Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Detection of extracellular vesicles in the mouse vaginal fluid: Their delivery of sperm proteins that stimulate capacitation and modulate fertility.

Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as "Vaginosomes" (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca2+ -ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10-30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics.

Exosomal L1CAM Stimulates Glioblastoma Cell Motility, Proliferation, and Invasiveness.

Immunoglobulin superfamily protein L1CAM (L1, CD171) normally facilitates neuronal migration, differentiation, and axon guidance during development. Many types of cancers, including glioblastoma (GBM), also abnormally express L1, and this has been associated with poor prognosis due to increased cell proliferation, invasiveness, or metastasis. We showed previously that the soluble L1 ectodomain, which is proteolyzed from the transmembrane form, can stimulate proliferation and motility of GBM cells in vitro by acting through integrins and fibroblast growth factor receptors (FGFRs). Minute L1-decorated exosomal vesicles also are released by GBM cells and potentially could stimulate cell motility, proliferation, and invasiveness, but this needed to be demonstrated. In the present study, we aimed to determine if minute L1-decorated extracellular vesicles (exosomes) were capable of stimulating GBM cell motility, proliferation, and invasiveness. L1-decorated exosomes were isolated from the conditioned media of the human T98G GBM cell line and were evaluated for their effects on the behavior of glioma cell lines and primary tumor cells. L1-decorated exosomes significantly increased cell velocity in the three human glioma cells tested (T98G/shL1, U-118 MG, and primary GBM cells) in a highly quantitative SuperScratch assay compared to L1-reduced exosomes from L1-attenuated T98G/shL1 cells. They also caused a marked increase in cell proliferation as determined by DNA cell cycle analysis and cell counting. In addition, L1-decorated exosomes facilitated initial GBM cell invasion when mixed with non-invasive T98G/shL1 cells in our chick embryo brain tumor model, whereas mixing with L1-reduced exosomes did not. Chemical inhibitors against focal adhesion kinase (FAK) and fibroblast growth factor receptor (FGFR) decreased L1-mediated motility and proliferation to varying degrees. These novel data show that L1-decoratred exosomes stimulate motility, proliferation and invasion to influence GBM cell behavior, which adds to the complexity of how L1 stimulates cancer cells through not only soluble ectodomain but also through exosomes.

Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca2+ efflux pump, PMCA4. This is the major Ca2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca2+ ] in capacitated and Ca2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca2+ -dependent manner. In Pmca4-/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4-/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

STUDY QUESTIONS: Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? SUMMARY ANSWER: OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. WHAT IS KNOWN ALREADY: Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. STUDY DESIGN, SIZE, DURATION: Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP. MAIN RESULTS AND THE ROLE OF CHANCE: TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-. WIDER IMPLICATIONS OF THE FINDINGS: Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

STUDY QUESTION: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation? SUMMARY ANSWER: JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro. WHAT IS KNOWN ALREADY: JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation. STUDY DESIGN, SIZE, DURATION: The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF. MAIN RESULTS AND THE ROLE OF CHANCE: Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate. WIDER IMPLICATIONS OF THE FINDINGS: Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.

Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca(2+)]c in sperm to down-regulate them, and thus prevent elevated levels of NO, known to induce asthenozoospermia via oxidative stress. Our studies point to the potential underlying cause of infertility in PMCA4's absence, and suggest that inactivating mutations of PMCA4 could lead to asthenozoospermia and human infertility. Screening for these mutations may serve both diagnostic and therapeutic purposes.

Hyaluronidase 2: a novel germ cell hyaluronidase with epididymal expression and functional roles in mammalian sperm.

To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ~57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization.

Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation.

Plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is the primary Ca(2+) efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca(2+)/CaM-dependent serine kinase (CASK) in regulating Ca(2+) homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT-PCR, that both the splice variants of Pmca4 mRNA are expressed in murine testis and throughout the epididymis. Immunofluorescence localized PMCA4a to the apical membrane of the epididymal epithelium, and Western analysis not only confirmed its presence but showed for the first time that PMCA4a and PMCA4b are secreted in the epididymal luminal fluid (ELF), from which epididymosomes containing PMCA4a were isolated. Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5-fold compared to caput sperm (detected by Western blotting) and ~2-fold after incubation in ELF, revealing in vitro uptake and implicating PMCA4a in epididymal sperm maturation. Coimmunoprecipitation using pan-PMCA4 antibodies, revealed that both variants associate with CASK, suggesting their presence in a complex. Because they have different kinetic properties for Ca(2+) transport and different abilities to bind to CASK, our study suggests a mechanism for combining the functional attributes of both PMCA4 variants, leading to heightened efficiency of the pump in the maintenance of Ca(2+) homeostasis, which is crucial for normal motility and male fertility.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

Promoter considerations in the design of lentiviral vectors for use in treating lysosomal storage diseases.

More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.

L1 stimulation of human glioma cell motility correlates with FAK activation.

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion.

BACKGROUND: Neural recognition molecule L1CAM, which is a key protein involved in early nervous system development, is known to be abnormally expressed and shed in several types of cancers where it participates in metastasis and progression. The distinction of L1CAM presence in cancerous vs. normal tissues has suggested it to be a new target for cancer treatment. Our current study focused on the potential role of soluble L1CAM in breast cancer cell adhesion to extracellular matrix proteins, migration, and invasion. RESULTS: We found L1 expression levels were correlated with breast cancer stage of progression in established data sets of clinical samples, and also were high in more metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435, but low in less migratory MDA-MB-468 cells. Proteolysis of L1 into its soluble form (sL1) was detected in cell culture medium from all three above cell lines, and can be induced by PMA activation. Over-expression of the L1 ectodomain in MDA-MB-468 cells by using a lentiviral vector greatly increased the amount of sL1 released by those cells. Concomitantly, cell adhesion to extracellular matrix and cell transmigration ability were significantly promoted, while cell invasion ability through Matrigel™ remained unaffected. On the other hand, attenuating L1 expression in MDA-MB-231 cells by using a shRNA lentiviral vector resulted in reduced cell-matrix adhesion and transmigration. Similar effects were also shown by monoclonal antibody blocking of the L1 extracellular region. Moreover, sL1 in conditioned cell culture medium induced a directional migration of MDA-MB-468 cells, which could be neutralized by antibody treatment. CONCLUSIONS: Our data provides new evidence for the function of L1CAM and its soluble form in promoting cancer cell adhesion to ECM and cell migration. Thus, L1CAM is validated further to be a potential early diagnostic marker in breast cancer progression and a target for breast cancer therapy.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm.

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule.

BACKGROUND: Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. RESULTS: L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. CONCLUSION: Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells.

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and ß3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers ß3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency.

Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract.

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model.

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.