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Female immunity protects from cutaneous squamous cell
carcinoma
Timothy Budden1,2, Caroline Gaudy-Marqueste3, Sarah Craig1,2, Yuan Hu4,5, Charles H.
Earnshaw1,2, Shilpa Gurung1,2, Amelle Ra6, Victoria Akhras6, Patrick Shenjere7, Ruth
Green8, Lynne Jamieson8, John Lear9, Luisa Motta8, Carlos Caulín4,5, Deemesh Oudit10,
Simon J Furney11,12, Amaya Virós1,2,9,*
1Skin Cancer and Ageing Lab, Cancer Research UK Manchester Institute, The University of
Manchester, Manchester, UK
2National Institute for Health Research Manchester Biomedical Research Centre, Manchester
3Aix Marseille Univ, APHM, CRCM Inserm U1068, CNRS U7258, CHU Timone, Dermatology and
Skin Cancer Department, Marseille, France
4Department of Otolaryngology, Head and Neck Surgery, University of Arizona, Tucson, AZ, USA
5 University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA
6Department of Dermatology, St. George’s NHS Foundation Trust, London, UK
7Department of Histopathology, The Christie Hospital NHS Foundation Trust, Manchester, UK
8Department of Histopathology, Salford Royal NHS Foundation Trust, The University of
Manchester, Manchester UK
9Department of Dermatology, Salford Royal NHS Foundation Trust, The University of Manchester,
Manchester UK
10Department of Plastic and Reconstructive Surgery, The Christie Hospital NHS Foundation Trust,
Manchester, UK
11Genomic Oncology Research Group, Department of Physiology and Medical Physics, Royal
College of Surgeons in Ireland, Dublin, Ireland
12Centre for Systems Medicine, Royal College of Surgeons in Ireland Dublin, Ireland
Abstract
Purpose—Cancer susceptibility and mortality are higher in males, and the mutational and
transcriptomic landscape of cancer differs by sex. The current assumption is that men are at higher
* Corresponding author: Amaya Virós, MD, PhD, Wellcome Trust Clinician Scientist, Skin Cancer and Ageing Lab, Cancer
Research UK Manchester Institute, Alderley Park. Alderley SK10 4TG, United Kingdom. Telephone: +44 (0) 7878794211, +44 (0)
161 306 6038 Amaya.viros@cruk.manchester.ac.uk.
Conflicts of interest: No competing interests
Author contributions
Conceptualization: A.V.; Writing of the original manuscript and generation of figures T.B. and A.V.; Collection of data: T.B., A.V.,
S.C., C.E., A.R., V.A., P.B., D.O., R.G., L.J., L.M., J.L., S.G., Y.H., C.C., C.G.-M.; Analysis of data: T.B., A.V., L.M., C.C, S.J.F.;
Tools and methods: T.B., A.V., S.J.F.; Software: T.B., S.J.F.; Funding acquisition, project administration, resources, supervision: A.V.
Europe PMC Funders Group
Author Manuscript
Clin Cancer Res. Author manuscript; available in PMC 2022 September 22.
Published in final edited form as:
Clin Cancer Res
. 2021 June 01; 27(11): 3215–3223. doi:10.1158/1078-0432.CCR-20-4261.
Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts
risk of epithelial cancers as they expose more to carcinogens and accumulate more damage than
women. We present data showing women present less aggressive primary cutaneous squamous cell
carcinoma (cSCC) and early strong immune activation.
Methods—We explored clinical and molecular sexual disparity in immunocompetent and
immunosuppressed primary cSCC patients (N=738, N=160), advanced stage cSCC (N=63, N=20)
and FVB/N mice exposed to equal doses of DMBA, as well as in human keratinocytes by whole
exome, bulk and single cell RNA sequencing.
Results—We show cSCC is more aggressive in men, and immunocompetent women develop
mild cSCC, later in life. To test if sex drives disparity, we exposed male and female mice to equal
doses of carcinogen, and found males present more aggressive, metastatic cSCC than females.
Critically, females activate cancer immune-related expression pathways and CD4 and CD8 T
cell infiltration independently of mutations, a response that is absent in prednisolone treated
animals. In contrast, males increase the rate of mitosis and proliferation in response to carcinogen.
