Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer.

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-ß5, Survivin, TGF-ß, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.

Prognostic significance of architectural subtypes of Gleason grade 4 prostate cancer in radical prostatectomy: A semiquantitative method of evaluation.

Studies have shown that Gleason grade 4 extent as well as architectural subtypes provide prognostic information. We aimed to evaluate the influence on biochemical recurrence following radical prostatectomy of patients with organ-confined tumor, Gleason score 7, and negative surgical margins. Total tumor extent, Gleason grade 4 total extent and the extent of each architectural subtype (fused glands, poorly defined glands, cribriform glands, and glomeruloid glands) were evaluated by a semiquantitative point-count method using different colors to identify each subtype. Microscopic morphology of glomeruloid glands was considered regardless of morphology: size (small or large), attachment (narrow or extensive), and cribriform or solid intraluminal protrusion. Gleason grade 4 total extent significantly predicted shorter time to biochemical recurrence in univariate and multivariate analysis. Stratifying extent, Gleason grade 4 with >30% of the total grade 4 extent was significantly predictive for time of recurrence. Considering architectural subtypes, cribriform and glomeruloid glands but not fused and poorly formed glands extent, significantly predicted shorter time to recurrence in univariate analysis. An important issue related to the studies on prognostic significance of Gleason grade 4 subtypes is the lack of uniformity in the definition of microscopic morphology of the subtypes particularly of the glomeruloid architecture.

The alpha-1,2 fucosylated tubule system of DU145 prostate cancer cells is derived from a partially fragmented Golgi complex and its formation is actin-dependent.

In previous work, we showed that highly proliferative cells and cancer cells, but not cells with normal growth rate, have tubules rich in alpha-1,2 fucosylated epitopes that extend radially from the nucleus to the cell periphery and form an unusual uptake system. The importance of alpha-1,2 fucosylation in forming tubules was demonstrated by proving that down-regulating the two corresponding fucosyltransferases (FUT1 and FUT2) causes tubule fragmentation. Here, we present evidence that in the prostate cancer cell line DU145, the tubules arise in actively growing cells from vesicles in the medial and trans elements of a partially fragmented Golgi complex, while in not actively growing cells the tubules become completely independent from the Golgi complex. Formation and elongation of the tubules proved to depend on the actin cytoskeleton, since the alpha-1,2 fucosylated protein(s) segregate with the cytoskeleton proteins, and not in the membrane fraction, as do the Golgi markers and other fucosylated proteins, while depolymerization of the actin filaments causes tubule fragmentation and shifting of the alpha-1,2 fucosylated proteins into the membrane fraction.

Electron Microscopic Analysis of Stem Cells in Human Prostate Cancer, Including Inverted Capsule Embedding Methods for Archival Sections and Falcon Films for Prostate Cancer Cell Lines.

BACKGROUND/AIM: Identification of prostatic stem cells in primary prostate tissue sections, organ cultures of prostate and cell lines requires a range of techniques that allows characterization of stem cells for their potential use in the treatment of patients. Isolated cells usually round-up and develop changes in shape, size and cellular characteristics. The aim of this study was to provide a range of methods for identifying prostatic stem cells and characterizing them with regard to ultrastructure, nuclear morphology, cytoplasmic organelles, and/or expression stem cell marker CD133. MATERIALS AND METHODS: Prostate biopsy and prostatectomy specimens were used for studying prostatic stem cells and their known marker CD133 in tissue sections by light and/or electron microscopy. Inverted capsule embedding was used to study archival metastatic prostate in pelvic nodes and Du145 cell line in a monolayer culture. RESULTS: Staining for CD133 positively identified stem cells that were found in benign prostatic hyperplasia, benign prostate, and prostate cancer cells. Paraffin embedded sections showed a single type of stem cells, whereas methylene blue-stained Epon sections showed both light and dark stem cells. Electron microscopy showed that both basal and stem cells were closely associated with the basement membrane (basal lamina). Stem cells had smooth plasma and nuclear membranes, a prominent nucleolus, small mitochondria, and few ribosomes. Du145 cells were separated by intercellular spaces in monolayer culture. CONCLUSION: The inverted capsule embedding method allowed the study of metastasized prostate cancer in pelvic lymph nodes. Our approach enabled the assessment of stem cells in tissue sections by light and electron microscopy.

3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines.

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.

Gold-caged copolymer nanoparticles as multimodal synergistic photodynamic/photothermal/chemotherapy platform against lethality androgen-resistant prostate cancer.

