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It’s Moustache season

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Case studies Oncology Review

It’s Moustache season: how single cell sequencing and growing a moustache can help prostate and testicular cancer research

November is the eleventh month of the year, contains 30 days and usually marks the beginning of the winter season. For some, this month holds some significant dates. There is Thanksgiving (4th Thursday of November) and Veterans Day (11th November) in the US, world vegan day (1st November), constitution day of India (26th November), All Saint’s Day (1st November), and the stranger, world toilet day (19th November). Recently, November has also become known as Movember. This has now become an annual event to raise awareness of men’s health issues, such as prostate cancer, testicular cancer, and men’s suicide. The event encourages people to grow a moustache during the month of November with the goal of raising awareness and money for men’s health.

This mo-vement originated in Australia in 2003 and has become a world recognised event with over 5 million supporters. Organised by The Movember Charity (www.movember.com), over the years it has raised enough money to fund over 1,200 men’s health projects in over 20 countries. The relevance to research prostate and testicular cancers are becoming increasingly important due to the rising prevalence rates of both cancers which accounts for a large percentage of cancer morbidity in the US and around the world (1).

Figure 1. Our mascot, Simon (Single cell, Multi-Omics Navigator) ‘growing’ a moustache for Movember.

The latest cancer statistics in the US has revealed that prostate, lung and colorectal cancers contribute to nearly half of all cancer occurrences in men (48%), where the majority of incidences are from prostate cancer (27%) (1). The pathophysiology of prostate cancer is complex and still poorly understood. While various known factors including ageing, obesity, family history and certain mutations are associated with prostate cancer, it is still unclear what the precise mechanism of this cancer (2,3). The prostate comprises of various cell types, such as, epithelial, immune, and stromal cells, each expressing a unique genetic profile. Research has strongly indicated a complex interaction between tumour cells surrounding the epithelial and stomal cells, potentially arising from the accumulation of somatic mutations in the epithelial cell genome (4,5). These mutations can occur in oncogenes or tumour suppressor genes leading to transcriptional or translational defects and ultimately effecting cellular homeostasis. The majority of genes possessing these mutations are involved in cell growth, DNA damage repair mechanisms, cellular proliferation and cell death (6,7). Previous studies have elucidated these genetic alterations to include fusions of TMPRSS2 with ETS family gene, MYC oncogene amplification, deletions and/or mutations within the PTEN and TP53 genes, and in advanced cases, the androgen receptor is amplified and / or mutated (8).

Androgen receptor (AR) signalling operates through cis-regulatory regions to regulate gene expression and is the main determinate of prostate cancer proliferation. Differences in tissue and cellular environments govern the interaction of AR signalling at specific enhancers, guiding a multitude of phenotypes through epigenetic heterogeneity. This phenotypic complexity of prostate cancer is further enhanced by the multifocal characteristics presented with a high degree of intra- and inter- tumour heterogeneity (9). This deems research into the tumour microenvironment of prostate cancer a difficult obstacle to tackle. Fortunately, the advancements in single cell sequencing technology have opened up unprecedented opportunities for analysing thousands of cells simultaneously in one sample. This has enabled the complex heterogeneity of the TME to be revealed.

Prostate epithelial cells have been further characterised from 3 subtypes (basal/luminal epithelial cells and neuroendocrine cells) to include hillock and club cells (10). Using single cell analysis, Song et al, examined prostate cancer biopsies and discovered high AR signalling correlates with certain cellular states in prostate epithelial cells. They also uncovered a population of tumour associated club cells that are potentially linked to prostate carcinogenesis (11).

Figure 2. UMAP projections of the different cell clusters from epithelial cells (left), where club cells were identified in certain clusters (right) (Image modified from Song et al (11), http://creativecommons.org/licenses/by/4.0/).

A study from Chen et al profiled the transcriptomes of over 111,000 single cells from 27 tissues samples, including prostate tumours and normal prostate tissue, to construct a comprehensive single cell expression atlas for the human prostate. They revealed a subset of epithelial cells correlating with disease aggressiveness, as well as, discovering how the tumour microenvironment activated multiple progression-associate transcriptomic programs (12).

Another study adopted single cell sequencing to map the developmental states against the evolution of the tumour subclones. Distinct subclones were identified with genomic and transcriptional heterogeneity, where some subclones exhibited distinct plasticity and cellular differentiation states (13). Another research group studying neuroendocrine prostate cancer (NEPC) was able to identify two NEPC gene expression signatures using single cell sequencing from analysing over 19,000 malignant cells (14).

Figure 3. UMAP plots displays tumour subtypes, subclones and cycling cells, showing cell cycle heterogeneity and differentiation. (Adapted from Ge et al (13), https://creativecommons.org/licenses/by/4.0/).