Women’s skin and keratinocytes also activate immune-cancer fighting pathways and immune
cells at ultraviolet radiation-damaged sites. Critically, a compromised immune system leads to
high-risk, aggressive cSCC specifically in women.
Conclusions—This work shows the immune response is sex biased in cSCC, and highlights
female immunity offers greater protection than male immunity.
Key words
cancer sex bias; cutaneous SCC; cancer immunity activation; non-melanoma cancer; cancer and
immunosuppression
Introduction
Many diseases show sex disparity in their epidemiology, clinical course and outcome(1),
and cancer incidence is higher in men even after adjusting for risk factors. Cancer
mortality also affects men disproportionately and women respond better to some cancer
therapies(2–4). Squamous cell carcinomas (SCCs) arise in epithelia from the head and neck,
lung, bladder, esophagus and skin, have a strong male bias and are primarily caused by
carcinogens such as tobacco, alcohol and ultraviolet radiation (UVR). The higher SCC
male incidence and mortality is thought to reflect the increased lifetime exposure of men
to carcinogens and delayed clinical care; and molecular studies show a higher burden of
carcinogen-driven mutations in male tumors(5–9). SCCs arising on the skin, cutaneous SCC
(cSCC)(10–12), are more frequent in men, the 2nd most frequent malignancy in humans and
the most common malignancy in patients with a compromised immune system(10,13,14).
Previous studies indicate male mice may be inherently more susceptible to cSCC(15,16),
so here we examined if the higher incidence and mortality of cSCC in men is due to
increased vulnerability to epithelial neoplasia reflected in the molecular landscape, rather
than due to increased exposure to a carcinogen. To test this hypothesis, we used the
most frequent cSCC mouse model driven by the topical cutaneous application of DMBA,
a polycyclic aromatic hydrocarbon that induces epidermal
Hras
mutations, followed by
topical tetradecanoyl-phorbol acetate (TPA), which induces inflammation and epidermal
proliferation, leading to epidermal papillomas and cSCC(17). The advantages of this model
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are that the genomic landscape of DMBA/TPA-induced cSCC displays significant overlap
with human cutaneous SCC, and that it allows the
in vivo
study of cSCC controlling for age,
strain, susceptibility and dose of carcinogen(18). We explored the clinical and molecular
sex bias of cSCC carcinogenesis in animals exposed to the same dose of carcinogen,
and confirmed the molecular findings in human skin and keratinocytes. Additionally, we
examined the relationship between age at diagnosis, histological grade of cSCC and immune
status in human cSCC patient cohorts to explore the role of immunity by sex.
Methods
Animal experiments
Experiments were performed in 4-week-old FVB/N male and female mice, and DMBA
(25mg/ml) and TPA (0.02mg/ml) in acetone applied once a week, two days apart for 6
weeks, followed by TPA weekly for 10 weeks or until tumor development. All procedures
involving animals were performed under the Home Office approved project license
P8ADED6C8, and UK Home Office regulations under the Animals (Scientific Procedures)
Act 1986. The study received ethical approval by the Cancer Research UK Manchester
Institute’s Animal Welfare and Ethics Review Body (AWERB).
Molecular analyses
DNA from the largest sized mouse tumor, adjacent treated skin and a kidney was sequenced,
patterns of single nucleotide variation, insertion and deletions in tumors and skin were
analysed and oncoplots generated of the top 20 frequently mutated genes by sex and
histology to identify patterns of mutations. Paired-end RNA sequencing was performed from
fresh whole DMBA/TPA treated skin from the back and normal skin from the abdomen.
Human single cell RNA-seq data from male and female keratinocytes was analysed from a
study of squamous cell carcinoma(19), downloaded from GEO database (GSE144236).
Histology
Mouse tumors were classified as papillomas, keratoacanthoma, well, moderately and poorly
differentiated cSCC, and visceral organs stained with H&E and pan-keratin to confirm
metastasis. Immunohistochemistry of tumors and skin was performed with anti-phospho-
Histone H3 (Ser10) (06-570) CD4 Antibody (14-9766-82) ThermoFisher / eBioscience and
CD8a Antibody (14-0808-82) ThermoFisher / eBioscience.
