One hundred and eight patients that underwent either prostate surgery or needle biopsy were selected for this study. The clinical and pathological features of the patient cohort are shown in Table 1. Fresh tissue specimens obtained from surgical procedures (TURP or HOLEP) or from ultrasound needle biopsies were used for the propagation of primary cultures of stromal cells (HPSCs) using the tissue explant technique (Fig. 1). We established HPSCs primary cultures from a total of 67 trans-rectal needle biopsies obtained from separate individuals or from surgical specimens; 23 samples from TURP and 18 samples from HOLEP (Table 1). Prostate tissue explants were evenly distributed over the surface of tissue culture-treated dishes in a minimal volume of culture medium, to preserve the tissue in contact with the culture surface (Fig. 1C). This procedure allowed maintaining the adherence of the tissue wedges in close to 100% of the samples analyzed (Fig. 1C). After 7–10 days of explant culture, spindle-shaped cells started to migrate out of tissue explants onto the culture dish (Fig. 1C, 1E). Significant differences were observed in the average of sprouting days between the different samples. HPSCs obtained from benign transition zone using the TURP procedure took longer to start to migrate out of the explanted tissue (TZ-BAF-T; 23 days) when compared to HPSCs obtained from the HOLEP procedure (TZ-BAF-H, average 16 days) (Fig. 1E). On the other hand, in tissue samples obtained from peripheral zone through needle biopsies (PZ-CAF and PZ-BAF), the average time for the sprouting process to start was 10 days or less. HPSCs from PCa patients (PZ-CAF) took the shortest time to sprout out the explanted tissue of all groups (7 days). Statistically significant differences in the average sprouting time were found in PZ-CAF when compared to PZ-BAF. The efficiency (percentage) of the explant technique (i.e. the percentage of wedges that sprouted cells compared from the total that adhered to the culture dish) was nearly 97% in the tissue samples obtained through needle biopsies (22 in PZ-BAF and 45 in PZ-CAF) (Fig. 1F). However, from the tissue samples obtained through surgical procedures, 79% of HOLEP (TZ-BAF-H) and 62% of TURP (TZ-BAF-T) specimens sprouted out, which indicated that the efficiency of the explant technique was significantly higher when samples were obtained from needle biopsies (94% PZ-BAF and 97% PZ-CAF). Samples obtained from TURP showed the lowest efficiency of all procedures (Fig. 1F).
Table 1
Clinicopathologic characteristics of the patient’s cohort
Procedure
|
HOLEP
(n = 18)
|
TURP
(n = 23)
|
Biopsy Pre-Qx (n = 22)
|
Biopsy Pre-Qx/RP
(n = 45)
|
Diagnosis
|
BPH
|
BPH
|
without neoplasia
|
PCa
|
Type of sample
|
TZ-BAF-H
|
TZ-BAF-T
|
PZ-BAF
|
PZ-CAF
|
Age (yr.)
|
54–77
|
55–84
|
52–74
|
41–85
|
Prostate volume (cc)
|
33–133
|
39–74
|
23–97
|
27–200
|
PSA (ng/ml)
|
0.8–21.3
|
0.5–75
|
3.2–8.7
|
3.3–4730
|
Gleason
|
N/A
|
N/A
|
N/A
|
6.0–9.0
|
Metastasis
|
N/A
|
N/A
|
N/A
|
M0-M1
|
TZ: transition zone; PZ: peripheral zone; BAF: benign-associated fibroblast; CAF: carcinoma-associated fibroblast; BPH: benign prostatic hyperplasia; CaP: prostate cancer; HOLEP: holmium laser enucleation of the prostate; TURP: transurethral resection of the prostate; RP: radical prostatectomy; N/A: not applicable. |
An immunohistochemical characterization of the HPSC cells isolated from human prostate tissue specimens revealed that, even when most migrating cells possessed a typical mesenchymal morphology, there was a mix of epithelial and stromal cells in all groups analyzed, as determined by expression of the epithelial and mesenchymal markers cytokeratin and vimentin, respectively (Fig. 2A, B). A detailed immunostaining analysis for the expression of vimentin, αSMA and pan-cytokeratin was performed for PZ-BAF and PZ-CAF at different cell culture passages (from passage 0 to passage 4, Fig. 2C, D). Data indicated that for benign and malignant primary cultures of peripheral zone at passages 0 and 1, a small proportion of cells were positive for cytokeratin (Fig. 2C, D). However, at passage 3, the primary cultures were positive only for stroma markers, indicating that from passage 3 and on, primary cultures isolated from prostate needle biopsies were composed mostly by fibroblasts without evident epithelial cell contamination (Fig. 2C, D). In summary, the needle biopsies explant technique allowed us to obtain highly enriched cultures of BAF from the peripheral zone, and CAF from either, the primary tumor or locally advanced/metastatic PCa.
