for CTC culture, and hypoxia enhances CTC growth. Genomic characterisation of CTC cultures DNA extracted from eight CTC cell lines with the fastest growth was subjected to WES. Somatic variants were sorted out and compared with variants in cancer databases. We analysed the mutations at the variant levels and gene levels. Although no common variants were identified at the variant level, we did find pan-cancer oncogenes at the gene level in each cell line (100%, 8/8, details shown in Table 2) 14 15 16 . HNSCC-specific cancer driver genes were found in 62.5% of the CTC cultures (5/8, QUT754, QUT759, QUT797, QUT775 and QUT780). Figure 3A summarizes the pan-cancer oncogenes and HNSCC-specific oncogenes in the heatmap. An analysis was also conducted on commonly mutated genes across the eight cultures (Figure 3B). Among the eight shared oncogenes identified ( CNTNAP5, FOXP1, ANKRD13A, HERC2, ISR1, COL9A3, PLEC , and SDK1 ), FOXP1 was recognized as a pan-cancer oncogene. However, no driver mutations were identified, nor mutations in oncogenes were found shared among the CTC cultures. This evidence supports the presence of individual tumour-related genomic features in CTC cultures. CTC cultures and functional analysis To demonstrate that the CTC cultures possess key cellular characteristics of tumour cells, we conducted functional assays on 11 CTC cultures and two HNSCC cell lines (SCC9 and SCC2) for comparison. In cell proliferation assays (Figure 4A), 90.91% (10/11) of CTC cultures and 100% of HNSCC cell lines displayed growth trends. By comparing the rate constant (k value) with the logistic growth model, the highest growth rate was observed in SCC2 (k=0.022), followed by QUT780 (k=0.021), SCC9 (k=0.020), QUT774 (k=0.017), GU0029 (k=0.013), QUT1368 (k=0.008), QUT1012 (k=0.007), QUT797 (k=0.005), QUT775 (k=0.003), QUT898 (k=0.002), QUT1014 (k=0.001), and QUT795 (k<0.001). QUT897 exhibited no growth. In cell migration assays (Figure 4B), 85.71% (6/7) of CTC cultures demonstrated migration ability, defined as the process by which cells move actively from one location to another 17 . By comparing the rate constant with the logistic growth model, QUT797 (k=0.041) exhibited the highest migration capacity, followed by QUT1012 (k=0.036), SCC9 (k=0.031), SCC2 (k=0.021), QUT780 (k=0.020), QUT897 (k=0.011) and QUT898 (k=0.003), and QUT774 exhibited no migration. CTC cultures exhibited proliferation and migration abilities based on the tumour functional assays mentioned before. Our observations indicate that CTC cultures share comparable tumour functions with ATCC HNSCC cell lines. Tumourigenicity in CTC cultures To test the tumorigenicity of the CTC cell lines (n=1), 1*10 7 CTCs from the MCTC-07 cultures were subcutaneously injected into NSG mice and successfully formed a 2mm×4mm tumour after five months (Figure 5A). Immunohistological analysis of the subcutaneous tumour revealed atypical cell infiltration (Figure 5B), strong positivity of cytokeratin A3AE3 (Figure 5C) and weak expression of p63 (Figure 5D). These findings indicate CTC-derived tumour is of epithelial origin, consistent with that of the primary tumour, as confirmed by a pathologist. Cytotoxic assays on CTC cell line cultures To demonstrate the suitability of CTC cultures as an in vitro drug testing model, cytotoxic assays were conducted using CTC cultures (N=8) and HNSCC cell lines (N=2, SCC9 and SCC2). Cisplatin and 5-fluorouracil (5-FU), two of the most common chemotherapy drugs used to treat patients with HNSCC, were tested in the CTC in vitro cultures (Figure 6A-B). Half-maximal inhibitory concentration (IC50) was utilized to assess the drug sensitivity of the cells. CTC cultures showed a higher sensitivity to 5-FU compared to HNSCC cell lines (IC50: 77.14 µM vs. 161.30µM, Mann-Whitney test, p=0.044), whilst a lower sensitivity to Cisplatin (IC50: 17.57µM vs. 9.98µM, Mann-Whitney test, p=0.044). Of the eight CTC cultures, QUT797, derived from a follow-up blood sample of a metastatic HNSCC patient, exhibited the highest IC50 values for both drugs (49.16µM in Cisplatin, 143.80µM