Human clinical data
A retrospective, non-interventional review of clinical data: age, sex, immune status and
histological grade of human primary cSCC, metastatic cSCC to the lymph node and
cSCC treated with systemic therapy from 3 UK NHS centers and one French hospital,
from immune-competent and immune-suppressed patients diagnosed by dermatologists
and pathologists under routine diagnostic practice were collected. The review at Salford
Royal is part of a larger skin cancer study IRAS approval: 216310, REC reference:
16/LO/2098. The Christie and St.George’s clinicians collected age, sex and immune status
within the SCC care audit. No patient consent of retrospectively obtained, anonymized
variables was required by local hospital and National Health Research Authority UK.
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The clinical data collection in the French cohort was approved by tumour board local,
national regulations, given Comite de Protection des Personnes Sud Méditerranée and Aix-
Marseile University Hospital approval. Publicly available genomic data from single cell
RNA seq analysis of human skin was accessed by GEO database (GSE144236). cSCC
were classified as keratoacanthoma (KA)/ well differentiated invasive SCC (1), moderately
differentiated SCC (2) and poorly differentiated SCC (3). The immune status of patients
was retrieved from clinical histories, and immunosuppressed patients were organ transplant
recipients on immunosuppressive medication, with white blood cell dyscrasia, systemic
cancer treatment with immunotherapy, radiotherapy or chemotherapy in the past 10 years,
chronic inflammatory disorders and autoimmune disorders on systemic immunosuppressive
medication.
Results
Immune competent men develop more aggressive cSCC than women
To explore if men are more susceptible to aggressive cSCC than women, we studied the
relationship between sex, age and histological grade in consecutive cSCC samples excised
from immunocompetent patients (n = 738; men: 459, women: 279, Supplementary Table
1A). We confirmed a male preponderance of cSCC in this cohort of immunocompetent
men(10) (62.2% men, Z-test p <0.0001), and found the median age of diagnosis to be similar
in men and women (median age men = 79, women = 81, Mann Whitney test p=0.10, Fig.
1A). Strikingly, men more frequently presented cSCC that was less differentiated, of higher
histological grade compared to cSCC of women (Fig. 1B, Mann Whitney test p=0.0002).
Furthermore, when we restricted our study by sex to the more aggressive variants of cSCC,
which have the highest risk of metastasis(14), we found that immunocompetent females
diagnosed with higher grade cSCC were older than men (median age men =80, median
women =83, p=0.04, Fig. 1C). Additionally, a review of the prevalence of metastatic cSCC
to the lymph nodes in a tertiary cancer hospital revealed male preponderance (men: 25,
women: 4, Z-test p = 0.0001, Supplementary Table 1B), and men developed aggressive
primary cSCC at a younger age than women (women: 86 (71-91), men 73 (41-91), p=0.01,
Figure 1D). Furthermore, we investigated sex bias in a cohort of patients with metastatic or
advanced stage cSCC that required systemic treatment, which showed more men received
systemic treatment than women (men: 62.2%, Z-test p = 0.0011; Supplementary Table 1C,
Figure 1E), and intriguingly, female cSCC was histologically more aggressive in this cohort
of poor outcome (Mann Whitney test p = 0.01, Figure 1F). These data show that men are at
higher risk of aggressive cSCC.
Male mice are more susceptible to chemically-induced aggressive cSCC
To determine if the increased susceptibility to aggressive cSCC in men is due to a greater
exposure to carcinogens or due to biological sex differences, we exposed immunocompetent
male and female mice, matched for age and strain, to equal doses of the carcinogen DMBA/
TPA, which promotes cSCC in mice. We recorded earliest lesion incidence and burden, and
found males developed more papillomas and cSCC earlier than female animals, although
this difference was not statistically significant (p=0.10, Fig. 2A). Animals presented a
range of squamoproliferative lesions, including epidermal hyperplasia, papillomas, well-
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differentiated, invasive SCC and more aggressive, undifferentiated SCC. However, compared
to females, males presented more advanced, aggressive subtypes of disease (Fisher exact
test, p=0.04) (Fig. 2B). Specifically, male cSCC presented more mesenchymal, spindle
features, compared to female cSCC. Importantly, only males presented metastatic lung
cSCC deposits (Fig. 2B, 2C). Taken together, these data show male mice developed more
aggressive primaries and metastatic cSCC than females exposed to the same dose of
carcinogen.
