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Abstract and Figures

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2–5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.
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International Journal of
Molecular Sciences
Comprehensive Mutational and Phenotypic
Characterization of New Metastatic Cutaneous
Squamous Cell Carcinoma Cell Lines Reveal Novel
Drug Susceptibilities
Jay Perry 1,2,3 , Bruce Ashford 1,2,3,4,5 , Amarinder Singh Thind 2,5 , Marie-Emilie Gauthier 6,
Elahe Minaei 1,2,3 , Gretel Major 1, 2, , Narayanan Gopalakrishna Iyer 7, Ruta Gupta 4,8,
Jonathan Clark 4and Marie Ranson 1,2,3,4,*
1Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong,
Wollongong, NSW 2522, Australia; (J.P.); (B.A.); (E.M.); (G.M.)
2Illawarra Health & Medical Research Institute, Wollongong, NSW 2522, Australia;
3CONCERT Translational Cancer Research Centre, Liverpool, NSW 2170, Australia
4Sydney Head and Neck Cancer Institute, Chris O’Brien Lifehouse, Camperdown, NSW 2050, Australia; (R.G.); (J.C.)
5School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia
6Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research,
Darlinghurst, NSW 2010, Australia;
7Department of Surgical Oncology, National Cancer Centre Singapore, Singapore 169610, Singapore;
8Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital,
NSW Health Pathology, Camperdown, NSW 2050, Australia
*Correspondence:; Tel.: +61-2-4221-3291
Current address: Department of Orthopaedic Surgery and Musculoskeletal Medicine, University of Otago,
Christchurch 8140, New Zealand.
Received: 3 November 2020; Accepted: 3 December 2020; Published: 15 December 2020
Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who
develop metastases (2–5%) present with advanced disease that requires a combination of radical
surgery and adjuvant radiation therapy. There are few eective therapies for refractory disease.
In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and
neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically
resembled the original patient tumor and were tumorogenic in mice. Dierences in cancer-related
gene expression between the tumor and cell lines after various culturing conditions could be largely
reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an
irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage
pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries,
which correlate with either copy number variations, targetable mutations and/or the upregulation of
gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs
supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines
are thus useful preclinical models for determining targetable pathways and candidate therapeutics.
cSCC; metastasis; skin cancer; PI3K; WGS; ultraviolet; cancer; cell culture; xenograft;
gene expression
Int. J. Mol. Sci. 2020,21, 9536; doi:10.3390/ijms21249536
Int. J. Mol. Sci. 2020,21, 9536 2 of 16
1. Introduction
Cutaneous squamous cell carcinoma (cSCC) is a common non-melanoma skin cancer and the
most common malignancy worldwide [1,2]. Primary cSCC is typically treatable, although in 2–5% of
cases metastatic spread occurs [
], resulting in the majority of disease-specific deaths. Other than
surgery and radiotherapy, the only systemic therapy approved for locally advanced and metastatic
cSCC is Cemiplimab immunotherapy, which resulted in a 50% response rate and was associated with
adverse events [9,10].
Recent eorts are helping to identify the biological processes underpinning the tumorigenesis
and metastasis of cSCC [
]. However, candidate biomarkers and therapeutics have seldom been
examined in pre-clinical models of metastatic disease. Cell lines remain powerful pre-clinical tools
to study biological behavior, as well as being amenable to high-throughput drug screening [
Cell lines derived from primary cSCC have been reported [
], but there are few cell lines derived
from metastatic cSCC (Table S1). The metastatic cell lines UT-SCC7, UT-SCC59A, and UT-SCC115 have
not been validated molecularly, nor has evidence of tumorigenicity been published. This is also the
case for an undesignated metastatic cSCC cell line recently developed [
]. MET4 and IC1MET form
tumors in mice but phenotypic comparisons to their originating tumors are limited. The mutational
profile of IC1MET was, however, analyzed by whole-exome sequencing and shown to be comparable to
its originating tumors [
]. Regardless, there is a need for additional high-fidelity models of metastatic
cSCC to cover the mutational spectrum observed between patients.
In this current study, we report the establishment of novel patient-derived cSCC cell cultures from
nodal metastases, designated UW-CSCC1 and UW-CSCC2. The originating tumors and
in vitro
derivatives underwent whole-genome sequencing (WGS), gene expression analysis, and other
phenotypic analyses to characterize the fidelity of the cell lines and the implications of these findings
for therapeutic investigations. The benefits of these cell lines are shown through high-throughput
screening of anti-cancer molecules, revealing candidate therapeutics for this disease.
2. Results
2.1. Phenotypic Validation of Novel Cell Lines from Patients with Metastatic cSCC
Long-term continuous cell lines were established from parotid node metastases from two
patients (Table S2), designated UW-CSCC1 and UW-CSCC2. To model changes that may be
incurred by tumor cells that survive radiation therapy, an irradiated sub-clone was expanded from
passage 13 UW-CSCC1, designated UW-CSCC1-R. The doubling times of UW-CSCC1, UW-CSCC1-R,
and UW-CSCC2 as monolayers were 47, 36, and 82 h, respectively. Morphologically, UW-CSCC1-R
appeared more mesenchymal-like compared to UW-CSCC1 (Figure 1a,b). The UW-CSCC2 were
morphologically dissimilar to UW-CSCC1/1-R, demonstrating rounder borders and a smaller diameter
(Figure 1c). UW-CSCC1 and UW-CSCC2 produced tight spheres in ultralow binding plates within
48 h, whereas UW-CSCC1-R only formed aggregates (Figure 1d–f).
All cell lines stained positive for the epithelial marker CK-5 (Figure 1g–i), but were negative
for the mesenchymal markers
-SMA and vimentin (data not shown), confirming their epithelial
purity. UW-CSCC1 and UW-CSCC1-R demonstrated invasion using an organotypic assay (Figure 1j,k).
UW-CSCC2 was not assessed in this assay. The tumorigenicity of UW-CSCC1 and UW-CSCC2 was
confirmed by xenografting in NOD scid gamma immunocompromised mice. Tumors arose at the site of
inoculation within three months and largely retained the tissue architecture of the clinical specimen
(Figure 1l–o), including unequivocal squamous epithelial cell morphology and malignant cytological
features (Figure 1l inset).
Int. J. Mol. Sci. 2020,21, 9536 3 of 16
Figure 1.
Tumor and cell line characterization by microscopy. Photomicrographs (10
objective) of
cell lines grown in (
) monolayer (2D) culture or (
) spheroid (3D) cultures. Immunocytochemical
images (
) of cell lines stained with CK-5 antibody (green): nuclei stained with RedDot2
(red). Organotypic assays showing UW-CSCC1 (
) and UW-CSCC1-R (
) directional invasion into
fibroblast contracted collagen I matrices at eight days after being placed onto an air–liquid interface.
Representative micrographs of hematoxylin and eosin (H&E)-stained sections (n=3 matrices).
Magnification 20
objective. Dotted—Patient 1 and (
) UW-CSCC1 xenotransplant in NSG mouse or
) clinical tumor from Patient 2 and (
) UW-CSCC2 xenotransplant in NSG mouse. Enlarged inset
panel (l) highlights polyploidy. Arrow heads denote papilliform architecture.
2.2. Genotypic Validation of Novel Cell Lines from Patients with Metastatic cSCC
The genomic characteristics of parent tumors and cell lines analyzed by whole genome sequencing
are summarized in Figure 2a–e. The low levels of variant detection for the Patient 2 tumor (Figure 2d)
were likely a result of the low tumor cellularity of the sample taken for DNA extraction, as confirmed
after WGS (14% by Purple analysis; Table S2), and thus could not be compared genomically to
its corresponding cell line UW-CSCC2. Major structural variation was present in UW-CSCC2,
including deletions, amplifications, inversions and translocations (Figure 2e), similar to that observed
for the Patient 1 tumor and its derived cell line (Figure 2a–c).
The structural variation patterns (copy number gains and losses and minor allele copy number)
were well conserved between UW-CSCC1/1-R and the originating tumor (Figure 2a–c), with the
exception of some UW-CSCC1-R-specific reductions in minor allele copy number present in
chromosomes 9 and 11. The UV-associated (C >T) mutational signature was conserved in the
cell lines (Figure 2f), accounting for >70% of small nucleotide variants (SNVs) across all samples.