The morphological characteristics and proliferative rate of TZ-BAF, PZ-BAF and PZ-CAF cultures were evaluated in vitro using growth curve and Ki-67 expression analyses. Fibroblasts maintained their phenotypic characteristics during culture showing a uniform spindle-shaped morphology (Fig. 3A). Quantitation of cell growth was performed using the trypan blue exclusion method. Our results indicated that primary cultures of fibroblasts obtained from explants of malignant PZ tissue showed higher level of proliferation compared to both, benign TZ or PZ tissues (Fig. 3B). Based on the data presented in Fig. 3B, we determined the doubling time (Dt) for each group. Our results indicated doubling times of 3.1, 7.9 and 8.7 days for PZ-CAF, TZ-BAF and PZ-BAF, respectively. No significant difference in doubling time were observed between TZ-BAF and PZ-BAF (p < 0.05). To confirm the differences found between CAF and BAF, we performed an immunofluorescence analysis for the expression of the cell proliferation marker, Ki-67. Percentages of Ki-67-positive cells were significantly higher in PZ-CAF (∼80%) compared to both, TZ-BAF and PZ-BAF (less than 20% for both groups, Fig. 3C, D) primary cultures. Based on these observations, we hypothesized that prostatic fibroblast cultures of different histological and pathological origins have distinct transcriptional profiles. To test this hypothesis, we performed a transcriptomic analysis of a pool of 10 cDNA samples from each origin (TZ-BAF, PZ-BAF and PZ-CAF) using an Illumina Platform array technology. Prostate fibroblasts samples were analyzed by hierarchical clustering[13] using 18,392 genes, or considering only the 393 genes differentially expressed (Fig. 3E). Dendrogram analysis (Fig. 3E) showed that TZ-BAF cultures clustered differentially from both, PZ-BAF and PZ-CAF, which indicates that the most striking difference of the transcriptional programs was associated with the histological origin of the fibroblasts (transitional vs peripheral zones). Heatmap analysis of the genes expressed 10-fold higher or 10-fold lower (393 in total) in each zone is showed in Fig. 3F. Figure 3G and 3H represents the genes that are most differentially expressed in benign PZ fibroblasts compared to benign TZ fibroblasts. Gene Ontology (GO) analyses revealed that many of those genes were involved in biological processes that are known to be important in the development of benign prostatic diseases, such as smooth muscle contraction, cytoskeleton organization, lipid metabolic process and immune response. Since PCa originates mostly in the peripheral zone, the comparison of the transcriptional profiles of fibroblasts obtained from benign or malignant PZ tissues revealed a set of differentially expresses genes (Fig. 3G, H). GO analyses indicated that many of those genes were involved in processes that are associated with the activation of biological programs such as transcription, inflammatory response, cell adhesion and motility. To confirm the gene expression changes observed by microarray analyses, real-time RT-PCR and western blot were performed in TZ-BAF, PZ-BAF and PZ-CAF for the typical activated markers genes associated to CAF. qRT-PCR analyses indicated that the activation markers α-smooth muscle actin (αSMA), tenascin C (TNC) and collagen 1 (Col1), were significantly up-regulated in CAF compared to BAF (Fig. 4A). No significant changes were observed for vimentin and fibroblast activation proteins (FAP; Fig. 4A). Western blot analyses of the smooth muscle cell markers calponin and αSMA, and the fibroblast marker vimentin, revealed that TZ-BAF cultures were heterogeneous and composed mostly of smooth muscle cells (calponin positive), while PZ-BAF cultures exhibited less expression of the smooth muscle marker calponin (Fig. 4B, C). In contrast, PZ-CAF cultures showed a phenotypic switch to a predominantly myofibroblast population, a typical feature of CAF (Fig. 4B, C).
As studied extensively, CAF showed an increased ability to affect tumor progression in initiated epithelial cells compared to BAF [4, 14]. Therefore, we decided to test whether our CAF cultures, obtained from needle biopsies, could increase tumor formation and growth in vivo. The PCa cell line (PC3) was grafted alone, or in combination with PZ-BAF or PZ-CAF, subcutaneously in the flank of immunocompromised mice. After 30 days, tissue recombinants composed of PC3-CAF tumors exhibited striking growth with a maximum wet weight of 2942 mg, and an average weight of 2118 mg. In contrast, PC3-BAF tissue recombinants, and PC3 alone, demonstrated low growth after 30 days of the co-engraftment. PC3-BAF showed a maximum wet weight of 1080 mg, and an average weight of 738. PC3 alone showed a maximum wet weight of 1087 mg, and an average weight of 765 mg (Fig. 4C, D). These results confirmed that the tissue explant procedure from needle biopsies allowed establishment of CAF cultures that expressed typical activation markers and stimulated tumor growth of PCa cells in vivo.