Introduction: Early stages of prostate cancer (PCa) are treated with surgery and androgen-deprivation therapy, but PCa can turn castration resistant possibly due to stem cell-like cells that re-initiate PCa growth and lead to metastasis. Given that Cripto is highly expressed in human tumors, we hypothesise that Cripto might play a role in tumor initiation and late progression, so we knockout Cripto in PCa GEMMs models with the aim of studying its oncogenic role. Methods: N (Nkx3.1CreERT2, R26 LSL-YFP/ LSL-YFP), NP (Nkx3.1CreERT2; Ptenflox/flox, R26 LSL-YFP/ LSL-YFP) and NPK (Nkx3.1CreERT2; Ptenflox/flox; KrasLSL-G12D/+, R26 LSL-YFP/ LSL-YFP) were crossed with conditional Cripto knockout (CRIPTOflox/flox) to generate NC, NPC and NPKC. N mice mirror normal epithelium, NP mimic the first stage of the disease, with high-grade prostatic intraepithelial neoplasia (PIN)/carcinoma lesions with local invasive epithelium, while NPK have invasive prostate adenocarcinoma with metastasis. In vivo experiments’ workflow is the following: castration of 8-weeks old mice and induction after 1 month with 5 daily injections of tamoxifen, after 2.5 weeks mice were treated with weekly testosterone (10 weeks). Single cells were isolated from prostate tissue and the YFP+/- population recovered by FACS sorting and cultured as organoids. Results: Histopathological evaluation of NPC and NPKC showed presence of mPIN (100%) with a dominant cribriform morphology. NPKC feature invasive PCa with an extent dominant pattern of 100% and portions with dense stroma forming whorl-like structures and occasional sheet-like accumulations of polygonal in mPIN regions. NPC and NPKC, compared to NP and NPK, present a more reactive stroma with a mild/moderate inflammation reaction. Organoids express luminal and basal markers mirroring molecular features of matched tissues. Morphology varies consistently: NP, NPC, NPK and NPKC organoids are solid which is consistent with an oncogenic transformation, whereas N and NC organoids present a cystic morphology, with lower densities, which correlates with low-grade PIN phenotypes. NC, NPC and NPKC organoids feature a higher percentage of hollow organoids compared to N, NP and NPK respectively. Conclusions: Stromal alterations in NPC and NPKC might suggest a role for Cripto not only restricted to prostate epithelium. For this reason, studies on secreted Cripto inhibition with ALK4-Fc are planned. Our finding support that organoids are an efficient in vitro model replicating in vivo phenotypes. SOURCE OF Funding: DoD (U.S. Department of Defence)