Some discordance was evident with respect to signature 7c and signature 58 between the various cell
lines and Patient 1 tumor.
The numbers of SNVs in the non-coding regions were similar between the samples, despite some
additional variants in the cell lines (Figure 2g). The up- and down-stream 2 kbp regions were analyzed
more discretely, revealing a similar pattern of fidelity (Figure S1), which has implications for gene
transcription. A detailed analysis of the coding variants in the Patient 1 tumor, its associated cell
lines and UW-CSCC2 identified 6518 exonic SNVs across 4335 genes (Figure S2a,b). Most of these
mutations functionally relate to poly(A) RNA binding (Figure S2c). In the Patient 1 tumor and related
cell lines, a substantially higher number of variants was observed compared to UW-CSCC2. Of the
2992 genes featuring exonic variants in the Patient 1 tumor, 2924 (97.73%) were shared with matched
cell lines UW-CSCC1 and UW-CSCC1-R (Figure S2d). A further 165 aected genes were identified and
Int. J. Mol. Sci. 2020,21, 9536 4 of 16
shared between UW-CSCC1 and UW-CSCC1-R only. Either this reflects new mutations or, more likely,
it demonstrates the increased purity in the cell lines, facilitating greater variant calling confidence.
There were 35 and 56 genes containing exonic variants exclusive to UW-CSCC1 and UW-CSCC1-R,
respectively. The majority (>97.5%) of the exclusive variants in UW-CSCC1-R were silent or missense
SNVs. Others included a frameshift truncation in PDGFRL and a stop-gained variant in DYNLL1
(Figure S2d). Moreover, the highest SNVs variance was observed in AHNAK2,AHNAK,MUC12,
and KMT2C across the Patient 1 tumor, UW-CSCC1 and UW-CSCC1-R (Figure S2e).
Figure 2.
Genomic landscape of cell lines and matched tumors. Circos plots showing overall pattern
of genetic aberrations between (
) the tumor of Patient 1 and matched cell lines, (
) UW-CSCC1 and
) UW-CSCC1-R; or (
) the tumor of Patient 2 and the matched cell line (
) UW-CSCC2. The layers
indicate the following (going from outside in): (i) chromosomes, with the darker shaded areas
representing large gaps in the reference genome due to regions of centromeres, heterochromatin,
and missing short arms; (ii) the purity-adjusted allelic frequency of all observed SNV (including introns
and intergenic regions); (iii) all observed copy number changes; with losses indicated in red and copy
number gain shown in green; (iv) the minor allele copy number (minor allele losses are indicated in
orange, whilst blue shows regions of minor allele gain); (v) the observed structural variants within or
between the chromosomes (translocations are indicated in blue, deletions in red, insertions in yellow,
tandem duplications in green and inversions in black). (
) Mutational signature frequency of cell lines
and tumor from Patient 1. (
) Total number of non-coding variants detected amongst patient 1 tumor,
UW-CSCC1, and UW-CSCC1-R. The unique non-coding SNV specific to each sample is overlain.
Of the 1309 known oncogenes, tumor suppressor genes or cancer-associated genes previously
examined for somatic variation in a wider group of 15 cSCC lymph node metastases, which included
the parent tumor for UW-CSCC1/1-R [
], 60 genes shared short variants between UW-CSCC1/1-R
and UW-CSCC2 (Table S3). Of these 60 genes, the Patient 1 tumor and its cell lines, along with
UW-CSCC2, showed similar alterations across seven drug-targetable genes (Figure 3). Not surprisingly,
TP53 inactivating mutations were common across these cell lines (and the lymph node metastases,
Table S3), suggesting susceptibility to DNA-damaging drugs. While NOTCH1 mutations often
occur concomitantly with TP53 in cSCC [
], this was not evident in the Patient 1 tumor and its
derivative cell lines (Figure 3). Further, the Patient 1 tumor, its derivative cell lines and UW-CSCC2
all harbored copy number amplifications in genes of the PI3K/mTOR signaling pathway (Table S4),
Int. J. Mol. Sci. 2020,21, 9536 5 of 16
as well as mutations in AKT3 (Figure 3), supporting this as a key targetable pathway in metastatic
cSCC. Altogether, this suggests that as these cell lines share several targetable alterations in cancer
driver genes, they are thus broadly representative of the metastatic cSCC of the head and neck in the
context of drug susceptibility testing.
Figure 3.
Recurrent coding short nucleotide variants of druggable targets shared across Patient 1
tumor, UW-CSCC1/-R and UW-CSCC2. Stop-gained and missense variants are considered high- and
medium-impact, respectively. Synonymous variants are considered low impact. Refer to Supplementary
Dataset 1 for detailed variant information.
2.3. Eect of In Vitro Culture on Cancer Gene Expression Patterns
The unbiased clustering analysis of gene expression from the nCounter panels separated the
Patient 1 tumor-derived
in vitro
cell lines from
in vivo
(patient tumor and xenograft) samples (Figure 4).
Of the various
in vitro
conditions tested, passage number resulted in the most impact, with low
passage number separating UW-CSCC1 away from the cells under the other
in vitro
(i.e., high passage, irradiated, normoxic and spheroid), which otherwise exhibited very similar pathway
score profiles. Indicators of stemness and DNA damage repair were upregulated in UW-CSCC1-R
relative to UW-CSCC1 (Figure S3a,b). Cell adhesion gene set expression was mostly downregulated in
UW-CSCC1-R (Figure S3c), which was reflected physiologically in the inability of UW-CSCC1-R to
form tight spheroids compared to UW-CSCC1.
Many pathways’ gene set expressions under
in vitro
conditions were downregulated relative
to the
in vivo
samples, with dierences between xenograft samples and the Patient 1 tumor evident
only for a subset of pathways in the PanCancer progression analysis (Figure 4a). In the PanCancer
pathways analysis, the gene set expression in all biological categories was very similar between the
xenograft and the Patient 1 tumor (Figure 4b). A comparison of the overall gene expression of a very
early passage of xenograft-derived UW-CSCC1 (Xenograft 2
UW-CSCC1 cell line) to the xenograft
(Figure 4a) revealed similar pathway score profiles.
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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 17
Figure 4. Cancer gene expression analysis. (a) nCounter PanCancer progression and (b) nCounter
Pancancer pathways panel gene expression pathway score profiles for Patient 1 and UW-CSCC1
culture derivatives. Nanostring heatmaps showing how pathway scores (fit using the first principal
component of each gene set’s data) change across samples or culture conditions. Pathway scores
condense each sample’s gene expression profile into a small set of pathway scores. Orange indicates
high scores; blue indicates low scores, i.e., samples that exhibit similar pathway score profiles. Scores
are displayed on the same scale via a Z-transformation. Sample details including passage number are
provided in Table S5.
2.4. High-Throughput and Secondary Drug Screens Reveal Novel Therapeutic Targets in Metastatic cSCC
Cell Lines
The novel drug susceptibilities of UW-CSCC1 and UW-CSCC1-R were identified using single-
dose anti-cancer compounds and kinase inhibitor screening libraries. The top 40 most efficacious
drugs for both libraries, along with their respective targets, are listed in Tables S5.1–S5.4. Both
libraries demonstrated a bimodal distribution, partitioning the majority of tested compounds as
either ineffective or highly effective at 1 µM (Figure 5a,b). Between the cell lines there was a strong
and moderate positive correlation for the anti-cancer and anti-kinase drug response, respectively.
The majority of the effective drugs (with > 70% inhibition) in the anti-cancer library targeted cell cycle,
cytoskeletal signaling, DNA damage, as well as protease pathways (Tables S6.1–S6.2). Efficacious
compounds included topoisomerase or cyclin-dependent kinase (CDK) inhibitors. The highly
efficacious compounds in the kinase-inhibitor library largely pertained to targeting the cell cycle
(particularly via CDKs), the cytoskeleton, or the PI3K/Akt/mTOR pathways (Tables S6.3–S6.4) in both
cell lines.
Figure 4.
Cancer gene expressionanalysis. (
) nCounter PanCancer progression and
(b) nCounter Pancancer
pathways panel gene expression pathway score profiles for Patient 1 and UW-CSCC1 culture derivatives.
