Cannabinoids as an environmental risk factor of testicular cancer: Assessment using cell culture models of human and mouse primordial germ cells and by exposure of pregnant mice - 1 Testicular cancer (TC) is the most common malignancy among young men aged 15 to 44 in the Unites States 2 and many other countries. Whereas TC has strong genetic risk factors such as race or family history, the 3 recent and global increase in TC incidence rate suggests the importance of the environmental risk factors in 4 TC etiology. Multiple epidemiological studies independently found a significant link between marijuana use and 5 increase in the TC risk. TC is derived from embryonic germline precursor cells – namely, primordial germ cells 6 (PGCs) or gonocytes, and its tumorigenesis initiates prenatally to form pre-malignant precursor lesions known 7 as Germ Cell Neoplasia In Situ (GCNIS) in testes before birth although diagnosis of TC peaks 25-35 years 8 after birth. Because all available studies link the increased TC risk to adolescence/adult marijuana use, critical 9 knowledge on effects of prenatal exposure to marijuana on TC risk is missing. The endocannabinoid system 10 regulates development of mammalian germ cells via paracrine actions through the cannabinoid receptors CB1 11 and/or CB2 expressed in germ cells and/or testicular somatic cells. We therefore hypothesize that the major 12 marijuana cannabinoids D9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD) may disrupt the normal 13 development of male PGCs/gonocytes via the cannabinoid receptors expressed by these germ cells and/or the 14 testicular somatic cells, leading to germ cell maldevelopment and transformation. To test this hypothesis, our 15 Specific Aim 1 will use human PGC-Like Cells (PGCLCs), a pluripotent stem-cell derived cell culture model of 16 PGCs, and the xenogeneic reconstituted testis (xrTestis), an organoid culture model in which human PGCLCs 17 are supported by mouse embryonic testicular somatic cells and differentiate to gonocyte-like cells (GoLCs). We 18 will expose normal or TC patient-derived PGCLCs (which harbor the genetic TC risk) with or without CRISPR- 19 knockout of CB1 and/or CB2, or xrTestes involving these PGCLCs, to THC, CBD, endocannabinoids, and 20 selective agonists/antagonists of the two cannabinoid receptors followed by bulk/single-cell deep sequencing 21 and various standard assays to obtain evidence that exposure of PGCLCs/xrTestis to cannabinoids causes 22 germ cell responses relevant to transformation, such as enhanced growth, delayed mitotic arrest of GoLCs, 23 suppression of apoptosis, chromosomal aneuploidy, and/or impaired differentiation. To distinguish the whole- 24 body effects of marijuana and its direct actions on prenatal testes, Specific Aim 2 will expose timed-pregnant 25 mice to THC and CBD during 12.5-17.5 dpc and collect fetal testes. Organ cultures of E12.5 embryonic testes 26 will also be exposed to THC and CBD for 5 days in vitro. Testicular samples after the in vivo or ex vivo 27 exposure to marijuana cannabinoids will be subjected to assays as described for Specific Aim 1 to obtain 28 evidence of germ cell transformation. The proposed research may obtain mechanistic evidence supporting the 29 link between marijuana and TC risk via direct gonadal effects of the cannabinoids, urging epidemiological 30 studies on marijuana use by pregnant women and TC risk of their male children.
