Operational classification of cutaneous squamous cell carcinomas based on unsupervised clustering of real cases by experts.

BACKGROUND: There is currently no staging system for cutaneous squamous cell carcinoma (cSCC) that is adapted to decision-making and universally used. Experts have unconscious ability to simplify the heterogeneity of clinical situations into a few relevant groups to drive their therapeutic decisions. Therefore, we have used unsupervised clustering of real cases by experts to generate an operational classification of cSCCs, an approach that was successful for basal cell carcinomas. OBJECTIVE: To generate a consensual and operational classification of cSCCs. METHOD: Unsupervised independent clustering of 248 cases of cSCCs considered difficult-to-treat. Eighteen international experts from different specialties classified these cases into what they considered homogeneous clusters useful for management, each with freedom regarding clustering criteria. Convergences and divergences between clustering were analysed using a similarity matrix, the K-mean approach and the average silhouette method. Mathematical modelling was used to look for the best consensual clustering. The operability of the derived classification was validated on 23 new practitioners. RESULTS: Despite the high heterogeneity of the clinical cases, a mathematical consensus was observed. It was best represented by a partition into five clusters, which appeared a posteriori to describe different clinical scenarios. Applicability of this classification was shown by a good concordance (94%) in the allocation of cases between the new practitioners and the 18 experts. An additional group of easy-to-treat cSCC was included, resulting in a six-group final classification: easy-to-treat/complex to treat due to tumour and/or patient characteristics/multiple/locally advanced/regional disease/visceral metastases. CONCLUSION: Given the methodology based on the convergence of unguided intuitive clustering of cases by experts, this new classification is relevant for clinical practice. It does not compete with staging systems, but they may complement each other, whether the objective is to select the best therapeutic approach in tumour boards or to design homogeneous groups for trials.

Regulation of PDE5 expression in normal prostate, benign prostatic hyperplasia, and adenocarcinoma.

BACKGROUND: Type 5 phosphodiesterase (PDE5) expression in the normal and pathological prostate is controversial. OBJECTIVES: This study aimed at identifying the cell type/s, if any, expressing PDE5 in human healthy or pathological prostate sections in order to further validate the rationale of PDE5 inhibitor (PDE5i) treatment of benign prostatic hyperplasia (BPH) and their safety in the treatment of erectile dysfunction following prostate cancer (PCa) surgery. MATERIALS AND METHODS: By immunohistochemical analysis, we studied PDE5 expression in tissue microarrays containing sections obtained from healthy, BPH, and PCa samples. RESULTS: Our results showed that PDE5 is barely expressed in the epithelial or stromal compartment of normal human prostates, but it is highly expressed in the stromal compartment of BPH sections. We also found that a low but significant number of PCa samples (22%) expressed PDE5 in the epithelial cancer cells but not in stromal cells and that such expression was not correlated with the tumor aggressiveness, according to their Gleason score. DISCUSSION AND CONCLUSION: PDE5 overexpression in the stromal compartment of BPH samples supports the rationale of PDE5 as a target in lower urinary tract symptoms of BPH. PDE5 expression in a significant percentage of PCa samples but the lack of correlation with the Gleason score suggests that this enzyme is not correlated with tumor aggressiveness; however, a role of PDE5 in the minimal residual disease of PCa cannot be excluded.

Sulodexide counteracts endothelial dysfunction induced by metabolic or non-metabolic stresses through activation of the autophagic program.