Nanostring heatmaps showing how pathway scores (fit using the first principal component of each
gene set’s data) change across samples or culture conditions. Pathway scores condense each sample’s
gene expression profile into a small set of pathway scores. Orange indicates high scores; blue indicates
low scores, i.e., samples that exhibit similar pathway score profiles. Scores are displayed on the same
scale via a Z-transformation. Sample details including passage number are provided in Table S5.
2.4. High-Throughput and Secondary Drug Screens Reveal Novel Therapeutic Targets in Metastatic cSCC
Cell Lines
The novel drug susceptibilities of UW-CSCC1 and UW-CSCC1-R were identified using single-dose
anti-cancer compounds and kinase inhibitor screening libraries. The top 40 most ecacious drugs
for both libraries, along with their respective targets, are listed in Tables S5.1–S5.4. Both libraries
demonstrated a bimodal distribution, partitioning the majority of tested compounds as either ineective
or highly eective at 1
M (Figure 5a,b). Between the cell lines there was a strong and moderate positive
correlation for the anti-cancer and anti-kinase drug response, respectively. The majority of the eective
drugs (with >70% inhibition) in the anti-cancer library targeted cell cycle, cytoskeletal signaling,
DNA damage, as well as protease pathways (Tables S6.1–S6.2). Ecacious compounds included
topoisomerase or cyclin-dependent kinase (CDK) inhibitors. The highly ecacious compounds
in the kinase-inhibitor library largely pertained to targeting the cell cycle (particularly via CDKs),
the cytoskeleton, or the PI3K/Akt/mTOR pathways (Tables S6.3–S6.4) in both cell lines.
Given the response to the PI3K inhibitor PIK-75 and the associated gene alterations in the
PI3K/AKT/mTOR pathway in all three cell lines, a secondary dose-response screen was performed
to elucidate IC
values. The UW-CSCC1 and UW-CSCC1-R sensitivity to PIK-75 did not dier
substantially, with IC
values being 0.122
M and 0.204
M, respectively. PIK-75 was
more potent against UW-CSCC2 compared to the other cell lines, exhibiting an IC
0.026 ±0.012 µM.
The dual PI3K/mTOR-targeting compound dactolisib was also strongly potent against the cell lines.
The inhibition of pAKT was observed with UW-CSCC1 and UW-CSCC1-R in response to IC
concentrations of PIK-75 and dactolisib, and the total AKT expression seemingly reduced in a
time-dependent manner (Figure 5d). IC
values for other common chemotherapeutics against the
cell lines are provided in Table 1for comparison. Of note, UW-CSCC2 appears to be more resistant
than UW-SCC1/1-R to carboplatin, which may reflect its dual TP53/NOTCH1 high impact mutations
(refer to Figure 3).
Int. J. Mol. Sci. 2020,21, 9536 7 of 16
Figure 5.
Drug screen response of UW-CSSC1/1-R. Overall distribution of percentage inhibition is shown
for the anti-cancer (
) and kinase-inhibitor (
) libraries. Negative inhibition implies a pro-proliferative
event has occurred. Lines of best fit and coecients of determination are shown. (
) Heatmap showing
the relative viability eect on the cell lines of the topmost eective PI3K/AKT/mTOR inhibitors.
) Western blots demonstrating total AKT and phosphorylated AKT (pAKT) levels in response to
pretreatment with the respective IC
value of the PI3K inhibitors PIK-75 or Dactolosib for the times
shown for both cell lines. The housekeeping gene GAPDH was used as a total protein loading control.
The densitometry analysis shows the ratios of pAKT/total AKT for each treatment.
Table 1.
values of chemotherapeutic agents against UW-CSCC1 and UW-CSCC2. Compounds are
listed in order of lowest IC
presented either as a mean or mean
standard error of the mean (SEM)
from >2 independent experiments each performed in triplicate.
Compound Drug Class UW-CSCC1 UW-CSCC1-R UW-CSCC2
IC50 µM
Dactosilib Dual PI3K/mTOR inhibitor 0.022 ±0.003 0.080 ±0.023 0.028 ±0.012
PIK-75 PI3K inhibitor 0.122 ±0.012 0.204 ±0.05 0.026 ±0.012
Antimetabolite 4.47 ±0.4 7.56 ±0.1 -
Platinum analogue 22.40 22.90 200.00
Compound not present in HTS drug screen.
3. Discussion
This is the first study to apply WGS and expression analysis on metastatic cSCC cell lines,
and to correlate those findings with small molecule drug responsiveness. Our results show that the
predominance of cSCC tumors and UV-associated mutation signature 7 are retained in the
cell lines. UW-CSCC1 almost completely retained the mutational landscape of the clinical tumor,
as has been witnessed in other cell lines [
], suggesting that genetic drift is minimal following 2D
culture. The additional coding and non-coding variants called in UW-CSCC1/1-R are not explicit
evidence of genetic drift, but rather a result of the culture purity enhancing variant detection through
the elimination of the stromal background. Adjustments in copy number were to be expected given
Int. J. Mol. Sci. 2020,21, 9536 8 of 16
that they are generally seen to be in greater numbers in cell lines [
]. The identification of SNVs
and CNVs among the cell lines pertaining to the PI3K/AKT/mTOR pathway builds upon previous
observations [
], supporting its prognostic utility, as well as being an indicator of therapeutic
responsiveness to corresponding selective inhibitors, as assessed by our compound screening.
The downregulated cancer gene expression profiles observed in the cell lines compared to the
original tumor are unsurprising given this eect has been seen in many cell lines of other cancer
types [
]. Contrastingly, Barretina et al. [
] observed a strong positive correlation between
947 cell lines and primary tumors, suggesting cell lines possessed many of the genomic aberrations
found in tumors. They proposed the dierences between cell lines and clinical tumors are a result of
background tumor microenvironment (TME)-related gene expression that could be simply subtracted,
leaving purely tumor cell-associated gene signatures.
High passage cultures were found to downregulate pathways further, highlighting the vulnerability
of high passage commercial cell lines. However, unpublished work from our group has shown
that changes in drug responsiveness over passage numbers were subtle. The spheroid derivative
demonstrated a slight change in expression profile towards the original tumor, although global changes
were found to be negligible, as found with other cell types [
]. It was observed in the normoxic
derivative that the genes involved in metastasis response were influenced by normoxic-inducible
factors, aligning with previous literature [41], supporting hypoxic culture conditions.
Cells surviving radiation may acquire new mutations or expose previously undetected mutations
present within the now-expanded subclone. The current study noted an increase in PCNA,MAD2L2,
and FEN1 gene expression in UW-CSCC1-R relative to UW-CSCC1; all of which contribute majorly
to DNA damage repair [
]. In addition, cell adhesion and stem cell-associated genes were also
altered in UW-CSCC1-R, contributing to a more mesenchymal physiology and likely contributing to
the changes observed in proliferation and drug responsiveness. In a clinical context, this may result in
more aggressive local recurrence; therefore, continued investigation into pre-clinical models based on
UW-CSCC1-R that imitate this scenario are warranted.
The cSCC secondary xenograft mostly regained the original tumor phenotype (indicative of
genome stability), with the exception of the following pathways: collagen family, ECM structure,
metastasis response, and regulation of angiogenesis. This may be due to the mouse stroma inhibiting
transcription of the relevant genes in the human cells, or species incompatibility as murine growth
factors do not activate certain human-specific pathways, e.g., human MET [
]. A similar study
compared cell lines with their successive xenografts and found a number of gene classes becoming
enriched in the xenograft tumors [
]. In contrast, it has been observed for small cell lung cancer that
many of the changes incurred by going onto plastic were irreversible [
]. We infer that this restorative
ability is cell line-dependent.
Thus, altogether, the WGS and gene expression data indicate that genotype was preserved in
the cell lines and that dierences in gene expression profiles due to
in vitro
culture can be largely
in vivo
. This property permits the highly reproducible and cost-eective derivation of cell
in vitro
for ecient biological assays, with the capability of resembling in situ tumor biology,
given the appropriate environment [45].
The establishment and anti-cancer drug library screening of UW-CSCC1 and its derivatives
also revealed ecacious agents and drug classes that will draw our focus in future investigations.