Given that there is no effective treatment method for lethality androgen-resistant prostate cancers (ARPC), herein we report a multifunctional gold-caged nanoparticle (PTX-PP@Au NPs) against ARPC through integrating functional organic/inorganic materials to exploit the superiors of gold particles such as photothermal effects (PTT), generating reactive oxygen species (photodynamic effects, PDT), carrying chemotherapeutic agents (chemotherapy effects, CT), and inhibiting ion channel. This synergistic PTT/PDT/CT platform consists of three components: i) the Pluronic-polyethylenimine assembling into micelles to encapsulate drugs and providing reduction sites for gold cage formation through a "green" method, ii) the gold cage with surface plasmon resonance peak at near-infrared (NIR) region in a broad window qualifying the PTT/PDT potentiality, iii) a chemotherapeutic agent paclitaxel (PTX) arresting the tumor cell cycle. As demonstrated, the system has remarkable performance on controlling drug release, blocking TRPV6 cation channel, enhancing cell cycle arrest, elevating temperature and generating ROS, thus improving cellular toxicity along with apoptosis, enhancing tumor targeting, and achieving the therapy to ARPC with low toxicity on liver function and minimal side effects to normal organs. Notably, both PTT and PDT effect are generated under single irradiation situation because of the broad absorbance window, along with limited skin damages. As a specific synergistic platform creatively integrating multiple treatment protocols with negative toxicity, PTX-PP@Au NPs provide a facile, effective, and broadly applicable strategy to deadly ARPC.

Extracellular Electrophysiology in the Prostate Cancer Cell Model PC-3.

Although prostate cancer is one of the most common cancers in the male population, its basic biological function at a cellular level remains to be fully understood. This lack of in depth understanding of its physiology significantly hinders the development of new, targeted and more effective treatment strategies. Whilst electrophysiological studies can provide in depth analysis, the possibility of recording electrical activity in large populations of non-neuronal cells remains a significant challenge, even harder to address in the picoAmpere-range, which is typical of cellular level electrical activities. In this paper, we present the measurement and characterization of electrical activity of populations of prostate cancer cells PC-3, demonstrating for the first time a meaningful electrical pattern. The low noise system used comprises a multi-electrode array (MEA) with circular gold electrodes on silicon oxide substrates. The extracellular capacitive currents present two standard patterns: an asynchronous sporadic pattern and a synchronous quasi-periodic biphasic spike pattern. An amplitude of ±150 pA, a width between 50⁻300 ms and an inter-spike interval around 0.5 Hz characterize the quasi-periodic spikes. Our experiments using treatment of cells with Gd³âº, known as an inhibitor for the Ca²âº exchanges, suggest that the quasi-periodic signals originate from Ca²âº channels. After adding the Gd³âº to a population of living PC-3 cells, their electrical activity considerably decreased; once the culture was washed, thus eliminating the Gd³âº containing medium and addition of fresh cellular growth medium, the PC-3 cells recovered their normal electrical activity. Cellular viability plots have been carried out, demonstrating that the PC-3 cells remain viable after the use of Gd³âº, on the timescale of this experiment. Hence, this experimental work suggests that Ca²âº is significantly affecting the electrophysiological communication pattern among PC-3 cell populations. Our measuring platform opens up new avenues for real time and highly sensitive investigations of prostate cancer signalling pathways.

Advances in the computational and molecular understanding of the prostate cancer cell nucleus.

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular-level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy-induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning-based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular-level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3-dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.

Sulforaphane metabolites cause apoptosis via microtubule disruption in cancer.

Sulforaphane (SFN) inhibited growth in many cancers, but its half-life is 2 h in circulation. However, its metabolites, sulforaphane-cysteine (SFN-Cys) and sulforaphane-N-acetyl-cysteine (SFN-NAC) had longer half-lives and decreased the cell viability in both dose- and time-dependent manners in human prostate cancer. Flow cytometry assay revealed that these two SFN metabolites induced apoptosis with the features such as vacuolization, disappeared nuclear envelope, nuclear agglutination and fragmentation via transmission electron microscopy observation. Western blot showed that the sustained phosphorylation of ERK1/2 mediated by SFN metabolites caused activation and upregulation of cleaved Caspase 3 and downregulation of α-tubulin. High expression of α-tubulin was demonstrated to be positively correlated with cancer pathological grading. Both co-immunoprecipitation and immunofluorescence staining implicated the interaction between SFN metabolite-induced phosphorylated ERK1/2 and α-tubulin, and Caspase 3 cleavage assay showed that α-tubulin might be the substrate for cleaved Caspase 3. More, the SFN metabolite-mediated reduction of α-tubulin increased the depolymerization and instability of microtubules by microtubule polymerization assay. Reversely, microtubule-associated protein Stathmin-1 phosphorylation was increased via phosphorylated ERK1/2 and total Stathmin-1 was reduced, which might promote over-stability of microtubules. Immunofluorescence staining also showed that SFN metabolites induced the 'nest-like' structures of microtubule distribution resulting from the disrupted and aggregated microtubules, and abnormal nuclear division, suggesting that the disturbance of spindle formation and mitosis turned up. Thus, SFN-Cys and SFN-NAC triggered the dynamic imbalance of microtubules, microtubule disruption leading to cell apoptosis. These findings provided a novel insight into the chemotherapy of human prostate cancer.

