Reconstituting human testicular germ cell neoplasia in situ (GCNIS) in vitro using iPSC-derived primordial germ cell-like cells (PGCLCs) and genetically engineered human testicular cancer cells

Massachusetts General Hospital, Boston MA

Investigators

Abstract

Testicular cancers (TCs) are most common malignancies in young adult men. Most TCs are Type 2 germ cell tumors, which arise from primordial germ cells (PGCs) or prospermatogonia, and the initial steps of TC carcinogenesis occur in prenatal testes. Germ Cell Neoplasia In Situ (GCNIS) are the common prenatally formed precursors of all subtypes of TCs. GCNIS are found in seemingly disease-free testes at the time of birth, and more than 90% of adult-onset TCs are associated with GCNIS located in non-malignant portions of the affected testes. GCNIS are also found in adult testes harboring high TC risk such as cryptorchidism or abnormal sexual differentiation. When GCNIS are found in non-cancerous testicular biopsy, ~50% cases develop TC in five years. Despite the obvious importance in TC pathogenesis, our knowledge of GCNIS is still very limited, due primarily to difficult access to live human GCNIS or the lack of adequate animal models or cell culture surrogates. Taking advantage of the latest progress in human germ cell biology, this R21 project will attempt to establish in vitro models recapitulating carcinogenic process of human GCNIS in the environment of xenobiotic testicular organoid culture or transplantation. We will produce human PGC-like cells (PGCLCs) from induced pluripotent stem cells (iPSCs) derived from TC patients. These PGCLCs or their precursor iPSCs will be genetically engineered to recapitulate the GCNIS-associated genetic events – namely, loss-of-function CHEK2 mutations, gain-of-function KIT mutations, deficiency of pro-apoptotic BAK1/BAX genes, and tetraploidization as our Forward approach converting normal PGCLCs to GCNIS cells. On the other hand, as our Reverse approach converting invasive TC cells to GCNIS cells, we will deplete amplified isochromosome 12p, which are found in practically all cases of TCs but only rarely in GCNIS, by CRISPR-aided targeted chromosome elimination. The engineered human PGCLCs and TCs will be subjected to two approaches to recapitulate GCNIS, which fill the lumen of seminiferous tubules without invading through its wall. Our Specific Aim 1 will construct the xenogeneic reconstituted testis (xrTestis) organoid culture, which consists of human germ cells and mouse embryonic gonadal somatic cells. In Specific Aim 2, the human PGCLCs/TC cells will be transplanted into the lumen of seminiferous tubules of genetically sterile W/Wv mice by microinjection, followed by explant culture of the mouse testes. We expect that successfully reconstituted human GCNIS will fill the lumen of the tubules-like structures in xrTestis (Aim 1) mouse testicular seminiferous tubules (Aim 2), expressing PGC/GCNIS markers OCT4 and NANOG. Immunofluorescence studies and single cell RNA-seq will be used to examine the resemblance of the GCNIS models to in vivo GCNIS, PGCs, gonocytes, and TCs as well as cultured PGCLCs, TC cells, and gonocyte-like cells emerged from PGCLCs. The unprecedented models of human GCNIS will be useful for studying molecular mechanisms of TC carcinogenesis and developing strategies for eradicating GCNIS or suppression of GCNIS progression to TC.