The moderate correlation in drug sensitivity between UW-CSCC1 and UW-CSCC1-R highlights the
eect that irradiation can have upon drug response. Nonetheless, the cells are still quite responsive to
chemotherapy post-radiation which, for the case
in vivo
, implies the potential benefit for chemotherapy
in the adjuvant setting or in cases of local recurrence. Drugs targeting cytoskeletal signaling were
particularly ecacious and may be related to mutations in SYNE1 which were notably present in
our cell lines as well as in 14/15 of the clinical samples assessed (Table S3). This gene has a role
in cytoskeletal arrangement and has been proposed as a biomarker of colorectal cancer in liquid
biopsies [
]. Dose-response screening and western blot analysis verified the sensitivity of the cell lines
Int. J. Mol. Sci. 2020,21, 9536 9 of 16
towards PIK-75, as well as the dual mTOR/PI3K-targeting dactolisib, thus permitting further pre-clinical
studies on the PI3K/AKT/mTOR oncogenic pathway in metastatic cSCC. A recent study similarly
identified sensitivity to dactolisib for invasive cell lines of cSCC [
]. According to the Genomics
of Drug Sensitivity in Cancer Database (; Accessed 1 December 2020)
the mean IC
for head and neck SCC cell lines using dactolisib (PIK-75 data not available) was
notably higher (311 nM) than the range observed with our cell lines (22–80 nM), further supporting the
sensitivity of our cell lines to this drug. Whilst cell cycle and cytoskeleton-targeting drugs were also
unsurprisingly highly ecacious in our study [
], these compounds do not oer the same degree
of specificity as PI3K/AKT/mTOR-targeting agents. Interestingly, some PI3K inhibitors produced
little to no response against the cell lines, a likely result of isoform specificity that requires further
investigation [
]. The anti-EGFR biologic Cetuximab is sometimes given to patients with advanced
cSCC, achieving overall response rates approaching 50% [
]. However, there are multiple mechanisms
by which downstream eectors of EGFR may become activated, including via PI3K [
]. Therefore,
the continued examination of such pathways in advanced cSCC is necessary.
Not surprisingly, TP53 inactivating mutations were common across these cell lines and the clinical
specimens assessed, which may explain their sensitivity to DNA-damaging drugs. The direct target
of p53, NOTCH1, is also often disrupted in cSCC and generally occurs early in cSCC [
]. However,
the Patient 1 tumor and its derivative cells lines did not harbor NOTCH1 mutations, suggesting this is
not a universal feature required for metastases. Concomitant NOTCH1 and TP53 stop-gain mutations
(high impact loss of function mutations) in UW-CSCC2 may explain the cell line’s resistance towards
carboplatin relative to UW-CSCC1 and UW-CSCC1-R.
In conclusion, the generation of the UW-CSCC cell lines with detailed genomic characterization
and HTS drug-screen data has provided important new insights into the biology of metastatic cSCC
and revealed new candidate therapeutic targets for this disease. Whilst much of the transcriptomic
dierence noted may prove experimentally and clinically irrelevant, caution must be exercised when
undertaking assays dependent upon the mechanisms provided by any significantly altered genes
and/or pathways. However, these dierences may be negated once they are re-established
in vivo
. It is
hoped that the provision and validation of these novel cell lines will stimulate further investigations in
this field.
4. Materials and Methods
4.1. Patient Characteristics and Specimen Selection
An intraparotid lymph-node metastasis of cSCC was resected from a 73-year-old male (
See Table S2
for patient characteristics). Clinical staging of the tumor was performed in accordance with the 8th
edition of the American Joint Committee on Cancer (AJCC) TNM staging manual. An area of the
tumor sample with 70% neoplastic content without necrosis, haemorrhage, high keratin content or
significant inflammation was selected for cell culture and nucleic acid extraction. A parotid nodal
metastasis from another patient was also processed (86-year-old male), however this sample forwent
extensive genomic and phenotypic analysis for reasons noted below. Tissue and blood were obtained
with written informed patient consent in accordance with the declaration of Helsinki and University of
Wollongong Human Research Ethics Committee’s approval (HE14/397; approval date: 26 May 2015).
4.2. Cell Culture Development and Maintenance
Tumor samples were dissected within 1 h of surgical resection into 1 mm
pieces, with some
fractions of the bulk tumor mass snap-frozen in liquid nitrogen for subsequent nucleic acid extraction.
Samples were either dissociated using the MACS Miltenyi human tumor dissociation kit in conjunction
with the gentleMACS
dissociator (Patient 1), or simply plated as an explant onto the tissue culture
surface (Patient 2). Resultant cultures were designated UW-CSCC1 and UW-CSCC2, respectively.
Dissociated tissue was passed through a 70
m MACS SmartStrainer, the filtrate was centrifuged,
Int. J. Mol. Sci. 2020,21, 9536 10 of 16
and the pellet was re-suspended in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with
10% heat-inactivated foetal calf serum (FCS), glucose (4500 mg/mL), and penicillin/streptomycin
(50 U/mL). Explant cultures were grown in Advanced DMEM/F12, supplemented with hEGF (20 ng/mL),
1% L-glutamine, 2% FCS, and penicillin/streptomycin (50 U/mL). Cells/explants were placed into tissue
culture flasks according to cell density and incubated at 37 C under a 5% CO2, 3% O2atmosphere.
Dierential trypsinization was employed to deplete the competing fibroblast population [
Pure metastatic cSCC cell cultures were confirmed within 13 (UW-CSCC1) and 4 (UW-CSCC2) passages
and could be frozen and thawed successfully. These cultures were henceforth regarded as low passage,
whilst a high passage culture of UW-CSCC1 was also established (UW-CSCC1-high passage) following
an additional 41 rounds of subculturing. To confirm the cultures as epithelial, antibodies specific
to cytokeratin 4/5/6/8/10/13/18 (CK223; Abcam, Cambridge, UK, Cat # ab115974) and the epithelial
cell adhesion molecule (EpCAM; Abcam, Cambridge, UK, Cat # ab7504) were used, as well as the
mesenchymal marker
-smooth muscle actin (
-SMA; Abcam, Cambridge, UK, Cat # ab7817) serving
as a negative control.
To obtain spheroid cultures (UW-CSCC1 Spheroid), cells were seeded in Corning
ultra-low binding plates (Sigma-Aldrich, St. Louis, MO, USA) at a density of 375 cells per well
using the aforementioned media formulation. UW-CSCC1 cells at passage 13 were also exposed
to the clinically relevant dose of 2 Gy orthovoltage X-ray radiation (Radiation Oncology Medical
Physics, Prince of Wales Hospital, Sydney, Australia) and surviving radio-resistant cells expanded
(UW-CSCC1-R). A normoxic acclimated population of cells (UW-CSCC1-Normoxic) was also derived
by incubating under standard atmospheric oxygen levels. A list of the cell lines generated along with
experimental derivations is provided in Table S5.
4.3. Generation of Mouse Xenografts
NOD scid gamma (NOD.Cg-Prkdc <scid >IL2rg <tm1Wjl >/SzJAusb) mice (aged 4–5 weeks old,
Australian Bioresources, Australia) were subcutaneously inoculated with cell lines at a density of
cells into the rear flanks. Upon reaching 10
10 mm
, tumors were harvested from the
sacrificed mice for cell culture, RNA extraction, and histological analysis. For culture from xenografts,
tumor tissues were cut into approximately 1 mm pieces and then incubated with tumor dissociation
enzymes (Tumor dissociation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) in culture media,
after which the tumor homogenate was centrifuged, and the pellet was resuspended and plated in
serum-free media and hypoxic conditions as above. All procedures were carried out in accordance
with the Australian Code for the Care and Use of Animal for Scientific Purposes 8th edition 2013,
and approved by the University of Wollongong’s Animal Ethics Committee (study AE15/17).
4.4. Organotypic Invasion Assay
Contractions of collagen I matrices using dermally derived telomerase induced fibroblasts (TIFs)
were performed as previously described [
]. Briefly, contraction to a 3D matrix was stimulated by
mixing a neutralized collagen I cocktail (8.8% v/v10
minimal essential media (Thermo Scientific,
Waltham, MA, USA); 75.8% v/v2 mg/mL collagen I; 8% v/v0.22 M NaOH (pH 7.4)) with TIFs
1×106 per 12 matrices
) resuspended in FCS. The TIFs used in this assay are required to be quiescent,
which was achieved by leaving the cells in culture for at least five days after confluency without a change
of media. Per 35 mm petri dish (Sigma-Aldrich, St. Louis, MO, USA), 2.5 mL of the collagen–fibroblast
cocktail was dispensed and allowed to polymerize for 30 min in an incubator at 37
C. Following this,
the petri dishes were topped with complete media (DMEM/10% FCS/P+S) and matrices permitted to
contract over a period of 5–12 days to give a diameter no smaller than 1 cm. The media was refreshed
every 2–3 days or as required depending on the state of the color indicator present within the media.