in 5-FU). Factors contributing to drug responsiveness were also analysed (Figure 6C-D). CTCs derived from post-treatment blood samples presented slightly higher median IC50s of both drugs (Mann-Whitney test, p>0.05). At the same time, CTCs derived from males, advanced tumour stage, or larger tumoral size presented higher median IC50s in 5-FU (Mann-Whitney test, p>0.05). This indicated that CTCs derived from the above samples might be more resistant to chemotherapy. Discussion CTCs can detach from the primary tumour and successfully colonize distant sites, contributing to cancer metastasis. CTCs are already established as prognostic biomarkers in metastatic breast cancer 18 , prostate cancer 19 and colorectal cancer 20 . However, this clinical validity was based solely on the CTC number 21 , which provides limited information about the tumour. CTCs are typically low in number and difficult to detect in most patients with early-stage cancer, and their presence is strongly affected by the cancer’s anatomical site 21 . Therefore, to perform more extensive analysis, such as sequencing and drug testing, it is necessary to increase their numbers in vitro . Hence, a protocol for the ex vivo expansion and characterization of CTCs is urgently needed. We successfully established 16 CTC cell lines from 107 blood samples using the immune based negative enrichment method. We found that the positive tumour p16 status significantly increased the chances of successful culture. These CTC cultures demonstrated comparable proliferation and migration abilities to secondary-HNSCC cell lines. One of the novel findings of our research is that CTCs injected into immunocompromised mice resulted in tumour formation, indicating that these CTCs have the potential for tumorigenicity. One of the striking findings from this work is that the CTC culturability increased if the patients were treatment naïve. This is plausible as treatments, especially chemotherapy, may negatively impact the CTC culturability. Like other stable HNSCC cell lines, CTC cultures can be used in cytotoxic assays for personalized drug modelling. We used a negative enrichment strategy targeting CD45 antigens (white blood cells) and reported a cultural success rate of 15%. Our CTC culture success rate matched with previous studies, which ranged from 1% to 17% 22 . Although there are several methods for CTC separation, these techniques primarily rely on either their physical properties or immunoaffinity characteristics, with the latter being one of the most widely used approaches for CTC culture. Positive enrichment targets CTCs directly by utilizing epithelial cell adhesion molecule (EpCAM), allowing for the elution of healthy blood cells 23 . In contrast, negative enrichment focuses on CD45 antigens, which are exclusively expressed in nucleated hematopoietic lineage cells. As some CTCs can be EpCAM-negative, especially those that underwent EMT, the positive enrichment isolation would miss a significant portion of CTCs. As such, negative enrichment is considered a more reliable approach for CTC culture studies. There are emerging novel microdevices that integrate both physical and immunoaffinity properties to enrich CTCs and, subsequently, for culture 24, 25 . Karabacak et al. combined size-based separation with EpCAM-based positive enrichment or CD45 negative depletion 24 , and these cells could be used for culture. Establishing CTC cultures relies on many factors, such as enrichment strategy, culture medium and culture conditions. Cancer subtypes and patient-specific characteristics can also influence the culture success rate. Patients with advanced-stage tumours or high tumour burden often have more circulating CTCs, increasing their potential to be cultured ex vivo 26 . Treatment status has also been shown to affect the success rate of CTC culture 26, 27 . Yu et al. reported that out of 36 breast cancer patients, 6 were able to generate CTC cultures, and these successful cultures were observed only in patients with progressing disease during treatment 28 . Our data presented, although