Impact of chemotherapy on the release and function of extracellular vesicles in the pre-pubertal testis

Abstract

INTRODUCTION: Childhood cancer affects nearly 2,000 children in the UK each year and encompasses a range of pathologies. The incidence of childhood cancer is rising, and the peak age of onset occurs between 0 - 4 years old. Despite more children being diagnosed with cancer year after year, advances in diagnostics and therapeutics mean five-year survivorship is over 80% and rising. This means that approximately 1 in 500 young adults in the UK today is a survivor of childhood cancer.

Rising incidence coupled with increasing survivorship means there is a growing population of childhood cancer survivors with unmet health needs secondary to the unwanted off-target effects of cancer treatment. One such treatment modality is chemotherapy, particularly cisplatin, which is used in a range of childhood and adult cancers.

One known impact of cisplatin treatment in boys is reduced fertility, as it damages the spermatogonial stem cell (SSC) niche within the testis. This niche is essential for future fertility as these SSCs will differentiate into sperm following the onset of puberty. In post-pubertal males, this can be somewhat mitigated by the use of sperm cryopreservation for future fertility treatment. However, in pre-pubertal males, there are currently no viable fertility preservation options. As such, there is a need to reduce the impact of cisplatin-mediated damage within the testis. One unexplored area is how cisplatin alters intercellular communication in the testis: specifically, how it impacts the release of extracellular vesicles (EVs) and what the impact of these EVs have on other cell populations in the testis and how this may impact fertility.

EVs are small lipid-bound structures released by all cells within the body and are a method of cell-to-cell communication. They mediate their effects on recipient cells through the delivery of cargoes and interaction with cell surface receptor ligands. The cargoes of EVs are numerous and include RNA, DNA, lipids, proteins, metabolites, and organelles. Cargo loading within EVs is a dynamic process and is influenced by the state of the parent cell with cargoes capable of being loaded at a greater magnitude than identified free within the cell of origin, suggestive of a highly regulated process. Little is known about the release of EVs within the testis, and what is known is focused on the adult testis.

I hypothesise that cisplatin alters EVs in the testis, and these EVs negatively impact the remaining SSCs and their supporting somatic Sertoli cells.

METHODS: Human SSCs and Sertoli cells are challenging to culture. Mouse cell lines were used as surrogates including GC1-spg cells (mouse type B spermatogonia) and TM4 cells (mouse pre-pubertal Sertoli). Cells were cultured in EV-depleted cell culture media and treated with cisplatin at a range of doses. The EVs released by these cells were then isolated from cell culture media using size exclusion chromatography.

EV characterisation from GC1-spg and TM4 cells was undertaken, comparing those released by cisplatin-treated cells and controls. Change in EV number was quantified using nanoparticle tracking analysis and impact on treatment-naïve recipient cells was assessed using in vivo cell imaging and detection of cleaved caspase-3 to identify apoptotic cells. EV uptake and movement within target cells were examined using super-resolution microscopy. Individual EVs were assessed for common EV surface markers CD9, CD63 and CD81 using dSTORM imaging and transmission electron microscopy (TEM) while bulk assessment of EVs for another common EV marker, TSG-101, was undertaken using Western blotting.

Assessment of EV release and impact of cisplatin-induced EVs on tissues were assessed using human fetal testis tissues as they represent a surrogate model for human pre-pubertal testis. Tissues were obtained from fetuses between 13 and 18 weeks gestation and cultured using a hanging drop culture system established in our laboratory previously.

Incubation of cisplatin-derived and control EVs were undertaken for eight days and immunofluorescence was undertaken to quantify Sertoli cell number and the number of apoptotic cells. EV release from human fetal testis tissues in response to cisplatin treatment was also undertaken, and the number of EVs released from these tissues was characterised.

Analysis of the protein cargoes of TM4 EVs was undertaken using mass spectrometry to compare EVs released by cells treated with cisplatin and control EVs. A focused assessment of pathways involved in apoptosis and reproduction was undertaken.

RESULTS: EV size assessed using NTA fell within the 50-250 nm range expected to be classified as small EVs and confirmed using TEM EV imaging for both GC1 and TM4 cells. dSTORM imaging of single EVs for CD9, CD63 and CD81 demonstrated EVs shared some of these markers, but not all EVs were positive for these tetraspanins. Bulk assessment of EVs for TSG-101 identified both GC1 and TM4 EVs expressed this marker.

Mouse Type-B spermatogonia (GC1) and Sertoli (TM4) cells treated with cisplatin release approximately twice as many EVs as control cells, (GC1, 7.2x108 / mL vs 16.4x108 / mL, p=0.0002; TM4, 2.8x108 / mL vs 6.5x108 / mL, p=0.0001). EVs released by cisplatin-treated cells were taken up at a higher rate by treatment-naïve cells, compared to control EVs, when assessed using the IncucyteZOOM system, with GC1 cisplatin-derived EVs being taken up 5.2 x compared to control EVs, p=0.007, and TM4 cisplatin-derived EVs being taken up 4.9 x compared to control EVs, p=0.006. However, this difference was not observed when using a different methodology to quantify individual EVs within cells.

Once internalised, tracking individual EV movements within the cell demonstrated peri-nuclear localisation in both TM4 and GC1 cells. We identified EVs released by cisplatin-treated TM4 cells induced a higher rate (2.4 x increase) of apoptosis in treatment-naïve TM4 cells as compared to control EVs, p=0.007. Increasing the concentration of cisplatin-derived TM4 EVs did not lead to a dose-response effect but did continue to show a significant increase in apoptotic cells.

When cisplatin-derived EVs from TM4 cells were incubated with treatment-naïve GC1 cells, they resulted in lower rates of apoptosis vs control EVs (0.6x the rate of apoptosis, p Human fetal testis tissues treated with cisplatin did release EVs. However, there was no significant difference in the number of EVs released between cisplatin-treated and control tissue. TM4 cisplatin-derived EVs, which induced apoptosis in treatment naïve TM4 cells, were incubated with human fetal testis tissues. However, immunofluorescence did not reveal any significant increase in apoptosis or reduction in Sertoli cell number compared to tissues incubated with control EVs.