From cells to tissue: How cell scale heterogeneity impacts glioblastoma growth and treatment response.

Glioblastomas are aggressive primary brain tumors known for their inter- and intratumor heterogeneity. This disease is uniformly fatal, with intratumor heterogeneity the major reason for treatment failure and recurrence. Just like the nature vs nurture debate, heterogeneity can arise from intrinsic or environmental influences. Whilst it is impossible to clinically separate observed behavior of cells from their environmental context, using a mathematical framework combined with multiscale data gives us insight into the relative roles of variation from different sources. To better understand the implications of intratumor heterogeneity on therapeutic outcomes, we created a hybrid agent-based mathematical model that captures both the overall tumor kinetics and the individual cellular behavior. We track single cells as agents, cell density on a coarser scale, and growth factor diffusion and dynamics on a finer scale over time and space. Our model parameters were fit utilizing serial MRI imaging and cell tracking data from ex vivo tissue slices acquired from a growth-factor driven glioblastoma murine model. When fitting our model to serial imaging only, there was a spectrum of equally-good parameter fits corresponding to a wide range of phenotypic behaviors. When fitting our model using imaging and cell scale data, we determined that environmental heterogeneity alone is insufficient to match the single cell data, and intrinsic heterogeneity is required to fully capture the migration behavior. The wide spectrum of in silico tumors also had a wide variety of responses to an application of an anti-proliferative treatment. Recurrent tumors were generally less proliferative than pre-treatment tumors as measured via the model simulations and validated from human GBM patient histology. Further, we found that all tumors continued to grow with an anti-migratory treatment alone, but the anti-proliferative/anti-migratory combination generally showed improvement over an anti-proliferative treatment alone. Together our results emphasize the need to better understand the underlying phenotypes and tumor heterogeneity present in a tumor when designing therapeutic regimens.

Comparative dynamics of microglial and glioma cell motility at the infiltrative margin of brain tumours.

Microglia are a major cellular component of gliomas, and abundant in the centre of the tumour and at the infiltrative margins. While glioma is a notoriously infiltrative disease, the dynamics of microglia and glioma migratory patterns have not been well characterized. To investigate the migratory behaviour of microglia and glioma cells at the infiltrative edge, we performed two-colour time-lapse fluorescence microscopy of brain slices generated from a platelet-derived growth factor-B (PDGFB)-driven rat model of glioma, in which glioma cells and microglia were each labelled with one of two different fluorescent markers. We used mathematical techniques to analyse glioma cells and microglia motility with both single cell tracking and particle image velocimetry (PIV). Our results show microglia motility is strongly correlated with the presence of glioma, while the correlation of the speeds of glioma cells and microglia was variable and weak. Additionally, we showed that microglia and glioma cells exhibit different types of diffusive migratory behaviour. Microglia movement fit a simple random walk, while glioma cell movement fits a super diffusion pattern. These results show that glioma cells stimulate microglia motility at the infiltrative margins, creating a correlation between the spatial distribution of glioma cells and the pattern of microglia motility.

The addition of Sunitinib to radiation delays tumor growth in a murine model of glioblastoma.

OBJECTIVES: Recent preclinical studies suggest that treating glioblastoma (GBM) with a combination of targeted chemotherapy and radiotherapy may enhance the anti-tumor effects of both therapies. However, the effects of these treatments on glioma growth and progression are poorly understood. METHODS: In this study, we have tested the effects of combination therapy in a mouse glioma model that utilizes a PDGF-IRES-Cre-expressing retrovirus to infect adult glial progenitors in mice carrying conditional deletions of Pten and p53. This model produces tumors with the histological features of GBM with 100% penetrance, making it a powerful system to test novel treatments. Sunitinib is an orally active, small molecule inhibitor of multiple receptor tyrosine kinases critical for tumor growth and angiogenesis, including PDGF receptors. We investigate the addition of Sunitinib to radiotherapy, and use bioluminescence imaging to characterize the effects of treatment on glioma growth and progression. RESULTS: Treating our PDGF-driven mouse model with either Sunitinib or high-dose radiation alone delayed tumor growth and had a modest but significant effect on survival, while treating with low-dose radiation alone failed to control glioma growth and progression. The addition of Sunitinib to low-dose radiation caused a modest, but significant delay in tumor growth. However, no significant survival benefit was seen as tumors progressed in 100% of animals. Histological analysis revealed a reduction in vascular proliferation and a marked increase in brain invasion. An additional study combining Sunitinib with high-dose radiation revealed a fatal toxicity despite individual monotherapies being well tolerated. DISCUSSION: These results show that the addition of Sunitinib to radiotherapy fails to significantly alter survival in GBM despite enhancement of the effects of radiation. Furthermore, an enhanced risk of toxicity associated with combined therapy must be considered in the design of future clinical studies.

Convection-enhanced delivery of topotecan into a PDGF-driven model of glioblastoma prolongs survival and ablates both tumor-initiating cells and recruited glial progenitors.