limited, suggests that CTC cultures may be more successfully established in treatment-naive patients when using the negative enrichment strategy targeting CD45 antigens. To confirm that the cultured CTCs are of tumour origin, most researchers have focused on morphological characterisation using cell-surface markers 11, 25, 29, 30 , and genomic analysis 25, 29 . CTCs are primarily characterized based on epithelial and mesenchymal markers. Epithelial markers, such as the ’universal EpCAM’ and Cytokeratin (CK) 31 , are used to characterise epithelial CTCs. As epithelial cancer cells typically undergo EMT prior to intravasation, they often express mesenchymal markers such as Vimentin, Twist and N-Cadherin 32 . Additionally, some markers are specific to certain cancer types. For instance, HER2 (human epidermal growth factor receptor 2), ER (estrogen receptor) and PR (Progesterone receptor) usually could be detected in CTCs derived from reproductive system cancers 33 . Since many of these cell markers are also expressed in normal, healthy epithelial and mesenchymal cells, it is important to employ additional techniques to confirm that CTCs are of tumour origin. Therefore, we employed whole exome sequencing (WES) to identify somatic variants in CTCs and discovered mutations in oncogenes, providing evidence that CTCs contained cancer-related mutations. We also conducted functional assays on CTC cell lines along with ATCC HNSCC cell lines to prove that CTC cultures exhibited similar proliferation and migration ability, as also shown by other studies 31 . Additionally, xenograft mouse models demonstrated the tumorigenic potential of CTCs, and subsequent immunopathological analysis confirmed that the tumours in the mice shared the same cellular origin as the primary tumour. Applying CTC cultures in drug trials is a current trend. Yu et al. tested the drug susceptibility of the tumour by using CTC cultures from breast cancer patients 28 . They concluded that evaluating the drug sensitivity of CTCs can assist in determining the most suitable treatment for individual cancer patients. Chen et al. tested anti-cancer drugs on CTCs derived from metastatic breast cancer patients and reported that CTC mirrored the drug’s impacts on metastatic tumours 34 . Though our study did not show any gene mutations related to targeted drugs, we demonstrated drug sensitivity using two common HNSCC chemotherapy drugs and highlighted the potential of CTC cultures in personalized drug testing. Conclusion We report a 15% CTC culture rate in HNSCC; the successful cultural rate is not as high as in breast, lung, colorectal or prostate cancers. The tumoral p16 status is notably affected the cultural rate. CTC cultures shared similarities with the ATCC-HNSCC cell lines in proliferation, migration and tumorigenicity. They can be applied in ex vivo cytotoxic assays, showcasing their potential ability to formulate a personalized therapeutic strategy. Future research should seek to integrate standard protocols for long-term CTC cultures with comprehensive characterisation. Genomic features, including gene expression level, mutational landscape, as well as tumoural functions, should be tracked to illustrate the evolution of CTC cultures. These would allow CTC cultures to become a standardised model for drug screening assays, which leads the way towards precision oncology. Ethics approval and consent to participate The study complies with the 2013 Declaration of Helsinki and the Australian Code for Responsible Conduct of Research. Ethics approvals were gained from Metro South Health District’s human research ethics committee (approval number: HREC/12/QPAH/381). The study was also approved by the Queensland University of Technology (approval number: 1400000617) and Griffith University (approval number: 2022/009). Written informed consent was obtained from all patients before enrolment in the study. Acknowledgements The authors acknowledged the above funding and clinical coordinators at Royal Brisbane Women’s Hospital. This study is funded by Cancer Australia [Grant number