Contracted matrices were moved into 24-well plates (Greiner Bio-One, Kremsmunster, Austria)
and 100
L of complete media containing 3.0
105 of the cells under investigation was seeded atop
each matrix. After 15 min in the incubator at 37
C to allow the cells to settle to the matrix, a further
Int. J. Mol. Sci. 2020,21, 9536 11 of 16
L of media was added to each well and left to grow until confluent. The matrices were then
transferred onto the top of sterile 40 mm mesh screens in 60 mm petri dishes. Fresh media was added
until surface tension was created between the mesh grid and media, thereby creating an air–liquid
interface to promote invasion across the chemotactic gradient (in this case the underlying media).
After 7–14 days, the matrices were fixed in 4% neutral buered formalin for 24 h followed by another
24 h in 10% formalin. The fixed samples were dehydrated in 70% ethanol and processed overnight in an
ASP200 vacuum tissue processor (Leica Biosystems, Wetzlar, Germany). Each matrix was then sliced in
half dorsoventrally and embedded in paran using the EG1150 Modular Tissue Embedding Centre and
EG1150 Cold Plate (Leica Biosystems, Germany). The resultant paran-embedded tissue block was
sectioned at a thickness of 4
m using a RM2255 Fully-Automated Rotary Microtome (Leica Biosystems,
Germany) and transferred onto glass slides by floating sectioned tissue in a dH
O water-bath at 40
Slides were allowed to dry overnight prior to histological staining. Sectioned tissue was deparanized
in dipentene (POCD Healthcare, Sydney, NSW, Australia) and rehydrated using graded ethanol washes
(60–100% EtOH). Hematoxylin and eosin (H&E; POCD Sciences, Australia)-staining was performed
on a LeicaST4020 small linear stainer (Leica Biosystems, Germany) and slides mounted with DPX
(Sigma-Aldrich, St. Louis, MO, USA). Following an overnight drying period, the invasion incurred by
the cells was assessed through examination of the slides under a Leica DM4000 bright-field microscope
(Leica Biosystems, Germany).
4.5. Nucleic Acid Extraction
Tumor and cell line DNA and RNA were extracted as was previously described (Mueller et al., 2019).
All samples were quantified using the NanoDrop spectrophotometer (ND1000, ThermoFisher scientific,
Waltham, MA, USA) and met the purity requirements for downstream applications (A260/280 between
1.7 and 2.3). Samples were further quality controlled prior to WGS by the sequencing facilities.
4.6. Gene Expression Analysis
Gene expression on the NanoString nCounter Sprint platform was assessed using total
RNA (25–50 ng) with two dierent panels (PanCancer Progression and PanCancer Pathways;
each containing 740 target and 30 “housekeeping” genes) as per the manufacturer’s instructions
(NanoString Technologies, Seattle, WA, USA). Table S5 summarizes the samples run, the passage
number from which extracts were made, and the panel used.
Data were analyzed using NanoString’s nSolver Analysis Software v4.0 (https://www.nanostring.
com; NanoString Technologies, Seattle, WA, USA) with raw counts normalized using house-keeping
genes, selected based on a low coecient of variation between samples and average counts above
negative controls. Dierential gene expression was derived using nCounter default settings and quality
control governed as per manufacturer’s instructions.
4.7. Whole-Genome Sequencing (WGS) and Bioinformatics Analysis Pipeline
WGS was performed on an Illumina HiSeqX instruments (Illumina, San Diego, CA, USA) by
Genome.One Pty Ltd. (Sydney, NSW, Australia) and at Macrogen (Seoul, Korea). Germline DNA
(blood) was sequenced to a depth 30–45X and the experimental samples to 60–90X depending on the
sequencing provider.
Using a Burrows–Wheeler Aligner (BWA-MEM v0.7.10-r789) [
], paired-end sequencing reads
were aligned to Genome Reference Consortium Human Build 37 (GRCh37/hg19) and improved using
realignment around known indels using the Genome Analyser toolkit (GATK) version 3.3.0. PCR
duplicates were removed using SAMtools v1 and Picard metrics have been used to evaluate the run
quality. Somatic single nucleotide variants (SNVs) were called by Strelka v1.0.15 [
] from tumor-normal
pairs. Calculations of the tumor cellularity, ploidy, and copy number alterations were performed by
Purple and Sequenza 2.1.2 [
]. Major structural variants (SVs) were inferred with Manta 0.27.1 [
The annotation and further filtering of Strelka quality-passed SNVs and indels were done based on
Int. J. Mol. Sci. 2020,21, 9536 12 of 16
two dierent platforms, i.e., OpenCravat [
] and Gemini-Seave pipeline [
]. Mutational signatures
were determined for each specimen as per the method described by
Mueller et al. [18].
Using the
Integrated Genomics Viewer (IGV; Broad Institute, Cambridge, MA, USA) somatic variants of interest
were verified to ensure coverage and call accuracy. Unique and shared somatic mutation sets were
obtained for each sequenced sample using Bedtools v2.27.0.
4.8. Chemical Compound Library Assays and Analysis
Cells were screened using the CHiP 2G anti-cancer and kinase-inhibitor libraries (SelleckChem,
Houston, TX, USA), at a concentration of 1
M. A total of 1000 cells were seeded per well into 384-well
plates (PerkinElmer, Waltham, MA, USA, Cat. no. 6007558). Cells were cultured at 37
C, 5% CO
treated with anti-cancer (Selleckchem, Houston, TX, USA, Cat. no. L3000) and anti-kinase (Selleckchem,
USA, Cat. no L1200) small molecule libraries 24 h after cell seeding. Cell viability was measured after
72 h using the CellTiter-Glo
Luminescent assay (Promega, Madison, WI, USA, Cat no. G7570) as per
manufacturer’s protocol. Percentage viability was calculated from replicates relative to the average
DMSO control reading.
4.9. Secondary Dose-Response Screening
Based on clinical use and the results of the high-throughput screen, cell lines were screened with a
suite of chemotherapeutic agents including PIK-75 (Selleckchem, USA, Cat no. S1205) at a titrated
range. The metabolic activity of cells was determined using a CellTitre 96
Aqueous One Solution Cell
Proliferation Assay (Promega, USA, Cat no. G3581) according to the manufacturer’s instructions and
as previously described [
]. The raw data of treated cells were normalized against vehicle controls
with background absorbance values subtracted. Half-maximal inhibitory concentration (IC
) values
were derived with GraphPad Prism 6.0 using a logarithmic sigmoidal dose–response curve with the
variable slope parameter. The cell viability of treated cells was normalized against vehicle controls.
4.10. Cell Lysate Preparation and Western Blot Analysis
Near-confluent UW-CSCC1 and UW-CSCC1-R were treated with IC
concentrations of PIK-75
(120 nM or 200 nM, respectively) or dactolisib (22 nM or 80 nM, respectively) for 3 and 6 h, along with
a 6 h DMSO vehicle control treatment. Lysates were prepared by washing the cells in PBS, followed by
a 20 min incubation with RIPA buer (50 mM Tris-HCl (pH =7.4), 150 mM NaCl, 1% Triton-x100,
5 mM EDTA, 1 mM PMSF, 1 mM sodium orthovandate) on ice. The crude lysate was transferred
to a microfuge tube and centrifuged at 12,000
gfor 5 min at 4
C to pellet debris and genomic
DNA. The proteinaceous supernatant was subsequently aliquoted for long-term storage at
The protein concentration of lysates was determined using a Pierce Bicinchoninic Acid (BCA) Protein
Assay Kit (ThemoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
Protein extracts (20
g) were resolved on a 4–20% gradient SDS-polyacrylamide gel (Invitrogen,
Carlsbad, CA, USA, Cat no. NP0324BOX) under reducing conditions. Proteins were transferred
to a PVDF membrane and blocked in 5% skim milk powder containing Tris-buered saline with
0.2% tween (TBST) for one hour at room temperature. Membranes were rinsed with TBST and
then incubated overnight at 4
C with primary antibodies from Cell Signalling (Danvers, MA, USA),
including: rabbit anti-human AKT ([11E7] 1:1000, Cat no. 46855), rabbit anti-human phospho-AKT
(Ser473; D9E, 1:1000, Cat no. 4060S), and the housekeeping mouse anti-human GAPDH (D4C6R,
1:2000, Cat no. 97166S) diluted in TBST containing 2% skim milk powder. After three rinses with TBST,
membranes were incubated for 90 min at room temperature with horseradish peroxidase-conjugated
anti-rabbit (Cell Signalling, Danvers, MA, USA, Cat no. 7074), and anti-mouse (Abcam, Cambridge, UK,
Cat no. ab205719) IgG secondary antibodies at a dilution of 1:2000 in TBST containing 2% skim
milk powder. Chemiluminescence was generated using Pierce
ECL Western Blotting Substrate
(ThermoFisher Scientific, Waltham, MA, USA, Cat no. 32109) and visualized on the Amersham
Imager 600 (GE Healthcare, Chicago, IL, USA). Captured blots were subsequently analyzed using
Int. J. Mol. Sci. 2020,21, 9536 13 of 16
ImageJ (version 1.53) to obtain densitometry. The resulting densitometry units were then graphically
interpreted using GraphPad Prism5 v6.0.