Compared to prostate cancer, testicular cancer is less common, however in men aged between 15-35 years, it is the most common diagnosed cancer (15) where the incidence has doubled over the past 40 years (16). Testicular cancer is defined into two distinct classes: germ cell neoplasia in situ related germ cell tumours, and non-germ cell neoplasia in situ related germ cell tumours. The majority of testicular cancers are germ cell tumours which are further sub classed into seminoma and nonseminoma, based on their histomorphology (17). Numerous environmental factors contribute to the development of testicular cancer, with the most common risk factor to be cryptorchidism, followed by family history, infections, and testicular trauma (18). Genetic factors include alterations in p53, polymorphisms in the PTEN tumour suppressor gene, dysregulation in the pluripotent program of foetal germ cells (18), as well as various single nucleotide polymorphisms (SNPs) markers that correlate with an increased risk of developing testicular cancer (19).

Due to the variation in testicular tumours, this can vary the prognosis for different histological types of tumours. Single cell sequencing has only recently been used to uncover the characteristics of testicular cancer at a cellular level. To date, only one published paper performed single cell sequencing on a patient diagnosed with testicular seminoma (20). The authors analysed tissue from tumour, PMBC, pelvic and renal hilus lymph node, obtaining a total of over 18,000 cells for sequencing, to characterise the metastatic cell lineages. They constructed a single cell map of testicular seminoma and discovered gene expression patterns specific to metastatic tumour cells. They were also able to identify some molecular markers specifically expressed by the metastatic cell lineage.

Figure 4. Violin plots showing the identified tumour specific markers (top). Distinct gene expression patterns of the tumour cells in primary testicular tumour tissue (bottom). (Image adapted from Mo et al (20), https://creativecommons.org/licenses/by/4.0/).

Singleron offers many different solutions if you are thinking of applying single cell sequencing analysis to your research. Get in touch with our expects at info@singleronbio.com.

References

  1. L. Siegel et al. Cancer statistics. CA Cancer J. Clin, 2022, 10.3322/caac.21708
  2. Sung, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin, 2021, https://doi.org/10.3322/caac.21660
  3. Olivas et al. Obesity, inflammation, and advanced prostate cancer. Nutr. Cancer, 2021, https://doi.org/10.1080/01635581.2020.1856889
  4. Tiwari, R et al. Dynamics of cellular plasticity in prostate cancer progression. Mol. Biosci. 2020, https://doi.org/10.1016/j.celrep.2022.111123
  5. Ahmed, M. & Eeles, R. Germline genetic profiling in prostate cancer: latest developments and potential clinical applications. Future Sci., 2016, https://www.future-science.com/doi/10.4155/fso.15.87
  6. Cancer Genome Atlas Research Network. The molecular taxonomy of primary prostate cancer.Cell, 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695400/
  7. Robinson, D. et al. Integrative clinical genomics of advanced prostate cancer. Cell, 2015, https://linkinghub.elsevier.com/retrieve/pii/S0092867415005486
  8. Rebello, R.J et al. Prostate cancer. Nat Rev Dis Primers, 2021, https://doi.org/10.1038/s41572-020-00243-0
  9. Kneppers, J et al. Prostate Cancer Epigenetic Plasticity and Enhancer Heterogeneity: Molecular Causes, Consequences and Clinical Implications. In: Campbell, M.J., Bevan, C.L. (eds) Nuclear Receptors in Human Health and Disease. Advances in Experimental Medicine and Biology, vol 1390. Springer, Cham, 2022. https://doi.org/10.1007/978-3-031-11836-4_15
  10. Henry, G. H. et al. A cellular anatomy of the normal adult human prostate and prostatic urethra. Cell Rep, 2018, https://doi.org/10.1016/j.celrep.2018.11.086
  11. Song, H et al.Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states. Nat Commun, 2022, https://doi.org/10.1038/s41467-021-27322-4
  12. Chen, S et al.Single-cell analysis reveals transcriptomic remodellings in distinct cell types that contribute to human prostate cancer progression. Nat Cell Biol,2021, https://doi.org/10.1038/s41556-020-00613-6
  13. Ge, G et al. Single-Cell RNA-seq Reveals a Developmental Hierarchy Super-Imposed Over Subclonal Evolution in the Cellular Ecosystem of Prostate Cancer. Sci.2022, https://doi.org/10.1002/advs.202105530
  14. Wang, Z et al. Single-cell transcriptional regulation and genetic evolution of neuroendocrine prostate cancer. iScience, 2022, https://doi.org/10.1016/j.isci.2022.104576
  15. Filippou P et al. Epidemiology of Prostate and Testicular Cancer. Semin Intervent Radiol, 2016, doi: 1055/s-0036-1586146
  16. Boccellino M et al. Testicular cancer from diagnosis to epigenetic factors. Oncotarget,2017, DOI: 18632/oncotarget.20992
  17. Moch H et al. The 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs-Part A: Renal, Penile, and Testicular Tumours. Eur Urol, 2016, DOI: 1016/j.eururo.2016.02.029
  18. Gaddam SJ and Chesnut GT. Testicle Cancer. [Updated 2022 Oct 3]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK563159/
  19. Loveday C et al. Validation of loci at 2q14.2 and 15q21.3 as risk factors for testicular cancer.Oncotarget, 2018, DOI: 18632/oncotarget.23117
  20. Mo L et al. Single-Cell RNA Sequencing of Metastatic Testicular Seminoma Reveals the Cellular and Molecular Characteristics of Metastatic Cell Lineage. Oncol, 2022, https://doi.org/10.3389/fonc.2022.871489
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