Adaptive-scanning, near-minimum-deformation atomic force microscope imaging of soft sample in liquid: Live mammalian cell example.

In this paper, an adaptive-scanning mode (ASM) of atomic force microscope (AFM) with near-minimum sample deformation is proposed for imaging live biological samples in liquid. Conventional contact mode (CM) imaging of live cells is rather slow (scan rate  <  0.2 Hz), and as the imaging speed increases, significant deformation of the soft and highly corrugated cell membrane is induced. Such a low speed CM imaging of live biological samples is not only time consuming, but also incapable of capturing dynamic biological evolutions occurring in seconds to minutes. The proposed ASM approach aims to address these issues through two synergetic efforts integrated together. First, an adaptive-scanning technique is proposed to optimally adjust the lateral scanning speed to accommodate the sample topography variation and the probe-sample interaction force, so that the scanning-caused sample deformation is maintained below the threshold value while the overall imaging time is minimized. Secondly, a data-driven iterative feedforward control is integrated to the vertical feedback loop along with a gradient-based optimization of the deflection set-point to substantially improve the tracking of the sample topography while maintaining the vertical sample deformation around the minimal. The ASM technique is experimentally validated through imaging live human prostate cancer cells on AFM. The experimental results demonstrate that compared to the conventional CM imaging, the imaging speed is increased over eight times without loss of tracking the topography details of the live cell membrane, and the probe-sample interaction force is substantially reduced.

In vitro design of mesenchymal to epithelial transition of prostate cancer metastasis using 3D nanoclay bone-mimetic scaffolds.

Nanocomposite scaffolds show extensive applications in regenerative medicine and have shown promise as in vitro analogues of human tissue that can be used for the study of diseases. The complex nature of cancer metastasis is recently investigated using several 3D scaffold models. Herein, we report a polymer-nanoclay-based in vitro tumour model that recapitulates early stage of prostate cancer (PCa) colonization during skeletal metastasis on bone mimetic scaffolds. A unique cell culture system termed as "sequential culture (SC)" has been applied to create a bone-mimetic niche for colonization of PCa cells. Human mesenchymal stem cells (MSCs) were seeded on the bone-mimetic scaffolds, where they differentiated into bone cells and then formed mineralized bone matrix without osteogenic supplements. Further, PCa was seeded on MSCs-seeded scaffolds. Sequentially cultured PCa cells with MSCs formed self-organized multicellular tumoroids with distinct tight cellular junctions and hypoxic core regions. Extensive quantitative reverse transcription-polymerase chain reaction experiments were performed to evaluate the expressions of genes related to osteotropic bone metastasis of PCa. On the nanoclay scaffolds, the MSCs differentiated to mature osteoblasts and epithelial to mesenchymal transition was inhibited whereas mesenchymal to epithelial transition was enhanced, as also the hypoxia increased angiogenesis, and finally, PCa cells initiated osteoblastic lesion. Further, the SC technique has significant effects on expression of key metastasis-related genes. Therefore, the SC-based tumour model can be applied to recapitulate more consistent osteotropic cancer cell behavior in understanding tumour biology. This model also can be implemented for drug screening to target colonization stage of PCa cells in the bone microenvironment.

AIM1 is an actin-binding protein that suppresses cell migration and micrometastatic dissemination.

A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components ß-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasive phenotypes that becomes dysregulated in primary and metastatic prostate cancer.

Leukocyte telomere length and renal cell carcinoma survival in two studies.

BACKGROUND: Leukocyte telomere length (LTL) is a potential biomarker of cancer prognosis; however, evidence for renal cell carcinoma (RCC) is inconsistent. METHODS: We investigated LTL and RCC-specific survival among 684 cases from the US kidney cancer study (USKC) and 241 cases from the prostate, lung, colorectal, and ovarian cancer screening trial (PLCO). Leukocyte telomere length was measured by quantitative polymerase chain reaction, and hazard ratios (HRs) and 95% confidence intervals (CIs) computed using multivariable Cox models. RESULTS: Short LTL was associated with poorer disease-specific survival in both USKC (lowest vs highest quartile: HR: 2.3, 95% CI: 1.2-4.4; P for trend=0.02) and PLCO (HR: 2.4, 95% CI: 1.0-5.4; P=0.04). Among USKC cases, the association was strongest for stage-I RCC (HR: 5.5, 95% CI: 1.6-19.0; P=0.006). CONCLUSIONS: Our findings suggest that shorter LTL is an independent marker of poor RCC prognosis, particularly for stage-I disease.