4.11. Data Repository
The variant call format files have been deposited at the European Genome-Phenome
Archive (, which is hosted by the EMBL-European Bioinformatics Institute
(Cambridgeshire, UK) and the Center for Genomic Regulation (Barcelona, Spain), under accession
number EGAD00001004530.
Supplementary Materials: The following are available online at 0067/21/24/9536/s1.
Author Contributions:
Conceptualization: M.R., B.A., J.P., N.G.I.; formal analysis: J.P., M.R., B.A., A.S.T.,
M.-E.G.; methodology: J.P., E.M., G.M., M.R., A.S.T., M.-E.G., N.G.I.; investigation: J.P., E.M., G.M., A.S.T.;
funding acquisition: M.R., B.A., J.C.; supervision: M.R., B.A., N.G.I., J.C., R.G.; writing—original draft preparation:
J.P., M.R.; writing—review and editing: All; project administration: M.R. All authors have read and agreed to the
published version of the manuscript.
This research received financial support from the Illawarra Cancer Carers and NHMRC Ideas Grant
We thank the Kinghorn Centre for Clinical Genomics for assistance with production and
processing of whole-genome sequencing data and the Illawarra Cancer Carers for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
cSCC Cutaneous squamous cell carcinoma
NER Nuclear excision repair
SNV Small nucleotide variant
UV Ultraviolet radiation
WGS Whole genome sequencing
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... It provides an overview of the complete gene expression landscape and enables detailed single gene expression analyses of therapeutically relevant targets [10,21,40,41]. Specifically, the comparative characterization of the transcriptome of cell lines and their original tumor tissues as performed here has been proved a powerful approach to identify similarities and discrepancies between them [42]. Alas, for many established cell lines, the original tumor tissue is not available or its quality is unsuitable for RNA sequencing and therefore, transcriptome level comparisons are rare [42]. ...
... Specifically, the comparative characterization of the transcriptome of cell lines and their original tumor tissues as performed here has been proved a powerful approach to identify similarities and discrepancies between them [42]. Alas, for many established cell lines, the original tumor tissue is not available or its quality is unsuitable for RNA sequencing and therefore, transcriptome level comparisons are rare [42]. Comparing cell lines to the original tumor tissue in a whole-transcriptome sequencing approach, this study revealed conserved patient-specific characteristics, EMT-properties and downstream PI3K-AKT signaling, along with unique therapeutic target signatures and cell-culture-related adaptations. ...
... The optimal reference for characterizing a cell line is the original tissue from which it was initially derived. However, in most cases, such a sample is either not available or not suitable for RNA-seq, and thus, such studies are scarce [42]. The sample set analyzed herein, consisting of canine cell lines derived from four PAC, two PAC metastases Fig. 8 The KEGG prostate cancer pathway hsa05215 (A) and bladder cancer pathway hsa05219 (B) [67]. ...
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Background Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. Methods This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. Results Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. Conclusion Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.
... Having such cell lines from the same patient allow the study of tumor progression and help in the identification of the molecular events leading to cSCC development. Together with the previous, IC1, a cSCC cell line from a 77-years-old male immunocompetent patient and the T11 cSCC cell line, derived from a 48-years-old male transplanted patient has been used for path discovery and drug susceptibility studies (82,83). ...
... In fact, those cSCC patients that develop advanced disease, with metastasis, are treated with a combination of surgery and radiation therapy, but there are few successful therapies for the refractory tumors. Therefore, several laboratories work extensively for the establishment of both primary and metastatic cSCC cell lines (83,84). Furthermore, the use of the skin derived SCC cells assumes a significant meaning also in the context of those SCCs arising from a genetic condition, such as the RDEB (recessive dystrophic epidermolysis bullosa) where a targeted therapy approach is required (85). ...
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Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients’ care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.
... The effect of EGF on cell migration was assessed in a scratchwound assay using the IncuCyte ® Zoom Kinetic Imaging System (Essen BioScience, Ann Arbor, MI, USA). Patient-derived metastatic cSCC cell line UW-CSCC2 [described in detail in (52)] was seeded onto collagen 1-coated 96-well ImageLock plates (Essen). After 24 h incubation in low serum containing media (DMEM supplemented with 1% FCS, no EGF), the cells were scratched according to manufacturer's instructions using the 96-pin Essen Woundmaker ™ . ...
... Given that uPAR levels were increased on metastatic vs. non metastatic tumors and that EGF was identified as a master regulator leading to PLAUR upregulation, we sought to confirm this relationship in vitro using a metastatic cSCC cell line derived from a lymph node deposit, UW-CSCC2 (Patient 40, Table S3) (52). These cells constitutively express EGFR ( Figure 6A) (but did not harbor EGFR mutations or copy number variations, data not shown) and responded to exogenous 5-20 ng/ml EGF with increased cell migration ( Figure 6B) and uPAR protein levels ( Figure 6C) compared to untreated cells. ...
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Cutaneous squamous cell carcinoma (cSCC) of the head and neck region is the second most prevalent skin cancer, with metastases to regional lymph nodes occurring in 2%–5% of cases. To further our understanding of the molecular events characterizing cSCC invasion and metastasis, we conducted targeted cancer progression gene expression and pathway analysis in non-metastasizing (PRI-) and metastasizing primary (PRI+) cSCC tumors of the head and neck region, cognate lymph node metastases (MET), and matched sun-exposed skin (SES). The highest differentially expressed genes in metastatic (MET and PRI+) versus non-metastatic tumors (PRI-) and SES included PLAU , PLAUR , MMP1 , MMP10 , MMP13 , ITGA5 , VEGFA , and various inflammatory cytokine genes. Pathway enrichment analyses implicated these genes in cellular pathways and functions promoting matrix remodeling, cell survival and migration, and epithelial to mesenchymal transition, which were all significantly activated in metastatic compared to non-metastatic tumors (PRI-) and SES. We validated the overexpression of urokinase plasminogen activator receptor (uPAR, encoded by PLAUR ) in an extended patient cohort by demonstrating higher uPAR staining intensity in metastasizing tumors. As pathway analyses identified epidermal growth factor (EGF) as a potential upstream regulator of PLAUR , the effect of EGF on uPAR expression levels and cell motility was functionally validated in human metastatic cSCC cells. In conclusion, we propose that uPAR is an important driver of metastasis in cSCC and represents a potential therapeutic target in this disease.
... Many commercial drug libraries are available to screen for targeted inhibitors that may impact survival of cell lines in vitro. Multiple array-based drug screens have been performed in HNSCC cell lines and primary HNSCC cells in 2D tissue culture, to identify synergistic drug combinations or new targets for therapy [139][140][141][142][143][144]. These screens prominently identified many cell cycle and DNA damage response inhibitors impacting survival of HNSCC cells. ...