OBJECTIVE: Endothelial dysfunction (ED) predisposes to venous thrombosis (VT) and post-thrombotic syndrome (PTS), a long-term VT-related complication. Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrombotic, pro-fibrinolytic and anti-inflammatory activity used in the treatment of chronic venous disease (CVD), including patients with PTS. SDX has recently obtained clinical evidence in the "extension therapy" after initial-standard anticoagulant treatment for the secondary prevention of recurrent deep vein thrombosis (DVT). Herein, we investigated how SDX counteracts ED. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were used. Metabolic and non metabolic-induced ED was induced by treating with methylglyoxal (MGO) or irradiation (IR), respectively. Bafilomycin A1 was used to inhibit autophagy. The production of reactive oxygen species (ROS), tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, Real-time PCR and Western blot analysis for gene and protein expression were used. RESULTS: SDX protected HUVEC from MGO- or IR-induced apoptosis by counteracting the activation of the intrinsic and extrinsic caspase cascades. The cytoprotective effects of SDX resulted from a reduction in a) ROS production, b) neo-synthesis and release of pro-inflammatory cytokines (TNFα, IL1, IL6, IL8), c) DNA damage induced by MGO or IR. These effects were reduced when autophagy was inhibited. CONCLUSIONS: Data herein collected indicate the ability of SDX to counteract ED induced by metabolic or non-metabolic stresses by involving the intracellular autophagy pathway. Our experience significantly increases the knowledge of the mechanisms of action of SDX against ED and supports the use of SDX in the treatment of CVD, PTS and in the secondary prevention of recurrent DVT.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Correction to: The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma.

The original article [1] contained an error whereby Fig. 4 displayed incorrect magnification scales.

Erectile function recovery in men treated with phosphodiesterase type 5 inhibitor administration after bilateral nerve-sparing radical prostatectomy: a systematic review of placebo-controlled randomized trials with trial sequential analysis.

The impact of phosphodiesterase type 5 inhibitor (PDE5I) treatment modality (on-demand vs. daily), PDE5I half-life and time from surgery to PDE5I prescription on the achievement of drug-assisted erectile function (EF) recovery is uncertain. We systematically reviewed published randomized clinical trials (RCTs). We performed meta-analyses of data on 2317 men treated with PDE5Is after nerve-sparing radical prostatectomy (NSRP). A PubMed and SCOPUS search was performed for trials published from 1 January 1969 to 30 June 2016. PDE5Is are effective in achieving drug-assisted recovery of erectile function (EF). From a statistical standpoint, these studies were subjected to Trial Sequential Analysis to determine whether the pooled data were adequately powered to verify the study outcomes. On-demand treatment with PDE5Is was significantly better than daily treatment in recovering drug-assisted EF. This effect was maintained even when the drugs were stratified according with half-life. Although not based on head-to-head trials, Avanafil used on-demand was the most effective PDE5I in recovering drug-assisted EF. Whereas tadalafil was equally effective when used both on-demand and daily, vardenafil significantly improved drug-assisted EF recovery only when used on-demand. The start of PDE5I treatment six months or more after surgery compared to treatment started earlier did not negatively affect the rate of drug-assisted EF recovery or the possibility to have successful intercourse based on the Sexual Encounter Profile question-3 (SEP-3). Current trials do not support the hypothesis that PDE5I use recovers drug-unassisted EF, although chronic low-dose tadalafil administration may help to preserve erectile tissue integrity. Potential shortcomings in the trials design may partially explain these disappointing results and several questions concerning the recovery of drug-unassisted EF remain unanswered. Thus, there is a need for well-designed new RCTs requiring changes in the timing of PDE5I administration as well as in the dose and the treatment duration.

c-Myc Sustains Transformed Phenotype and Promotes Radioresistance of Embryonal Rhabdomyosarcoma Cell Lines.

We have previously reported that the MEK/ERK pathway sustains in vitro and in vivo transformed phenotype and radioresistance of embryonal rhabdomyosarcoma (ERMS) cell lines. Furthermore, we found that aberrant MEK/ERK signaling activation promotes c-Myc oncoprotein accumulation. In this study, the role of c-Myc in sustaining the ERMS transformed and radioresistant phenotype is characterized. RD and TE671 cell lines conditionally expressing MadMyc chimera protein, c-Myc-dominant negative and shRNA directed to c-Myc were used. Targeting c-Myc counteracted in vitro ERMS adherence and in suspension, growth motility and the expression of pro-angiogenic factors. c-Myc depletion decreased MMP-9, MMP-2, u-PA gelatinolytic activity, neural cell adhesion molecule sialylation status, HIF-1α, VEGF and increased TSP-1 protein expression levels. Rapid but not sustained targeting c-Myc radiosensitized ERMS cells by radiation-induced apoptosis, DNA damage and impairing the expression of DNA repair proteins RAD51 and DNA-PKcs, thereby silencing affected ERMS radioresistance. c-Myc sustains ERMS transformed phenotype and radioresistance by protecting cancer cells from radiation-induced apoptosis and DNA damage, while promoting radiation-induced DNA repair. This data suggest that c-Myc targeting can be tested as a promising treatment in cancer therapy.