The secretion and biological function of tumor suppressor maspin as an exosome cargo protein.

Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.

Formation of intracellular lumina in human prostate carcinoma (DU145) cells, maturation into signet cells, and the cribriform morphology of tumors.

The intracellular or intracytoplasmic lumen (IL) is an enigmatic histological structure that occurs in various tumor cells. A reassessment of diverse ILs fine-structure micrographs obtained out of previous studies encompassing the human prostate carcinoma (DU145) cell line and xenotransplanted carcinomas enabled us to propose aspects of ILs development in cancer cells: a combination of altered expressions in intercellular contacts and their cytoskeletal components would favor a disarray of self-apical polarity orientation; those defects, associated with a local, entwined enriched membranous structures growing as microvilli-like formations out of a disrupted endoplasm and trans-Golgi sorting, create ILs in cells' perikarya. These misplaced intracytoplasmic domains can become enlarged through spaces made between the finger-like structures by accruing membranes of coalescent intracytoplasmic vesicles then adding microvilli and glycocalyx to constitute ILs. Cationic mucins added with or without a progressive or total loss of microvilli and content generate signet or ring cell, while ILs enlarge. Variable build-ups of these cells' populations in carcinomas result in architectural mix-up of adjacent cells around these voids, misconstrued as new lumen, and establish a "cribriform" tumor pattern that often implies a poor cancer prognosis. Alternatively, cytotoxic changes caused by anticancer pro-oxidant treatment favor membrane alterations and exaggerate the ILs in xenotransplants into intracellular crypts that accompany other tumor degenerative changes.

Chromatin changes predict recurrence after radical prostatectomy.

BACKGROUND: Pathological evaluations give the best prognostic markers for prostate cancer patients after radical prostatectomy, but the observer variance is substantial. These risk assessments should be supported and supplemented by objective methods for identifying patients at increased risk of recurrence. Markers of epigenetic aberrations have shown promising results in several cancer types and can be assessed by automatic analysis of chromatin organisation in tumour cell nuclei. METHODS: A consecutive series of 317 prostate cancer patients treated with radical prostatectomy at a national hospital between 1987 and 2005 were followed for a median of 10 years (interquartile range, 7-14). On average three tumour block samples from each patient were included to account for tumour heterogeneity. We developed a novel marker, termed Nucleotyping, based on automatic assessment of disordered chromatin organisation, and validated its ability to predict recurrence after radical prostatectomy. RESULTS: Nucleotyping predicted recurrence with a hazard ratio (HR) of 3.3 (95% confidence interval (CI), 2.1-5.1). With adjustment for clinical and pathological characteristics, the HR was 2.5 (95% CI, 1.5-4.1). An updated stratification into three risk groups significantly improved the concordance with patient outcome compared with a state-of-the-art risk-stratification tool (P<0.001). The prognostic impact was most evident for the patients who were high-risk by clinical and pathological characteristics and for patients with Gleason score 7. CONCLUSION: A novel assessment of epigenetic aberrations was capable of improving risk stratification after radical prostatectomy.

Real Time Metastatic Route Tracking of Orthotopic PC-3-GFP Human Prostate Cancer Using Intravital Imaging.

The cellular basis of metastasis is poorly understood. An important step to understanding this process is to be able to visualize the routes by which cancer cells migrate from the primary tumor to various distant sites to eventually form metastasis. Our laboratory previously developed single-cell in vivo imaging using fluorescent proteins to label cancer cells. In the present study, using PC-3 human prostate cancer cells labeled with green fluorescent protein (GFP) and orthotopic tumor transplantation, we have imaged in live mice various highly diverse routes by which PC-3 cells metastasize superiorly and inferiorly to distant sites, including in the portal area, stomach area, and urogenital system. Imaging began at day 9, at which time distant metastasis had already occurred, and increased at each imaging point at days 10, 13, 14, and 16. Metastatic cells were observed migrating superiorly and inferiorly from the primary tumor as well as in lymphatic channels and trafficking in various organ systems demonstrating that PC-3 has multiple metastatic routes similar to hormone-independent advanced-stage prostate cancer in the clinic.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

BACKGROUND: Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. METHODS: Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. RESULTS: Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. CONCLUSIONS: The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention.

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models.

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumour-specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ∼60 nm near-monodisperse nanoparticles that increased the systemic exposure of PTX by sevenfold compared with free drug and twofold compared with the Food and Drug Administration-approved taxane nanoformulation (Abraxane). The tumour uptake of the CP-PTX nanoparticle was fivefold greater than free drug and twofold greater than Abraxane. In a murine cancer model of human triple-negative breast cancer and prostate cancer, CP-PTX induced near-complete tumour regression after a single dose in both tumour models, whereas at the same dose, no mice treated with Abraxane survived for >80 days (breast) and 60 days (prostate), respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for PTX delivery.