DNA damage accumulates equally in male and female animal skin and cSCC
Increased mutation burden underpins more aggressive forms of epithelial cancer(20), and
human male epithelial tumors have a higher mutation burden(5–9). Therefore, we examined
if the more aggressive male cSCCs in our animals are due to greater mutation accumulation,
or less DNA repair, leading to higher mutation burden. For this, we compared the tumor
mutation burden (TMB) in DMBA/TPA-induced mouse cSCCs (DT-cSCC), and found deep
targeted exome sequencing of DT-cSCC revealed the number of total mutations increased
with increasing histological grade, but did not differ by sex (Fig. 2D, 2E). Furthermore, we
found no differences in the pattern and types of mutations by sex (Supplementary Table 2,
Supplementary Fig. 1A).
Mouse tumors display a range of morphological features within the same histological grade,
and cancer-driving mutations can associate specific histological features(21). Therefore,
to ensure specimens were exactly comparable between males and females, we focused
on mutations accrued in clinically and histologically normal DMBA/TPA-treated skin (DT-
skin). We found males and females accumulate the same amount of genetic damage in
DT-skin (Fig. 2D), and similar to DT-cSCCs, there was no sex disparity in the number,
pattern and types of mutations (Supplementary Fig. 1B, Supplementary Table 2). These
data indicate carcinogens damage male and female DNA similarly, and the more aggressive
phenotype of male cSCC is independent of mutation burden.
Unique transcriptomic and immune changes by sex in mice
Male animals develop more aggressive cSCC independently of the mutation spectrum.
Therefore, we explored if the transcriptomic response to carcinogen exposure differed by
sex. To reduce the gene expression variability due to histological differences between
tumors, we compared the histologically normal, carcinogen exposed DT-skin of males
and females. We first investigated the autosomal gene expression changes in DT-skin
and normal skin, and then studied whether DT-skin gene expression varied by sex. We
found the most significant changes in DT-skin involved critical cancer immune regulatory
pathways including the interferon gamma and interferon alpha response pathways; as well
as the inflammation-related and allograft rejection genes (Fig. 3A, Supplementary Table
3A). In contrast, normal, untreated skin expressed genes involved in RAS signaling (Fig.
3A, 3B). We then investigated sex-specific changes in DT-skin, and found female DT-skin
presented more transcriptomic changes than male skin overall, including higher expression
of critical genes involved in cancer immunity, compared to males. We noted specifically the
cytokine interferon gamma (
Ifng
), which is known to drive potent antitumor activity(22–24),
to be increased in female carcinogen-treated skin. Intriguingly, we additionally observed
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female upregulation of the G1 to S-phase tumor suppressor, senescence-inducing cell
cycle regulator
Cdkn2a
(Fig. 3C, Supplementary Table 3B). Cdkn2a can exert ample anti-
tumor effects(25,26), and when expressed in keratinocytes restricts proliferation, increases
senescence and differentiation, and reduces tumor growth(27), consistent with its tumor-
suppressive roles. Furthermore, recent work shows natural and immune checkpoint cancer
immune control is achieved via Cdkn2a-dependent signaling, and interferons directly
activate
Cdkn2a
(28). We therefore examined cSCC incidence, histological grade and
metastasis in an available model of spontaneous carcinogenesis in
Cdkn2afl/fl
animals(29),
and found that although the study was not powered to detect sex differences,
Cdkn2afl/fl
female mice presented more spindle, aggressive primary tumour and metastatic cSCC than
Cdkn2awt
females (two-way Anova p =0.0796, Supplementary Fig. 1C, Supplementary
Table 4).
We next investigated if, in addition to immune-gene expression changes, female animals
present a unique immune cell landscape compared to males. For this, we studied the
proportion and subtypes of adaptive and innate immune cells by gene expression, which
revealed a trend in female skin for increased antigen-presenting CD1 cells and CD4 / CD8
tumor lymphocytes. In contrast, male animal skin tended to be enriched for macrophages
(Supplementary Fig. 1D) although these were not significant (p>0.5, Mann Whitney test).
We explored gene expression by immunohistochemistry staining of CD4+ and CD8+ T
cells in DT-cSCC and DT-skin by sex, and confirmed female tumors and female DT-skin
were more infiltrated with immune cells than male DT-cSCC and DT-skin (Fig. 3D, 3E. To
confirm the immune response to the DT challenge is sex-specific, we compared the early
inflammatory and CD4+ cell infiltration in immunocompetent and immunosuppressed mice.
