Human intermediate prostate cancer stem cells contribute to the initiation and development of prostate adenocarcinoma

Abstract

Background: Intermediate cells are present in the early stages of human prostate development and adenocarcinoma. While primary cells isolated from benign human prostate tissues or tumors exhibit an intermediate phenotype in vitro, they cannot form tumors in vivo unless genetically modified. It is unclear about the stem cell properties and tumorigenicity of intermediate cells.

Methods: We developed a customized medium to culture primary human intermediate prostate cells, which were transplanted into male immunodeficient NCG mice to examine tumorigenicity in vivo. We treated the cells with different concentrations of dihydrotestosterone (DHT) and enzalutamide in vitro and surgically castrated the mice after cell transplantation in vivo. Immunostaining, qRT-PCR, RNA sequencing, and western blotting were performed to characterize the cells in tissues and 2D and 3D cultures.

Results: We found intermediate cells expressing AR⁺PSA⁺CK8⁺CK5⁺ in the luminal compartment of human prostate adenocarcinoma by immunostaining. We cultured the primary intermediate cells in vitro, which expressed luminal (AR⁺PSA⁺CK8⁺CK18⁺), basal (CK5⁺P63⁺), intermediate (IVL⁺), and stem cell (CK4⁺CK13⁺PSCA⁺SOX2⁺) markers. These cells resisted castration in vitro by upregulating the expression of AR, PSA, and proliferation markers KI67 and PCNA. The intermediate cells had high tumorigenicity in vivo, forming tumors in immunodeficient NCG mice in a month without any genetic modification or co-transplantation with embryonic urogenital sinus mesenchyme (UGSM) cells. We named these cells human castration-resistant intermediate prostate cancer stem cells or CriPCSCs and defined the xenograft model as patient primary cell-derived xenograft (PrDX). Human CriPCSCs resisted castration in vitro and in vivo by upregulating AR expression. Furthermore, human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in PrDX tumors in vivo, which can dedifferentiate into CriPCSCs in vitro.

Conclusions: Our study identified and established methods for culturing human CriPCSCs, which had high tumorigenicity in vivo without any genetic modification or UGSM co-transplantation. Human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in the fast-growing tumors in vivo, which hold the potential to dedifferentiate into intermediate stem cells. These cells resisted castration by upregulating AR expression. The human CriPCSC and PrDX methods hold significant potential for advancing prostate cancer research and precision medicine.

Keywords: Cancer stem cell; Castration-resistant prostate cancer; Enzalutamide; Intermediate cell; Prostate adenocarcinoma.