Human prostate cancer initiating cells isolated directly from localized cancer do not form prostaspheres in primary culture

Abstract

BACKGROUND Recent experimental studies suggest that hierarchical expansion from a minor population of cancer cells with an unlimited self-renewal capacity, termed cancer initiating cells (CICs), drives both lethality and heterogeneity of prostate cancer. Human prostate CICs have been established from only two primary prostate cancer patients, with the remaining established CIC lines being derived from metastatic sites from <10 patients. This suggests that the established CIC lines are significant “outliers” and may not be representative of the prostate CICs seen clinically. Thus, there is an urgent need to develop new approaches to achieve the “routine” establishment of CIC containing lines, particularly derived from primary prostate cancers. METHODS In the present studies, we confirmed that in serum free, high Ca2+ (i.e., DMEN: F12) growth factor defined (GFD) media plus androgen, a large (n = 10) series of established human prostate cancer cell lines derived from both localized and metastatic sites characteristically self-associate in suspension and grow as unattached spheroids, termed prostaspheres which contain CICs based upon their self-renewal in vitro and tumorigenicity in vivo. RESULTS Unfortunately, however, while dissociated single cells from human primary prostate cancer tissues are viable, contain CICs as documented by their ability to take and proliferate as xenografts, and produce prostaspheres when plated with serum free, high Ca2+/GFD-media plus androgen onto standard tissue culture flask, these prostasphere do not contain CICs. CONCLUSION The development of reproducibly methods to culture CICs isolated directly from localized cancers is still an urgent unmeet need of the prostate cancer research community.

Bone Marrow-derived Mesenchymal Stem Cells (MSCs) as a Selective Delivery Vehicle for a PSA-Activated Protoxin for Advanced Prostate Cancer

“…Both hBM-MSCs and PrCSCs stained positive for alpha-smooth muscle actin (aSMA) ( Figure 1E, G) and vimentin (Vim) ( Figure 1F, H), but not cytokeratins 5 (CK5) ( Figure 1I, K) or 8 (CK8) ( Figure 1J, L). These results are the absolute opposite of those obtained for PrECs, which are negative for aSMA and Vim, but positive for CK5 and CK8 [25][26][27][28]. Similar to hBM-MSCs ( Figure 2Q, S, T), differentiation of PrCSCs into adipocytes (Oil Red O-positive) ( Figure 2B, G, L), osteoblasts (Alizarin Red-positive) ( Figure 2D, I, N), and chondrocytes (Safranin O-positive) ( Figure 2E, J, O) was observed if the cells were cultured under the appropriate induction conditions, but not in the uninduced controls (Figure 2A, C, F, H, K, M, P, R).…”

“…From these explanted cells, outgrowth of fibroblast-like prostate cancer-derived stromal cells (PrCSCs) ( Figure 1A, B) was observed that had a similar morphology to human bone marrow-derived MSCs (hBM-MSCs) ( Figure 1C, D). If a portion of the same cellular suspension was cultured in keratinocyte serum-free media (K-SFM), basal-like prostate-derived epithelial cells (PrECs) were obtained [25][26][27][28]. Both hBM-MSCs and PrCSCs stained positive for alpha-smooth muscle actin (aSMA) ( Figure 1E, G) and vimentin (Vim) ( Figure 1F, H), but not cytokeratins 5 (CK5) ( Figure 1I, K) or 8 (CK8) ( Figure 1J, L).…”

“…hBM-MSCs were obtained from Lonza (Walkerville, MD). Primary prostate epithelial and stromal cells from patient radical prostatectomy specimens were isolated at our institution in accordance with an Institutional Review Board approved protocol according to previously published protocols [25][26][27][28]62] for the cell cultures used in the differentiation assays, immunofluorescence staining, and cell surface expression studies. hBM-MSCs and PrCSCs were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin in a 5% CO 2 , 95% air humidified incubator at 37˚C.…”

Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

“…Recent successful attempts are based on the implantation of tumor fragments under the renal capsules of immunodeficient mice in the presence of androgen-releasing pellets (42). Yet generation of cell suspensions from clinical samples and cell sorting are known to affect viability of prostate cancer cells (42,43), therefore preventing successful tumor transplantation (reviewed in ref. 19).…”

Low p16INK4aExpression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone

“…Normal prostate tissue was isolated from non‐cancer containing tissue locations within radical prostatectomy specimens obtained from localized prostate cancer patients according to NA_00001575 protocol approved by the Johns Hopkins Institutional Review Board, as previously reported . Single cell suspensions were obtained by digesting the prostate tissue overnight at 37°C in collagenase solution containing 0.28% collagenase I from Sigma–Aldrich, 1% DNase I from Sigma–Aldrich, 10% fetal calf serum (FCS) and 1× concentration of Gibco ® Antibiotic‐Antimycotic solution, in RPMI 1640.…”