We confirmed that DT-skin from immunocompetent females presented a denser immune
cell (Supplementary Fig. 1E, 1F) and CD4+ infiltration (Supplementary Fig. 1G, 1H) across
all the layers of the skin, compared to males. However, immunosuppressed females and
males, treated with oral prednisolone in addition to DMBA/TPA, had an equally reduced
inflammatory cell response and CD4+ infiltration (Supplementary Fig. 1E-I). These data
indicate the early inflammatory response to carcinogen in immunocompetent animals differs
by sex, and this difference is lost in immunosuppressed animals.
Tumors in animals arise weeks after being exposed to the DMBA carcinogen, but the total
number of mutations per cSCC rises progressively in tumors of increasing histological grade
(Fig. 2E). We hypothesized the selection and growth of the more mutated clones leading to
more aggressive cSCC occurs due to increased cell division and a proliferation advantage
of these clones. As males present more aggressive disease, and female epidermis expresses
higher levels of the cell cycle regulator
Cdkn2a
, we investigated if a higher epidermal
proliferation rate in the male epidermis could underpin speedier clonal selection and disease
progression of male disease. For this, we compared the rate of the mitosis-specific marker
phospho-histone 3 (phospho-H3) in male and female epidermis and observed that DT-skin,
compared to animal-matched non-DT skin was thickened, presenting increased number of
layers, independently of sex (Supplementary Fig. 1J, p<0.0001). Next, we compared male
to female DT-epidermis and found that phosphoH3 in male epidermis was increased in
males (Fig. 3F, 3G, p<0.0001). We compared the expression of phospho-H3 in a subset of
cSCCs, and again observed a trend for higher expression in males (Supplementary Fig. 1K,
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1L). Thus, male and female mouse epidermis modulate the rate of epidermal proliferation
differently following exposure to a chemical carcinogen.
Taken together, these findings indicate the transcriptome and immune cell response of
male and female mice differs at the earliest stage of carcinogenesis, independently of
DNA damage. Males present higher rates of epidermal proliferation following chemical
carcinogen exposure compared to females. In contrast, females strongly upregulate
immune responses and cancer-linked lymphocytes, implicating immunity in delayed female
tumorigenesis.
Human female skin upregulates immune-related cancer defense pathways
To validate the role of immune-related expression changes and immune cells modulating
carcinogenesis in females, we compared the sex-specific changes in single cell RNA
of normal male and female human keratinocytes obtained from cSCC-adjacent skin(19).
Human cSCC is driven by ultraviolet light and arise in fields of severely sun-damaged skin,
so we reasoned cSCC-adjacent keratinocytes, from sun exposed anatomic sites of adults,
will accumulate significant carcinogen exposure. Indeed, we found that similar to animal
DT-skin, the most prominently upregulated pathways in human keratinocytes overlapped
with mouse skin. Strikingly, the most prominent pathway expression changes in all female
keratinocyte subtypes were up-regulation of interferon gamma (IFNG), interferon alpha and
allograft rejection pathway genes (Fig. 4A, Supplementary Table 5). In further agreement
with the mouse experiment, we found female human sun-exposed epidermis and cSCCs had
a trend to higher counts of CD4, CD8 T cells and CD1C dendritic cells, whilst male skin and
cSCC showed a trend for more macrophages (Mann-Whitney p=0.38, Supplementary Fig.
2A, 2B).
The use of the human cSCC single-cell RNAseq data allows expression patterns to be
isolated to specific cell types, and this reveals that in human skin and cSCC,
IFNG
is
almost exclusively expressed by T Cells. Cytokines
CXCL10
and
CXCL9
, which can
chemoattract T-cells, are expressed from multiple cell types including keratinocytes and
other immune cells such as Langerhans cells. The immune pathway enrichment of human
female keratinocytes (Fig. 4A) is particularly enriched for genes that play a role in antigen
presentation such as HLA genes and
TAP1
, suggesting a complex multicellular immune
response in human skin including alterations in cytokine production, T cell recruitment
and IFNG production, and increased antigen presentation of keratinocytes for immune
recognition in female skin after UV damage (Supplementary Fig. 2C, 2D). Overall, these
data indicate that the biological response to chemical and ultraviolet light carcinogen
exposure in keratinocytes varies by sex in mice and humans.