Vitamin D protects endothelial cells from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis.

PURPOSE: Radiotherapy toxicity is related to oxidative stress-mediated endothelial dysfunction. Here, we investigated on radioprotective properties of Vitamin D (Vit.D) on human endothelial cells (HUVEC). METHODS: HUVEC, pre-treated with Vit.D, were exposed to ionizing radiation (IR): ROS production, cellular viability, apoptosis, senescence and western blot for protein detection were performed. The role of MAPKs pathway was investigated by using U0126 (10 µM) MEKs/ERKs-, SB203580 (2.5 µM) p38-inhibitor or by over/expressing MKK6 p38-upstream activator. RESULTS: Vit.D reduced IR-induced ROS production protecting proliferating and quiescent HUVEC from cellular apoptosis or senescence, respectively, by regulating MAPKs pathways. In proliferating HUVEC, Vit.D prevented IR-induced apoptosis by activating ERKs while in quiescent HUVEC counteracted IR-induced senescence by inhibiting the p38-IR-induced activation. MEKs&ERKs inhibition in proliferating or MKK6/mediated p38 activation in quiescent HUVEC, respectively, reverted anti-apoptotic or anti-senescent Vit.D properties. SirT1 protein expression levels were up-regulated by Vit.D. ERKs inhibition blocked Vit.D-induced SirT1 protein up-regulation in proliferating cells. In quiescent HUVEC cells, p38 inhibition counteracted the IR-induced SirT1 protein down-regulation, while MKK6 transfection abrogated the Vit.D positive effects on SirT1 protein levels after irradiation. SirT1 inhibition by sirtinol blocked the Vit.D radioprotective effects. CONCLUSION: Vit.D protects HUVEC from IR induced/oxidative stress by positively regulating the MAPKs/SirT1 axis.

Squamous cell carcinoma of the rectum: The treatment paradigm.

PURPOSE: This study was planned to clarify the optimal treatment for squamous cell carcinoma of the rectum, an histological entity extremely rare. METHODS: Ten patients with histologically proven squamous cell carcinoma of the rectum were treated with concomitant radiochemotherapy. Radiation therapy was delivered with a 3Dconformational multiple field technique to a dose ranging from 45 to 76.5 Gy, with 6-15 MV energy photons. Chemotherapy consisted of an antimetabolite drug in association with mitomycin C or oxaliplatin. Overall survival and disease free survival were considered in months from the end of the concomitant treatment. RESULTS: All patients completed programmed radiochemotherapy treatment but two patients were excluded to the analysis. Six patients (75%) presented negative biopsy 6 months after the end of radiochemotherapy. Seven patients (87.5%) showed a tumour regression after initial treatment. Only 1 patient underwent salvage surgery. Considering a mean follow-up of 41.75 months, 7 patients are still disease free survivors. Only 1 patient developed local recurrence at 6 months and he died 14 months after abdomino-perineal resection. CONCLUSION: Primary radio chemotherapy, with a curative intent, could be considered the treatment modality of choice for squamous carcinoma of the rectum.

Bioclinical parameters driving decision-making of subsequent lines of treatment in metastatic castration-resistant prostate cancer.