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Head and neck squamous cell carcinomas (HNSCC) develop in the mucosal lining of the upper-aerodigestive tract. In carcinogen-induced HNSCC, tumors emerge from premalignant mucosal changes characterized by tumor-associated genetic alterations, also coined as ‘fields’ that are occasionally visible as leukoplakia or erythroplakia lesions but are mostly invisible. Consequently, HNSCC is generally diagnosed de novo at more advanced stages in about 70% of new diagnosis. Despite intense multimodality treatment protocols, the overall 5-years survival rate is 50–60% for patients with advanced stage of disease and seems to have reached a plateau. Of notable concern is the lack of further improvement in prognosis despite advances in treatment. This can be attributed to the late clinical presentation, failure of advanced HNSCC to respond to treatment, the deficit of effective targeted therapies to eradicate tumors and precancerous changes, and the lack of suitable markers for screening and personalized therapy. The molecular landscape of head and neck cancer has been elucidated in great detail, but the absence of oncogenic mutations hampers the identification of druggable targets for therapy to improve outcome of HNSCC. Currently, functional genomic approaches are being explored to identify potential therapeutic targets. Identification and validation of essential genes for both HNSCC and oral premalignancies, accompanied with biomarkers for therapy response, are being investigated. Attentive diagnosis and targeted therapy of the preceding oral premalignant (preHNSCC) changes may prevent the development of tumors. As classic oncogene addiction through activating mutations is not a realistic concept for treatment of HNSCC, synthetic lethality and collateral lethality need to be exploited, next to immune therapies. In recent studies it was shown that cell cycle regulation and DNA damage response pathways become significantly altered in HNSCC causing replication stress, which is an avenue that deserves further exploitation as an HNSCC vulnerability for treatment. The focus of this review is to summarize the current literature on the preclinical identification of potential druggable targets for therapy of (pre)HNSCC, emerging from the variety of gene knockdown and knockout strategies, and the testing of targeted inhibitors. We will conclude with a future perspective on targeted therapy of HNSCC and premalignant changes.
... Interestingly, clinically-relevant genomic alterations of PIK3CA have been reported in some recurrent, metastatic cSCC [57] and PIK3CA activating mutations have been detected in some cSCC lymph node metastases [58]. These observations, together with results from a recent study reporting the inhibitory effect of the PI3K inhibitor PIK-75 on cell lines derived from cSCC metastases of the head and neck [59], strongly suggest that sensitivity to BYL719 should be further investigated in a panel of metastatic cSCC cells. ...
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Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.
Some beta genus human papillomaviruses (β-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from β-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure.
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Metastatic cutaneous squamous cell carcinoma (CSCC) is a highly morbid disease requiring radical surgery and adjuvant therapy, which is associated with a poor prognosis. Yet, compared to other advanced malignancies, relatively little is known of the genomic landscape of metastatic CSCC. We have previously reported the mutational signatures and mutational patterns of CCCTC-binding factor (CTCF) regions in metastatic CSCC. However, many other genomic components (indel signatures, non-coding drivers, and structural variants) of metastatic CSCC have not been reported. To this end, we performed whole genome sequencing on lymph node metastases and blood DNA from 25 CSCC patients with regional metastases of the head and neck. We designed a multifaceted computational analysis at the whole genome level to provide a more comprehensive perspective of the genomic landscape of metastatic CSCC. In the non-coding genome, 3′ untranslated region (3′UTR) regions of EVC (48% of specimens), PPP1R1A (48% of specimens), and ABCA4 (20% of specimens) along with the tumor-suppressing long non-coding RNA (lncRNA) LINC01003 (64% of specimens) were significantly functionally altered (Q-value < 0.05) and represent potential non-coding biomarkers of CSCC. Recurrent copy number loss in the tumor suppressor gene PTPRD was observed. Gene amplification was much less frequent, and few genes were recurrently amplified. Single nucleotide variants driver analyses from three tools confirmed TP53 and CDKN2A as recurrently mutated genes but also identified C9 as a potential novel driver in this disease. Furthermore, indel signature analysis highlighted the dominance of ID signature 13 (ID13) followed by ID8 and ID9. ID9 has previously been shown to have no association with skin melanoma, unlike ID13 and ID8, suggesting a novel pattern of indel variation in metastatic CSCC. The enrichment analysis of various genetically altered candidates shows enrichment of “TGF-beta regulation of extracellular matrix” and “cell cycle G1 to S check points.” These enriched terms are associated with genetic instability, cell proliferation, and migration as mechanisms of genomic drivers of metastatic CSCC.
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Background Cemiplimab, a high-affinity, potent human immunoglobulin G4 monoclonal antibody to programmed cell death-1 demonstrated antitumor activity in a Phase 1 advanced cutaneous squamous cell carcinoma (CSCC) expansion cohort ( NCT02383212 ) and the pivotal Phase 2 study ( NCT02760498 ). Here we report the primary analysis of fixed dose cemiplimab 350 mg intravenously every 3 weeks (Q3W) (Group 3) and provide a longer-term update after the primary analysis of weight-based cemiplimab 3 mg/kg intravenously every 2 weeks (Q2W) (Group 1) among metastatic CSCC (mCSCC) patients in the pivotal study ( NCT02760498 ). Methods The primary objective for each group was objective response rate (ORR) per independent central review (ICR). Secondary endpoints included ORR by investigator review (INV), duration of response (DOR) per ICR and INV, and safety and tolerability. Results For Group 3 (n=56) and Group 1 (n=59), median follow-up was 8.1 (range, 0.6 to 14.1) and 16.5 (range, 1.1 to 26.6) months, respectively. ORR per ICR was 41.1% (95% CI, 28.1% to 55.0%) in Group 3, 49.2% (95% CI, 35.9% to 62.5%) in Group 1, and 45.2% (95% CI, 35.9% to 54.8%) in both groups combined. Per ICR, Kaplan–Meier estimate for DOR at 8 months was 95.0% (95% CI, 69.5% to 99. 3%) in responding patients in Group 3, and at 12 months was 88.9% (95% CI, 69.3% to 96.3%) in responding patients in Group 1. Per INV, ORR was 51.8% (95% CI, 38.0% to 65.3%) in Group 3, 49.2% (95% CI, 35.9% to 62.5%) in Group 1, and 50.4% (95% CI, 41.0% to 59.9%) in both groups combined. Overall, the most common adverse events regardless of attribution were fatigue (27.0%) and diarrhea (23.5%). Conclusion In patients with mCSCC, cemiplimab 350 mg intravenously Q3W produced substantial antitumor activity with durable response and an acceptable safety profile. Follow-up data of cemiplimab 3 mg/kg intravenously Q2W demonstrate ongoing durability of responses. Trial registration number, NCT02760498 . Registered May 3, 2016,
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Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans and its incidence continues to rise. Although CSCC usually display a benign clinical behavior, it can be both locally invasive and metastatic. The signaling pathways involved in CSCC development have given rise to targetable molecules in recent decades. In addition, the high mutational burden and increased risk of CSCC in patients under immunosuppression were part of the rationale for developing the immunotherapy for CSCC that has changed the therapeutic landscape. This review focuses on the molecular basis of CSCC and the current biology-based approaches of targeted therapies and immune checkpoint inhibitors. Another purpose of this review is to explore the landscape of drugs that may induce or contribute to the development of CSCC. Beginning with the pathogenetic basis of these drug-induced CSCCs, we move on to consider potential therapeutic opportunities for overcoming this adverse effect.
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Cytoskeletal proteins, which consist of different sub-families of proteins including microtubules, actin and intermediate filaments, are essential for survival and cellular processes in both normal as well as cancer cells. However, in cancer cells, these mechanisms can be altered to promote tumour development and progression, whereby the functions of cytoskeletal proteins are co-opted to facilitate increased migrative and invasive capabilities, proliferation, as well as resistance to cellular and environmental stresses. Herein, we discuss the cytoskeletal responses to important intracellular stresses (such as mitochondrial, endoplasmic reticulum and oxidative stresses), and delineate the consequences of these responses, including effects on oncogenic signalling. In addition, we elaborate how the cytoskeleton and its associated molecules present themselves as therapeutic targets. The potential and limitations of targeting new classes of cytoskeletal proteins are also explored, in the context of developing novel strategies that impact cancer progression.
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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.
