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Background: Accumulating evidence supports that prostate cancer stem-like cells (PCSCs) play significant roles in therapy resistance and metastasis of prostate cancer. Many studies also show that nitric oxide (NO) synthesized by NO synthases can function to promote tumor progression. However, the exact roles of NOSs and NO signaling in the growth regulation of PCSCs and castration-resistant prostate cancer (CRPC) are still not fully understood. Methods: The regulatory functions of NOS-NO signaling were evaluated in prostate cancer cells, especially in PCSCs enriched by 3D spheroid culture and CD133/CD44 cell sorting. The molecular mechanisms of NOS-NO signaling in PCSCs growth regulation and tumor metastasis were investigated in PCSCs and mice orthotopic prostate tumor model. Results: Endothelial NOS (eNOS) exhibited a significant upregulation in high-grade prostate cancer and metastatic CRPC. Xenograft models of CRPC exhibited notable increased eNOS expression and higher intracellular NO levels. PCSCs isolated from various models displayed significant enhanced eNOS-NO signaling. Functional analyses demonstrated that increased eNOS expression could promote in vivo tumorigenicity and metastatic potential of prostate cancer cells. Characterization of eNOS-NO involved downstream pathway which confirmed that enhanced eNOS signaling could promote the growth of PCSCs and antiandrogen-resistant prostate cancer cells via an activated downstream NO-sGC-cGMP-PKG effector signaling pathway. Interestingly, eNOS expression could be co-targeted by nuclear receptor ERRα and transcription factor ERG in prostate cancer cells and PCSCs. Conclusions: Enhanced eNOS-NO signaling could function to promote the growth of PCSCs and also the development of metastatic CRPC. Besides eNOS-NO as potential targets, targeting its upstream regulators (ERRα and ERG) of eNOS-NO signaling could also be the therapeutic strategy for the management of advanced prostate cancer, particularly the aggressive cancer carrying with the TMPRSS2:ERG fusion gene. [ABSTRACT FROM AUTHOR]

Intratumor heterogeneity is one of the major features of cancers, leading to aggressive disease and treatment failure. Cancer stem-like cells (CSCs) are believed to give rise to the heterogeneous cell types within tumors. Hence, understanding the regulatory mechanism underlying the recurrence process of heterogeneous tumor by CSCs could facilitate the development of CSC-targeted therapies. Here, utilizing single-cell transcriptomics, we present the molecular profile of osteosarcoma CSCs-derived heterogeneous tumors consisting of CSC clusters, osteoprogenitor and differentiated cell types, such as pre-osteoblasts, osteoblasts and chondroblasts. Furthermore, by constructing the comprehensive map of modulated genes during CSCs self-renewal and differentiation, we identify RAN exhibiting specific peak expression in osteosarcoma CSCs clusters which is transcriptionally up-regulated by MYBL2. Functionality, MYBL2-RAN pathway promotes the CSCs self-renewal by enhancing the nuclear accumulation of MYC protein, which in turn boosts the overexpression of RAN as a positive feedback. Importantly, blockage of MYBL2-RAN pathway sensitizes CSCs to cisplatin treatment and synergistically enhanced the cisplatin-induced cytotoxicity. Both MYBL2 and RAN are highly expressed in clinical osteosarcoma tissues which indicate poor prognosis. Collectively, our study provides advanced insights into the regeneration process of heterogeneous tumor originating from CSCs and highlights the MYBL2-RAN pathway as a promising target for CSC-based therapy in osteosarcoma. [Display omitted] • Molecular landscape of osteosarcoma CSCs-derived heterogeneous tumors profiled by scRNA-seq. • RAN and MYBL2 are amplified in CSCs clusters and predict worse patient prognosis. • MYBL2 transcriptionally up-regulates RAN to promote CSCs self-renewal. • RAN enhances MYC nuclear accumulation which in turn boosts RAN expression. • Knockdown of MYBL2 and RAN sensitizes CSCs to cisplatin treatment and decreases tumorigenesis. [ABSTRACT FROM AUTHOR]

Background: Non-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly. Methods: In this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated. Results: Our results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres. Conclusions: The described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres. [ABSTRACT FROM AUTHOR]