Female immunity inhibits aggressive cSCC
Because we found the immune response is critical to the carcinogen response in female
animals, and immune competent women have less aggressive forms of cSCC (Fig. 1B),
we reasoned women with compromised immune systems would present more aggressive
cSCC, comparable to immunocompetent men. To test this hypothesis, we investigated sex,
age and histological grade in cSCC specimens excised from a cohort of immunosuppressed
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patients (IS, n= 160, men =106 and women n=54, Supplementary Table 1, 6). Strikingly,
we discovered cSCC appeared at a younger age in both immunosuppressed men and
women, supporting the critical role of immunity in delaying cancer in both sexes (median
age men =72, women =71, p=0.25, Fig. 4B). We then compared the histological grade
in immunosuppressed and immunocompetent women, which revealed immunosuppressed
women present more histologically aggressive disease (p=0.029, Fig. 4C), of equivalent
grade to men. By contrast, men present aggressive disease regardless of their underlying
immune status (Fig. 4C).
Next, we restricted our study by sex to the more aggressive, risky variants of cSCC in
the immunosuppressed population, and found, as expected, that aggressive disease occurred
earlier in life in both sexes. However, the lower age at diagnosis particularly occurred in
women, suggesting the loss of immunity is more damaging to females (median age men
=73, median women =70, p=0.04, Fig. 4D). Taken together, these data show the immune
system is critical to contain carcinogenesis, and validate the immune response underpins less
aggressive disease in females.
Discussion
The molecular landscape of cancer differs by sex, and men are more frequently diagnosed
with cancer and die more of cancer than women. Men traditionally are exposed to more
carcinogens due to professional hazards and behavior, but in risk-adjusted epidemiological
studies, there is still an unexplained greater proportion of men with cancer compared to
women(2–4). Furthermore, there is significant variation in the DNA alterations and RNA
landscape by sex in most cancers, suggesting there are sex-specific susceptibilities and
biological processes driving female and male cancer(5–9). However, comparing human
tumors from different sexes is a limited approach as human tumors are not matched for age,
histological grade, underlying susceptibility to cancer, and dose of carcinogen.
In this study we show immunocompetent women have less aggressive cSCC than men.
We compared the development and the molecular hallmarks of cSCC in male and female
animals using a well-established mouse model of DMBA/TPA carcinogenesis, adjusted
for carcinogen exposure(18), showing that similar to other studies using UVB as the
carcinogenic challenge(15,16), males present histologically more aggressive cSCC and
metastasis.”
We show that the rate of DNA damage accumulation is equal in male and female mice
exposed to equal doses of DMBA. This indicates both sexes repair damage similarly
following exposure to the chemical carcinogen. Intriguingly, we observe the overall
mutation burden (TMB) increases as tumors become more aggressive, or with increasing
histological grade in both sexes; despite animals not receiving additional exposures to
carcinogen. This suggests that in DMBA-driven cSCC there is a selection for clones with
more mutations as cell division progresses and tumors advance. Previous work comparing
mutation burden in adjacent areas of early dysplasia, intermediate dysplasia and primary
cutaneous melanoma reveal increasing mutation burden with oncogenic stage(30); raising
the hypothesis that areas with early damage must receive additional carcinogenic exposure
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to drive cancer progression. However, our study indicates carcinogenic damage conferring
full oncogenic potential may occur at the early stage and will depend on successful
expansion of clones with higher TMB, as we discontinued carcinogen exposure before cSCC
onset.
Intriguingly, we find it is the transcriptional response to carcinogen exposure that differs
between the sexes. At the earliest stage of disease, female animals increase cancer immune-
related responses and recruit an immune cell landscape involved in cancer defense, which is
reversed in animals on prednisolone with a compromised immune system. Male animals,
by contrast, have more macrophages, which are linked to poor prognosis in cancer,
and comparatively show few immune-related gene expression changes. The macrophage
male sex bias, although from a small cohort, is mirrored in the human immune cell
cSCC landscape as well as in a mouse hepatocellular carcinogenesis model driven by
diethylnitrosamine(31), indicating they may be specifically involved in the male response to
extrinsic damage.