Different options are available as second-line treatment of metastatic castrate-resistant prostate cancer: cabazitaxel, abiraterone, and enzalutamide. Phase III studies evaluating cabazitaxel and the two hormonal agents have been shown to significantly prolong overall survival compared to mitoxantrone and placebo, respectively. Several studies have also demonstrated feasibility and activity of docetaxel rechallenge in case of a sufficient progression-free interval (3-6 months), good performance status, and previous acceptable safety profile, thus providing an additional treatment option in clinical practice. Clinical and biological parameters should be considered to tailor II line treatment. In clinical practice, we can primarily evaluate patients' fitness according to age, performance status, symptomatic disease, comorbidities, and expected safety profile of each drug. Different prognostic/predictive factors may be considered, such as presence of bone-limited or visceral metastases, length of androgen deprivation therapy (ADT) before chemotherapy, time to progression after docetaxel, Gleason score, PSA doubling time, and serum testosterone, even if their clinical relevance is still debated. This review will discuss current options of innovative drugs sequencing and selection according to bioclinical parameters.

A feasibility study of percutaneous Radiofrequency Ablation followed by Radiotherapy in the management of painful osteolytic bone metastases.

OBJECTIVES: To determine whether Radiofrequency Ablation (RFA) followed by Radiotherapy (RT) (RFA-RT) produces better palliation in terms of pain than RT alone in patients with osteolytic bone metastases. METHODS: Patients with solitary bone metastases and a pain score of least 5 or more on the VAS scale were selected. Fifteen patients were treated with RFA-RT (20 Gy delivered in 5 fractions of 4 Gy over 1 week) and were compared with a matched group (30 subjects) treated by RT. RESULTS: A complete response in terms of pain relief at 12 weeks was documented in 16.6% (5/30) and 53.3% (8/15) of the subjects treated by RT or RFA-RT, respectively (p = 0.027). The overall response rate at 12 weeks was 93.3% (14 patients) in the group treated by RFA-RT and 59.9% (18 patients) in the group treated by RT (p = 0.048). Although recurrent pain was documented more frequently after RT (26.6%) than after RFA-RT (6.7%) the difference did not reach statistical significance. The morbidity related to RT did not significantly differ when this treatment was associated with RFA. CONCLUSIONS: Our results suggest that RFA-RT is safe and more effective than RT. The findings described here should serve as a framework around which to design future clinical trials.

The impact of conventional or hypofractionated radiotherapy on voice quality and oncological outcome in patients with early glottic cancer.

The hypothesis being tested in this study is that hypofractionated radiotherapy is well tolerated and not lower in terms of oncological outcome than conventional radiotherapy. Forty patients with histologically proven glottic cancer were included in the analysis. Twenty-two were treated by hypofractionated radiotherapy (3D-HFRT) (25 fractions of 2.4 Gy delivered daily to a total dose of 60 Gy). This group was retrospectively compared to 18 subjects who met the same inclusion criteria and who were treated with conventional radiotherapy (3D-CRT) (33 fractions of 2 Gy delivered daily to a total dose of 66 Gy). One year after RT treatment in 10 patients (5 in the arm-1 and 5 in the arm-2) mild dysphonia persisted. The other patients achieved a complete recovery of the overall quality of voice with no significant difference documented between the two groups. At 3 years the local control rate was 100% for the patients treated with hypofractionated radiotherapy and 96% for the patients treated with conventional regimen. The statistical analysis did not show any significant difference in local control between the two groups (p=0.45). No significant acute and late toxicity was documented in both groups. Subjects with early glottic cancer seem to experience comparable levels of morbidity irrespective whether they were treated by hypofractionated or conventional conformal therapy without any worsening of the tumor local control. Thus, we provide clinical evidence to justify trends already emerging toward hypofractionated regimens in early glottic cancer.

Subjective and objective measures of late genitourinary morbidity following hypofractionated radiotherapy in men with prostate cancer.