Although cutaneous squamous cell carcinoma (cSCC) is treatable in the majority of cases, deadly invasive and metastatic cases do occur. To date there are neither reliable predictive biomarkers of disease progression nor FDA-approved targeted therapies as standard of care. To address these issues, we screened patient-derived primary cultured cells from invasive/metastatic cSCC with 107 small-molecule inhibitors. In-house bioinformatics tools were used to cross-analyze drug responses and DNA mutations in tumors detected by whole-exome sequencing (WES). Aberrations in molecular pathways with evidence of potential drug targets were identified, including the Eph-ephrin and neutrophil degranulation signaling pathways. Using a screening panel of siRNAs, we identified EPHA6 and EPHA7 as targets within the Eph-ephrin pathway responsible for mitigating decreased cell viability. These studies form a plausible foundation for detecting biomarkers of high-risk progressive disease applicable in dermatopathology and for patient-specific therapeutic options for invasive/metastatic cSCC.
Background Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer and is responsible for over one million cases annually. While only 3-5% of SCCs metastasize, those that do are associated with significant morbidity and mortality. Using gene mutations to help predict metastasis and select therapeutics is still being explored. Objective To present novel data from targeted sequencing of 20 case-matched localized and metastatic high-risk SCCs. Methods A cancer-associated gene panel of 76 genes was run from formalin-fixed paraffin-embedded samples of 20 case-matched localized (10) and metastatic (10) high-risk SCCs (Vela Diagnostics). Results Using spatial clustering analysis, primary driver mutations were identified asEGFR in localized SCC and CDH1 in metastatic SCC. ERBB4 and STK11 were found to be significant co-occurring mutations in localized SCC. Pathway analyses showed the RTK/RAS, TP53, TGF-b, NOTCH1, PI3K, and cell cycle pathways to be highly relevant in all high-risk SCCs with the Wnt pathway enhanced in metastatic SCC only. Conclusions This study compared gene mutations between localized and metastatic SCC with the intent of identifying key differences and new potential targeted treatment options. To our knowledge, the co-occurrence ofERBB4 and STK11 mutations has not been previously reported. Targeted inhibition of CDH1 and the Wnt pathway should be further explored in metastatic SCC.
PURPOSE The modern researcher is confronted with hundreds of published methods to interpret genetic variants. There are databases of genes and variants, phenotype-genotype relationships, algorithms that score and rank genes, and in silico variant effect prediction tools. Because variant prioritization is a multifactorial problem, a welcome development in the field has been the emergence of decision support frameworks, which make it easier to integrate multiple resources in an interactive environment. Current decision support frameworks are typically limited by closed proprietary architectures, access to a restricted set of tools, lack of customizability, Web dependencies that expose protected data, or limited scalability. METHODS We present the Open Custom Ranked Analysis of Variants Toolkit ¹ (OpenCRAVAT) a new open-source, scalable decision support system for variant and gene prioritization. We have designed the resource catalog to be open and modular to maximize community and developer involvement, and as a result, the catalog is being actively developed and growing every month. Resources made available via the store are well suited for analysis of cancer, as well as Mendelian and complex diseases. RESULTS OpenCRAVAT offers both command-line utility and dynamic graphical user interface, allowing users to install with a single command, easily download tools from an extensive resource catalog, create customized pipelines, and explore results in a richly detailed viewing environment. We present several case studies to illustrate the design of custom workflows to prioritize genes and variants. CONCLUSION OpenCRAVAT is distinguished from similar tools by its capabilities to access and integrate an unprecedented amount of diverse data resources and computational prediction methods, which span germline, somatic, common, rare, coding, and noncoding variants.
There is no standard of care for unresectable cutaneous squamous cell carcinoma (cSCC). Chemotherapy, alone or combined with radiotherapy, is commonly used mostly as palliative treatment; moreover, its poor safety profile limits its use most of the time, especially in elderly patients. Thus, alternative options are needed. Targeted molecular inhibitors, such as the epidermal growth factor receptor inhibitor cetuximab, seem promising, but data are limited. We retrospectively evaluated clinical outcomes of cetuximab as a single agent in this indication. The primary endpoint was the Disease Control Rate (DCR) at 6 weeks according to RECIST criteria. Secondary endpoints included DCR at 12 weeks, objective response rate (ORR) at 6 and 12 weeks, progression-free-survival (PFS), overall survival (OS), and safety profile. Fifty-eight patients received cetuximab as monotherapy. The median age was 83.2 (range, 47.4 to 96.1). The majority of patients was chemotherapy naïve. The median follow-up was 11.7 months (95% CI: 9.6-30.1). The DCR at 6 and 12 weeks was 87% and 70%, respectively. The ORR was 53% and 42%, respectively, at 6 and 12 weeks. The median PFS and OS were 9.7 months (95% CI: 4.8-43.4) and 17.5 months (95% CI: 9.4-43.1), respectively. Fifty-one patients (88%) experienced toxicity, and 67 adverse events related to cetuximab occurred. Most of them (84%) were grade 1 to 2. Our study shows that cetuximab is safe and efficient for the treatment of patients, even elderly ones, with advanced cSCC. These results indicate that cetuximab is a promising agent to test in new combinations, especially with immune checkpoint inhibitors such as anti-PD-1 agents.
Background: Cemiplimab has shown substantial antitumour activity in patients with metastatic cutaneous squamous cell carcinoma. Patients with locally advanced cutaneous squamous cell carcinoma have poor prognosis with conventional systemic therapy. We present a primary analysis of the safety and antitumour activity of cemiplimab in patients with locally advanced cutaneous squamous cell carcinoma. Methods: This pivotal open-label, phase 2, single-arm trial was done across 25 outpatient clinics, primarily at academic medical centres, in Australia, Germany, and the USA. Eligible patients (aged ≥18 years with histologically confirmed locally advanced cutaneous squamous cell carcinoma and an Eastern Cooperative Oncology Group performance status of 0-1) received cemiplimab 3 mg/kg intravenously over 30 min every 2 weeks for up to 96 weeks. Tumour measurements were done every 8 weeks. The primary endpoint was objective response, defined as the proportion of patients with complete or partial response, according to independent central review as per Response Evaluation Criteria in Solid Tumors version 1.1 for radiological scans and WHO criteria for medical photography. Data cutoff was Oct 10, 2018, when the fully enrolled cohort reached the prespecified timepoint for the primary analysis. Analyses were done as per the intention-to-treat principle. The safety analysis comprised all patients who received at least one dose of cemiplimab. This study is registered with, number NCT02760498. Findings: Between June 14, 2016, and April 25, 2018, 78 patients were enrolled and treated with cemiplimab. The median duration of study follow-up was 9·3 months (IQR 5·1-15·7) at the time of data cutoff. An objective response was observed in 34 (44%; 95% CI 32-55) of 78 patients. The best overall response was ten (13%) patients with a complete response and 24 (31%) with a partial response. Grade 3-4 treatment-emergent adverse events occurred in 34 (44%) of 78 patients; the most common were hypertension in six (8%) patients and pneumonia in four (5%). Serious treatment-emergent adverse events occurred in 23 (29%) of 78 patients. One treatment-related death was reported that occurred after onset of aspiration pneumonia. Interpretation: Cemiplimab showed antitumour activity and an acceptable safety profile in patients with locally advanced cutaneous squamous cell carcinoma for whom there was no widely accepted standard of care. Funding: Regeneron Pharmaceuticals and Sanofi.
The altered metabolic programme of cancer cells facilitates their cell-autonomous proliferation and survival. In normal cells, signal transduction pathways control core cellular functions, including metabolism, to couple the signals from exogenous growth factors, cytokines or hormones to adaptive changes in cell physiology. The ubiquitous, growth factor-regulated phosphoinositide 3-kinase (PI3K)–AKT signalling network has diverse downstream effects on cellular metabolism, through either direct regulation of nutrient transporters and metabolic enzymes or the control of transcription factors that regulate the expression of key components of metabolic pathways. Aberrant activation of this signalling network is one of the most frequent events in human cancer and serves to disconnect the control of cell growth, survival and metabolism from exogenous growth stimuli. Here we discuss our current understanding of the molecular events controlling cellular metabolism downstream of PI3K and AKT and of how these events couple two major hallmarks of cancer: growth factor independence through oncogenic signalling and metabolic reprogramming to support cell survival and proliferation. This Review discusses the PI3K–AKT signalling network and its control of cancer cell metabolism through both direct and indirect regulation of nutrient transport and metabolic enzymes, thereby connecting oncogenic signalling and metabolic reprogramming to support cancer cell survival and proliferation.