One critical gene expression difference we found between male and female animals is
the up-regulation of
Cdkn2a
in females, a fundamental cell cycle regulator that exerts
profound influence in both cell proliferation(25,26) and cancer-immune responses(27,28).
Intriguingly, our preliminary data show the histological grade and rate of metastasis is more
advanced in
Cdkn2afl/fl
than in
Cdkn2af
wild type females, suggesting
Cdnkn2a
expression
may play a critical role restricting female cSCC carcinogenesis. In further support for a
central role of
Cdnkn2a
, we found the mitotic rate of carcinogen-exposed epithelium was
higher in male epidermis, indicating greater proliferation, than female skin. Additionally,
epidemiological studies indicate that
CDKN2A
germline loss-of-function carriers, who
are at high risk for cSCC and melanoma, are particularly susceptible to tobacco-induced
lung cancers(32). This further supports a central role for CDKN2A in the response to
environmental damage beyond skin carcinogenesis.
We validated the relevance of the
in vivo
findings in human female and male epidermal
keratinocytes, which originated from a sun-exposed site(19), and in human cSCCs.
Keratinocytes from women mirror the response observed in female mouse epidermis,
up-regulating critical cancer-immune pathways, genes and immune cells following UVR,
compared to men. The use of human cSCC single-cell RNAseq data allows expression
patterns to be isolated to specific cell types, indicating the cytokine differences are expressed
by multiple cell types, including keratinocytes and immune cells, in keeping with a complex
multicellular immune response in human skin which requires further study.
Similar to the mouse model, although cSCC arises at a similar age in immunocompetent
men and women, men have significantly more aggressive disease, and the aggressive tumors
arise at a younger age in men. Restricting our analysis to immunosuppressed patients, we
show cSCC arises in younger men and women. Importantly, cSCC in immunosuppressed
women are significantly more aggressive, similar to male disease, whilst male disease
is aggressive regardless of immune status. Although our study includes a broad and
heterogeneous range of diagnoses and medication driving immune suppression in our
patients, these data show the immune response plays a critical role constraining cSCC
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progression at the earliest stage of disease in both sexes, as the age of incidence drops
in both men and women. However, the data indicate the mechanisms restricting tumor
progression are more robust in females. Thus, this study, adjusted for risk factors, implicates
distinct biology driving male and female cancer; and shows differences in immune responses
between the sexes. Future studies should be conducted to identify the potential regulators
of sex-linked SCC dimorphism, to understand if the sex bias in cSCC onset and outcome is
due to chromosomal content or due to hormonal causes, and if our findings extend to other
epithelial and non-epithelial cancers with a strong male bias.
These results are strongly aligned with clinical observations revealing higher incidence
of excess immunity-linked disease in females and unique immune responses to infectious
disease by sex(33). Personalized medicine approaches stratify cancer patients by genotype;
however, to date, the potential for cancer stratification and therapy by sex has not been
explored. Further work will be necessary to identify the potential regulators of sex-linked
cSCC dimorphism.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We acknowledge the contribution of CRUK Manchester Institute and Facilities, in particular
the Histology department led by Garry Ashton. We are grateful to the Khavari Lab for
advice and shared data. We acknowledge the support of the National Institute for Health
Research Manchester Biomedical Research Centre, Manchester.
Funding
AV: Wellcome Beit Fellow, personally funded by a Wellcome Intermediate Fellowship
(110078/Z/15/Z), additional work funded by Cancer Research UK (A27412) Leo
Pharma Foundation and Royal Society RGS\R1\201222. SJF: European Commission (FP7-
PEOPLE-2013-IEF – 6270270) and the Royal College of Surgeons in Ireland StAR
programme. The Irish Centre for High End Computing (https://www.ichec.ie/) provided
HPC infrastructure.
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Translational relevance
Sex bias affects cancer incidence, mortality and therapy response; and the molecular
landscape of cancer differs by sex. However, it is not known if the sex discrepancy
is due to a difference in behaviour and exposure to carcinogens, or due to sex-linked
susceptibility. This work reveals men are more susceptible to cutaneous aggressive
squamous cell carcinoma, in contrast to women who activate stronger immune responses
when challenged with the same carcinogens. The loss of immunity particularly affects
women.