APP1145657]. Chamindie Punyadeera is currently receiving research funding from the National Health and Medical Research Council [Grant number APP 2002576; APP 2012560], the Australian Research Council, the Garnett Passe and Rodney Williams Foundation, Gallipoli Medical Research Foundation, Metro North Collaborative Grant Scheme and the Royal Brisbane Women’s Hospital Foundation. Disclosure Conflict of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1. T.N. Seyfried, L.C. Huysentruyt, Crit Rev Oncog , 2013 , 18 , 43.2. T. Lozar, K. Gersak, M. Cemazar, C.G. Kuhar, T. Jesenko, Radiol Oncol , 2019 , 53 , 131.3. D. Lin, L. Shen, M. Luo, K. Zhang, J. Li, Q. Yang, F. Zhu, D. Zhou, S. Zheng, Y. Chen, J. Zhou, Signal Transduction and Targeted Therapy , 2021 , 6 , 404.4. J. Muller Bark, A. Kulasinghe, G. Hartel, P. Leo, M.E. Warkiani, R.L. Jeffree, B. Chua, B.W. 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Demographics of HNSCC patients Age (years) Median 64 59 Average 64 61 Gender Male 68 (89.47%) 13 (86.67%) Female 7 (9.21%) 2 (13.33%) Smoking history Ex-smoker 13 (17.11%) 5 (33.33%) Yes 8 (10.53%) 3 (20.00%) Never 8 (10.53%) 4 (26.67%) Drinking history Yes 19 (13.16%) 8 (53.33%) No 9 (11.84%) 4 (26.67%) Tumour stage I 21 (27.63%) 5 (33.33%) II 14 (18.42%) 2 (13.33%) III 15 (19.74%) 5 (33.33%) IV 25 (32.89%) 3 (20.00%) T stage 1 13 (17.11%) 2 (13.33%) 2 19 (25.00%) 5 (33.33%) 3 15 (19.74%) 2 (13.33%) 4 28 (36.84%) 6 (40.00%) N stage 0 18 (23.68%) 2 (13.33%) 1 25 (32.89%) 7 (46.67%) 2 27 (35.53%) 5 (33.33%) 3 5 (6.58%) 1 (6.67%) HPV/p16 status Positive 40 (52.63%) 12 (80%) Negative 21 (27.63%) 2 (13.33%) Tumour location Oral cavity 21 (27.63%) 3 (20.00%) Oropharyngeal 48 (63.16%) 12 (80%) Hypopharynx 2 (2.63%) 0 Larynx 4 (5.26%) 0 Note that the staging system for nodal disease differs in HPV+ve compared to HPV-ve cancers, which are recorded according to the clinical record Table 2 . Pan-Cancer Oncogenes and HNSCC-Specific Oncogenes in CTC Cultures not-yet-known not-yet-known not-yet-known unknown QUT754 ERBB4, FOXP1, TERT, FAM135B, FGFR2, ERCC4 FAM135B QUT759 SFPQ, FOXP1, SND1, A1CF, AKT1, NF1, STAT6B, PPP2R1A NF1 QUT795 EPS15 - QUT797 AFF1, NSD1, ETV6, TYRO3, CLTCL1 NSD1 QUT775 PTPRK, AKAP9, ERC1, CLIP1 AKAP9, CLIP1 QUT780 ACKR3 - QUT1368 SUZ12, CCND1, CSF3R CSF3R, CCND1 GU0029 NONO - Information & Authors Information Version history V1 Version 1 03 June 2025 Peer review timeline Published VIEW Version of Record 31 Oct 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection View Keywords circulating tumour cells head and neck squamous cell carcinoma in vitro cancer models liquid biopsy Authors Affiliations Xiaomin Huang Griffith University View all articles by this author Xi Zhang 0000-0001-6148-8837 Griffith University View all articles by this author Chameera Ekanayake Weeramange Griffith University View all articles by this author Paul Leo Queensland University of Technology View all articles by this author Brett Hughes Royal Brisbane and Women's Hospital View all articles by this author Riccardo Dolcetti The University of Melbourne View all articles by this author Bijun Zeng The University of Melbourne View all articles by this author Roberta Mazzieri The University of Melbourne View all articles by this author Gunter Hartel QIMR Berghofer Medical Research Institute View all articles by this author Rahul Ladwa Princess Alexandra Hospital View all articles by this author Rouraj Taheri Royal Brisbane and Women's Hospital View all articles by this author Omar Briek The University of Queensland View all articles by this author Lizbeth Kenny Royal Brisbane and Women's Hospital View all articles by this author Sarju Vasani Royal Brisbane and Women's Hospital View all articles by this author Chamindie Punyadeera 0000-0001-9039-8259 [email protected] Griffith University - Nathan Campus View all articles by this author Metrics & Citations Metrics Article Usage 419 views 189 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xiaomin Huang, Xi Zhang, Chameera Ekanayake Weeramange, et al. 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