Culturing conditions and gene expression

The fidelity of a cell line can vary depending on the culturing conditions it has been subject to. Whilst UW-CSCC1 (at a low passage) has been described above to be a reasonably faithful model of its parental tumour, retaining key oncogenic pathways, the caveat lies in the prolonging of cell culture – inducible of secondary genomic changes such as copy number alterations as well as transcriptomic drift. Li et al., (2014) found copy number alterations in

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HNSCC cell lines that were not present in the original tumours, presumably resulting from selection and propagation in vitro. This has likely been the case for the UW-CSCC series as well. The impacts of several culturing conditions on gene expression were assessed through the provision of UW-CSCC1 derivatives and the findings discussed below.

3.4.4.1 Xenografting of PDCCs largely restores clinical expression profile

Critics of in vitro cell lines suggest that cell line establishment results in the distinct and irreversible loss of important biological properties. A study by Daniel et al., (2009) attempted to compare the expression profiles between non-small cell lung cancer primary tumour xenografts, the cell lines derived from these xenografts, and a secondary xenograft created from these cell lines. Their experiment found that many of the changes incurred by going onto plastic were irreversible, unable to be regained in the secondary xenograft. This is contrary to the findings of the UW-CSCC1 xenograft, whereby the majority of examined genes/pathways regained the original tumour phenotype for both the progression and pathways panel. Unaffected gene annotations included: collagen family, ECM structure, metastasis response, and regulation of angiogenesis. This may be due to the mouse stroma accommodating the production of ECM components (including collagen) and the provision of a blood supply, thereby allowing the cell line to cease transcription of the associated genes. This may also be a function of species incompatibility as murine growth factors do not activate certain human- specific pathways, e.g. human MET (Wilding and Bodmer, 2014; Goodspeed et al., 2016). With the PanCancer pathways panel, the UW-CSCC1 xenograft similarly regained expression almost identical to that of the clinical tumour, with the exception of the pathways PI3K, transcriptional misregulation, and Notch. Again, this is very likely due to subtle differences and the interactions with mouse growth factors.

A similar study by Creighton et al., (2003) compared cell lines with their successive xenografts and found a number of gene classes becoming enriched in the xenograft tumours.

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It is inferred that the restorative ability is cell line dependent, explaining why Daniel et al., (2009) did not observe such a response. This restoration of function may suggest that the tumorigenic mutations have been retained (genomic stability), despite clonal expansion in plastic. This property allows for PDCCs to be derived in vitro in a cost-effective and highly reproducible manner for efficient biological assays with the capability of resembling in situ tumour biology once given the appropriate environment (Creighton et al., 2003). This confers significant advantages over long-term culture through mice.

NanoString is a highly sensitive technology with great specificity to human tissue. As such, practically no mouse stromal tissue would be detected; therefore the expression profile observed here is of authentic human carcinoma. Human stroma may be detected in the cognate tumour sample to a degree, although given the substantial tumour cellularity; this would have a minimal impact. If the xenograft truly has regained important functions, than perhaps subtracting the difference between the xenograft profile and that of the original tumour may reveal the degree of human stromal contamination.

Interestingly, upon culture of the harvested xenograft, the expression profile of this ‘secondary’ cell culture was maintained despite now being in a 2D plane. Albeit, mRNA was

extracted from a very early passage and therefore the effects of 2D culture may not have developed yet. Nonetheless, the sustained expression similar to the clinical tumour adds credence to the suggestion that the differences between the clinical tumour, the cell cultures, and the xenograft are due to stromal background noise.

It is recognised that whether an in vitro or in vivo model is used, both are incomplete mimics of original tumour physiology (Wilding and Bodmer, 2014). Whilst the in vitro PDCCs are missing expression of genes associated with stromal components and immune response, xenograft models also fail to express these genes to a human specificity (Wilding and Bodmer, 2014).

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3.4.4.2 Atmosphere

Given concentrations of oxygen within a tumour are far lower than atmospheric levels, ranging from 1-12 % (Goodspeed et al., 2016), all cell lines were cultured in a hypoxic atmosphere (3 % O2), with the exception of the normoxic acclimated UW-CSCC1 derivative. This derivative sought to showcase the effect of non-physiological levels of oxygen upon culture gene expression profile. It was observed in this normoxic derivative that the regulation of metabolism and metastasis response were influenced by normoxic-inducible factors. This aligns with previous literature reporting on alterations in the above pathways as well as angiogenesis and tumour cell growth in response to varied oxygen concentrations (Geraghty

et al., 2014). This evidence, paired with known physiological oxygen concentrations both

surrounding, and within cSCC in vivo, reveals the basis for culture in hypoxia.

3.4.4.3 Spheroid culture

On the basis of the PanCancer progression panel, the spheroid derivative demonstrated only a slight change in expression profile from the other PDCC derivatives. Although, much like a study by Kenny et al., (2007), global changes were found to be negligible. However, noteworthy alterations were observed, specifically the downregulation of the fibrosis pathway and upregulation of metabolism, reverting to a more in vivo tumour physiology.

The expression profile derived from the PanCancer pathways panel revealed that the spheroid culture better resembled the clinical tumour phenotype more than the other derivatives, including the low passage UW-CSCC1. The spheroid culture was also found to express the stem cell associated gene SOX2 at physiological levels, whilst the other PDCC derivatives demonstrated downregulation of this gene. However, SOX2 expression was low even in the originating tumour; therefore these differences may not be entirely relevant to physiology.

With the exception of upregulation of DNA damage repair genes, and the downregulation of Hedgehog and TGF-beta genes, the spheroid derivative produced an expression profile akin to

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the xenograft. These changes are consistent with those reported by Zschenker et al., (2012), noting changes in processes associated with immune system responses, tissue development, and response to cytokines.

3.4.4.4 Subculture/Passage number

The number of times a cell line is subcultured – or passaged – can result in differences in expression profiles, with serial passaging causing further genotypic and phenotypic variation (Hughes et al., 2007; Kaur and Dufour, 2012). Most pathways in UW-CSCC1 at passage 41 were significantly downregulated compared to an early passage (# 13) with respect to the clinical sample, albeit this was proportionally insignificant compared to the differences seen between the cell lines holistically and the tumour. Mouriaux et al., (2016) found that the effects of serial passage were most severe in the first four passages, with subsequent passages incurring additional, but less dramatic, modifications. This highlights the vulnerability that researchers may be facing by using commercial cell lines that may have been serially passaged over 100 times under various conditions (Hughes et al., 2007). UW-CSCC2 was less burdened by fibroblasts, and a pure culture was reached by passage two.