Personalised medicine approaches stratify cancer patients by genotype; however, to
date, the potential for cancer stratification, prevention strategies and therapy by sex
and immune competency has not been explored. We show men and immunosuppressed
women are at higher risk of aggressive disease and may benefit from targeted prevention
programmes, closer surveillance and stratification for adjuvant therapy.
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Figure 1. Men have more aggressive cSCC.
(A) Age of immunocompetent patients diagnosed with primary cSCC by sex, n.s
not significant, Mann-Whitney p = 0.1. (B) Histological grade of primary cSCC
by sex in immunocompetent patients (median, interquartile range (IQR). (C) Age of
immunocompetent women and men diagnosed with moderately and poorly differentiated
primary cSCC. (D) Lymph node metastatic cSCC by sex and age at time of primary cSCC
diagnosis in immunocompetent patients. (E) Median age at primary diagnosis of a cohort of
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advanced stage cSCC treated with systemic agents. (F) Histological grade of cSCC by sex at
the time of primary tumour diagnosis (all tests Mann-Whitney).
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Figure 2. Male animals are more susceptible to chemically-induced aggressive cSCC.
(A) Days to first lesion in male and female skin exposed to DMBA/TPA, n.s not significant
Mann Whitney test p=0.1 (B) Histological grade of cSCC tumors by sex (Fisher exact test,
p = 0.04). (C) Representative H&E of papilloma (scale bar: 400μm), keratoacanthoma (scale
bar: 500μm), well differentiated cSCC (scale bar: 500μm), poorly differentiated cSCC (scale
bar: 500μm) and insert (box, scale bar: 100μm) and lung metastasis stain for pan-cytokeratin
(brown, scale bar: 1mm). (D) Tumor mutation burden in DMBA/TPA exposed skin (Treated
Skin) and DMBA/TPA-induced cSCC (Tumor) by sex, Mann Whitney test p = 0.7, p = 0.8
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(E) Tumor mutation burden in DMBA/TPA lesions by histological grade and sex. Error bars
represent standard error of the mean (bar).
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Figure 3. Female mouse skin upregulates immune cancer pathways and cells in response to
chemical carcinogenesis.
(A) Gene pathways differentially expressed by sex in normal skin (left) and in DMBA/
TPA-exposed skin (treated skin, right). Size of spheres represent numbers of enriched
genes in pathway, color represents significance of enrichment (red most significant). (B)
Differentially expressed genes enriched in pathways in normal skin and (C) treated skin.
Negative fold change (blue) represents genes expressed higher in female skin, positive fold
change (red) represents genes expressed higher in male skin. (D) Quantification of CD4
and CD8 in tumors and DMBA/TPA tumors and treated skin by sex, Mann Whitney test.
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(E) Representative images of H&E and CD4 and CD8 T cell IHC in male and female
cSCC (scale bar: 200μm). (F) Quantification of mitotic cell marker phospho-histone 3 (Ph3)
immunohistochemistry (IHC) intensity score low (1), moderate (2), high (3) and very high
(4), Mann Whitney test. (G) Photomicrographs of H&E (scale bar: 50μm) and mitotic cells
(Ph3, scale bar 15μm) IHC by sex in normal and DMBA/TPA-skin.
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Figure 4. cSCC histological grade of human cSCC is tied to female immunity.
(A) Pathways enriched for genes differentially expressed by sex in human adult
keratinocytes from UVR-exposed, tumor-adjacent skin by sex. Female pathways (left)
represent gene pathways expressed higher in female keratinocytes. Male pathways (right)
represent gene pathways expressed higher in male keratinocytes. Keratinocytes grouped into
basal, cycling and differentiated cells. Size of spheres represent numbers of enriched genes
in pathway, color represents significance (- log10(False discovery rate) of enrichment (red
most significant). (B) Age comparison of immunocompetent (IC) and immunosuppressed
(IS) patients diagnosed with primary cSCC by sex, Mann-Whitney (C) Histological grade
comparison of primary cSCC by sex in immunocompetent (IC) and immunosuppressed
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(IS) patients (median, interquartile range (IQR), Mann-Whitney. (D) Age comparison
of immunocompetent (IC) and immunosuppressed (IS) women and men diagnosed with
moderately and poorly differentiated primary cSCC, Mann-Whitney.
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