To value the late genitourinary (GU) morbidity in men treated with a hypofractionated radiotherapy regimen for prostate cancer. Patients with intermediate risk factors according to D'Amico's criteria were selected. The hypofractionated schedule consisted of 15 fractions of 3.63 Gy delivered three times per week for a total dose of 54.3 Gy. Significant changes in storage-symptoms were not found. A significant transient worsening in the score of late effects of normal tissue late effects normal tissue task force (LENT)-subjective, objective, management, analytic (SOMA) urinary-function domain was observed at 12 months with subsequent improvement at 28 months. The assessment of voiding-symptoms and maximum urinary flow rate (Qmax) showed that no significant difference was measurable at 12 and 28 months. For PVR, a transient increase at 12 months with a subsequent decrease at 28 months was measured. No significant increase in alpha-blockers usage and in the percentage of men with pathological nonintubated uroflowmetry (NIF) was observed at 12 and 28 months. Finally, patients did not perceive any clinical worsening in their quality of life (QoL) as attested by the International Prostate Symptom Score (IPSS)-QoL. Our study seems to suggest that our hypofractionated radiotherapy schedule for the treatment of prostate cancer is safe in terms of late urinary morbidity. Further study will be required to confirm our results.

Foreign bodies aspirated in children: role of bronchoscopy.

OBJECTIVE: To examine the diagnostic and therapeutic role of fiber-optic and rigid bronchoscopy in pediatric patients with foreign body inhalations. METHODS: From January 1986 to December 2004, we observed 128 young patients with suspicion of foreign body aspiration. Patients were divided into 3 groups: group I, patients with negative chest X-ray; group II, patients with radiological direct signs; group III, patients with radiological indirect signs. RESULTS: Removal of the foreign body was effected in 105 patients by rigid bronchoscopy and in 13 patients by fiber-optic bronchoscopy. In 3 group II patients a thoracotomy with a bronchotomy was necessary. CONCLUSIONS: Fiber-optic bronchoscopy showed a diagnostic accuracy rate of 100 % but played a poor therapeutic role with a case resolution of 10.7 %. Rigid bronchoscopy was the main technique, permitting the removal of the tracheobronchial foreign body in 97.2 % of patients.

Molecular aspects of gefitinib antiproliferative and pro-apoptotic effects in PTEN-positive and PTEN-negative prostate cancer cell lines.

To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer (PCa) to more invasive forms. One of the major targets for the therapy in PCa can be epidermal growth factor receptor (EGFR), which signals via the phosphoinositide 3'-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, among others. Despite multiple reports of overexpression in PCa, the reliance on activated EGFR and its downstream signalling to the PI3K and/or MAPK/extracellular signal-regulated kinase (ERK) pathways has not been fully elucidated. We reported that the EGFR-selective tyrosine kinase inhibitor gefitinib (ZD1839; Iressa) is able to induce growth inhibition, G(1) arrest and apoptosis in PCa cells and that its effectiveness is associated primarily with phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression (and thus Akt activity). In fact PTEN-negative PCa cells are slowly sensitive to gefitinib treatment, because this molecule is unable to downregulate PI3K/Akt activity. PI3K inhibition, by LY294002 or after PTEN transfection, restores EGFR-stimulated Akt signalling and sensitizes the cells to pro-apoptotic action of gefitinib. The MAPK pathway seems to be involved primarily on cell-growth modulation because dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition (both not cell apoptosis) in PTEN-positive PCa cells and reduced EGF-mediated growth in PTEN-negative cells. Thus the effectiveness of gefitinib requires growth factor receptor-stimulated PI3K/Akt and MAPK signalling to be intact and functional. The loss of the PTEN activity leads to uncoupling of this signalling pathway, determining a partial gefitinib resistance. Moreover, gefitinib sensitivity may be maintained in these cells through its inhibitory potential in MAPK/ERK pathway activity, modulating proliferative EGFR-triggered events. Therefore, our data suggest that the inhibition of EGFR signalling can result in a significant growth reduction and in increased apoptosis in EGFR-overexpressing PCa cells with different modalities, which are regulated by PTEN status, and this may have relevance in the clinical setting of PCa.

Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Osteoblast-derived TGF-beta1 modulates matrix degrading protease expression and activity in prostate cancer cells.

Tumor progression and metastasis may result in part from the selection of cell clones competent for survival, invasion and growth at secondary sites and characterized by loss of growth inhibitory responses, acquisition of increased adhesiveness and enhanced motility and protease expression. Transforming growth factor-beta1 (TGF-beta1) is produced by osteoblasts (OB) in a latent form and is activated by proteases in a cell-dependent manner. We show here that OB conditioned medium (OB CM) modulates Matrigel invasion of a bone metastatic prostate cancer cell line (PC3) and that this effect is blocked by antibody against TGF-beta1 and by uPA/plasmin inhibitors, suggesting that TGF-beta1 can modulate OB-mediated cell recruitment and that PC3 cells can activate TGF-beta1. TGF-beta1 induces uPA and PAI-1 secretion and promotes binding of uPA at the external plasma membrane with increased membrane-associated plasmin activity. Matrix metalloprotease-9 (MMP-9) is induced both in the medium and in the membrane associated form. Moreover, the balance between proteolytic activity and inhibition is crucial in the metastatic event. Indeed, the increment of PAI-1 could have an important regulatory role on the extracellular proteolysis and might explain the decrease of net PA and gelatinolytic activities measured in the medium. In addition, PAI-1 plays a regulative role localizing matrix degradation in some specific sites, such as areas of cell-to-cell or cell-to-ECM contacts. In conclusion, TGF-beta1 enhances PC3 Matrigel invasion by a uPA/plasmin-dependent mechanism, also involving the MMP-9, and thus may play a central role in malignant prostate tumor progression as a result of stimulating bone matrix invasion.

Culture conditions modulate cell phenotype and cause selection of subpopulations in PC3 prostate cancer cell line.

PC3 cell line contains different cell variants. A first variant grows as spherical multicellular aggregates and shows anchorage-independent growth. A second variant grows as single small rounds and shows anchorage-dependent growth without cell spreading. A third variant, representing the most abundant population, grows as adherent cells. These populations differ in alpha 2 beta 1 and alpha 3 beta 1 integrin expression with low levels in the suspended (S) cells, intermediate in partially adherent (R) cells and high in adherent cells (A). TPA, which up-regulates the expression of beta 1 integrins, increases invasiveness of cells. In addition, PC3 variants differ in MMP9 and uPA secretion and activity. High levels of TIMP1 and PAI1 present in S variant reduce MMP9 and uPA activities, respectively. In conclusion, PC3 cell line shows variants with strong phenotypic heterogeneity reflecting also the in vitro culture condition. Our observations may explain some of the contradictions in the literature. Therefore, the data obtained with this line should be evaluated more carefully, considering morphological and functional characteristics of the possible variants in the cell population. However, this heterogeneity may represent a good model in the study of tumor progression.

Vesicle-associated urokinase plasminogen activator promotes invasion in prostate cancer cell lines.

The ability of a cell to modify the extracellular matrix is important in several pathophysiological alterations including tumorigenesis. Cell transformation is accompanied by changes in the surrounding stroma as a result of the action of specific proteases such as the urokinase plasminogen activator (uPA), which has been associated with invasive potential in many tumor types. In this study, we analyzed the release of vesicle-associated uPA by the aggressive prostatic carcinoma cell line PC3 and the implications of this release for the invasive behaviour of prostatic tumor cells. Zymography and Western blot analysis revealed the presence of vesicle-associated uPA in the high-molecular weight form. Vesicles adhered to and degraded both collagen IV and reconstituted basal membrane (Matrigel), and plasminogen enhanced the degradation in a dose-dependent manner. Addition of membrane vesicles shed by PC3 cells to cultures of the poorly invasive prostate cancer cell line LnCaP enhanced the adhesive and invasive capabilities of the latter, suggesting a mechanism involving substrate recognition and degradation. Together, these findings indicate that membrane vesicles can promote tumor invasion and point to the important role of vesicle-associated uPA in the extracellular compartment.