The contribution of microenvironment to tumor growth has important implications for optimizing chemotherapeutic response and understanding the biology of recurrent tumors. In this study, we tested the effects of locally administered topotecan on a rat model of glioblastoma that is induced by intracerebral injection of PDGF (platelet-derived growth factor)-IRES (internal ribosome entry site)-GFP (green fluorescent protein)-expressing retrovirus, treated the tumors by convection-enhanced delivery (CED) of topotecan (136 µmol/L) for 1, 4, or 7 days, and then characterized the effects on both the retrovirus-transformed tumor cells (GFP(+) cells) as well as the uninfected glial progenitor cells (GFP(-) cells) that are recruited to the tumor. Topotecan treatment reduced GFP(+) cells about 10-fold and recruited progenitors by about 80-fold while providing a significant survival advantage that improved with greater treatment duration. Regions of glial progenitor ablation occurred corresponding to the anatomic distribution of topotecan as predicted by MRI of a surrogate tracer. Histopathologic changes in recurrent tumors point to a decrease in recruitment, most likely due to the chemotherapeutic ablation of the recruitable progenitor pool.

The activity of medroxyprogesterone acetate, an androgenic ligand, in ovarian cancer cell invasion.

OBJECTIVES: An epithelial ovarian cancer cell line constitutively expressing the androgen receptor was created to evaluate the mechanism and effects of androgen receptor activation on epithelial ovarian cancer cell invasion. METHODS: Immunocytochemistry and Western blot analyses confirmed androgen receptor expression. Boyden chamber invasion assays were performed using cells treated with the androgen receptor ligands medroxyprogesterone acetate or dihydrotestosterone. The matrix metalloproteinases associated with invasion were investigated using zymographic assays. RESULTS: Androgen receptor-mediated invasion is ligand dependent. While both medroxyprogesterone acetate and dihydrotestosterone signal through androgen receptor, medroxyprogesterone acetate is more effective at stimulating invasion of epithelial ovarian cancer cells. Unlike the wild-type epithelial ovarian cancer cells, this increase in invasion in androgen receptor + epithelial ovarian cancer cells does not seem to be dependent on matrix metalloproteinase 2 or 9 activation. CONCLUSION: Although classified as a progestin, medroxyprogesterone acetate has significant androgenic activity unique from the pure androgen dihydrotestosterone. Our studies suggest that pharmacologic doses of medroxyprogesterone acetate may actually increase the invasive potential of epithelial ovarian cancer cells.

Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylation.

An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1-ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin-L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1-ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1-ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1-ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1.

Lysophosphatidic acid (LPA) promotes E-cadherin ectodomain shedding and OVCA429 cell invasion in an uPA-dependent manner.

OBJECTIVES: To evaluate the role of LPA in regulating E-cadherin cell surface expression, adhesion, and invasion in epithelial ovarian carcinoma (EOC) cells. METHODS: E-cadherin mRNA expression in OVCA429 and IOSE-29 cells was evaluated by real-time RT-PCR. Immunofluorescence and Western blot analysis were performed to determine cell surface expression and shedding of E-cadherin 80-kDa soluble fragment by LPA. Kinetics of LPA-induced uPA activity was followed with a colorimetric enzymatic assay. Invasion assays were performed in a modified Boyden chamber where cells were allowed to migrate to the bottom compartment through a porous filter coated with collagen. Additionally we measured the 80-kDa form from the ascites of women with stage III/IV EOC. RESULTS: LPA induces E-cadherin shedding of a soluble 80-kDa fragment. We found that this process is mediated by the uPA proteolytic cascade. High levels of soluble E-cadherin were found in the ascites from women with advanced stage EOC. LPA and a soluble recombinant E-cadherin-Fc chimera promotes invasion of OVCA429 cells. CONCLUSIONS: LPA induces shedding of an 80-kDa E-cadherin-soluble fragment in an uPA-dependent manner and promotes in vitro invasion. High levels of soluble E-cadherin in malignant ascites may also affect ovarian metastasis.

S1P and LPA have an attachment-dependent regulatory effect on invasion of epithelial ovarian cancer cells.

OBJECTIVES: We previously demonstrated the regulation of epithelial ovarian cancer (EOC) cell invasiveness by the bioactive phospholipid sphingosine 1-phosphate (S1P). Low-dose S1P stimulated invasion like lysophosphatidic acid (LPA), while high-dose S1P inhibited invasion. Here we investigate how cell attachment status affects response to S1P and examine the effects of S1P and LPA on cell-cell and cell-extracellular matrix (ECM) adhesion. METHODS: EOC Dov13 cell invasion, ECM attachment and cell adhesion were tested through in vitro assays of Matrigel invasion and attachment to Matrigel, collagen or cell monolayer. Fractionated membrane and cytoplasmic proteins and biotin-labeled surface proteins were analyzed by western analysis. Actin cytoskeleton and FAK were visualized by immunofluorescence. RESULTS: S1P (20 muM) inhibited invasion of sustained, attached cells but enhanced that of invading cells. Membrane N-cadherin was depleted upon reattachment to ECM. S1P pretreatment (20 muM) accelerated N-cadherin recovery, while 40 muM LPA or 0.5 muM S1P delayed recovery. Cell-cell adhesion and stress fibers were decreased by LPA and by 0.5 muM S1P but increased by 20 muM S1P. While S1P increased cellular attachment to Matrigel and collagen-I, LPA inhibited attachment to Matrigel. Surface N-cadherin, gamma- and beta-catenins, FAK and integrinbeta1 were altered by both reattachment and treatment with S1P or LPA. CONCLUSIONS: S1P inversely affects invasion of attached and invading cells, switching from inhibition to stimulation. This switch is associated with depletion of N-cadherin and membrane FAK. The recovery of membrane N-cadherin, change in cell-cell adhesion and actin stress fibers intensity in response to LPA and S1P inversely correlate with their effects on cellular invasiveness.

Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface.

The function of adhesion receptors in both cell adhesion and migration depends critically on interactions with the cytoskeleton. During cell adhesion, cytoskeletal interactions stabilize receptors to strengthen adhesive contacts. In contrast, during cell migration, adhesion proteins are believed to interact with dynamic components of the cytoskeleton, permitting the transmission of traction forces through the receptor to the extracellular environment. The L1 cell adhesion molecule (L1CAM), a member of the Ig superfamily, plays a crucial role in both the migration of neuronal growth cones and the static adhesion between neighboring axons. To understand the basis of L1CAM function in adhesion and migration, we quantified directly the diffusion characteristics of L1CAM on the upper surface of ND-7 neuroblastoma hybrid cells as an indication of receptor-cytoskeleton interactions. We find that cell surface L1CAM engages in diffusion, retrograde movement, and stationary behavior, consistent with interactions between L1CAM and two populations of cytoskeleton proteins. We provide evidence that the cytoskeletal adaptor protein ankyrin mediates stationary behavior while inhibiting the actin-dependent retrograde movement of L1CAM. Moreover, inhibitors of L1CAM-ankyrin interactions promote L1CAM-mediated axon growth. Together, these results suggest that ankyrin binding plays a crucial role in the anti-coordinate regulation of L1CAM-mediated adhesion and migration.

Developmental patterns of cadherin expression and localization in relation to compartmentalized thalamocortical terminations in rat barrel cortex.

The wiring of synaptic circuitry during development is remarkably precise, but the molecular interactions that enable such precision remain largely to be defined. Cadherins are cell adhesion molecules hypothesized to play roles in axon growth and synaptic targeting during development. We previously showed that N-cadherin localizes to ventrobasal (VB) thalamocortical synapses in rat somatosensory (barrel) cortex during formation of the whisker-map in layer IV (Huntley and Benson [1999] J. Comp. Neurol. 407:453-471). Such specific association of N-cadherin with one identified afferent pathway raises the prediction that other cadherins are expressed in barrel cortex and that these are, in some combination, also differentially associated with distinct inputs. Here, we first show that N-cadherin and three other classic cadherins (cadherin-6, -8, and -10) are expressed contemporaneously in barrel cortex with relative levels of postnatal expression that are highest during the first 2 weeks, when afferent and intrinsic circuitries are forming and synaptogenesis is maximal. Each displayed distinct, but partly overlapping laminar patterns of expression that changed over time. Cadherin-8 probe hybridization formed a particularly striking pattern of intermittent, columnar patches extending from layer V through layer III, which was first detectable at approximately postnatal day 3. The patches were centered precisely over regions of dysgranular layer IV and, in the whisker barrel field, over barrel septa. This pattern is similar to that formed by the terminal distribution of thalamocortical afferents arising from the posterior nucleus (POm), suggesting cadherin-8 association with the POm thalamocortical synaptic circuit. Consistent with this, cadherin-8 mRNAs were enriched in the POm nucleus, and cadherin-8 immunolabeling in layer IV was enriched in barrel septa and codistributed with labeled POm thalamocortical synaptic-like puncta. The striking molecular parcellation of at least two different cadherins to the two, converging thalamic pathways that terminated in non-overlapping barrel center and septal compartments in layer IV strongly suggested that cadherins provide requisite molecular recognition and targeting that enable precise construction of thalamocortical and other synaptic circuitry.

The cadherin family of cell adhesion molecules: multiple roles in synaptic plasticity.

Cadherins are cell adhesion molecules that are critically important for establishing brain structure and connectivity during early development. They are enriched at synapses and, by virtue of a number of properties including homophilic recognition and molecular diversity, have been implicated in the generation of synaptic specificity. Cadherins also participate in remodeling synaptic architecture and modifying the strength of the synaptic signal, thereby retaining an active role in synaptic structure, function, and plasticity, which extends beyond initial development. Cadherins have been implicated in the induction of long-term potentiation (LTP) of hippocampal synaptic strength, a cellular model for learning and memory. LTP is associated with the synthesis and recruitment of N-cadherin to newly forming synaptic junctions, induces molecular changes to N-cadherin indicative of augmented adhesive force, and can be prevented when cadherin adhesion is blocked. NMDA receptor activation, which is critically required for synaptic plasticity, may provide a signal that regulates the molecular configuration of synaptic N-cadherin, and therefore the strength of adhesion across the synaptic cleft. Additionally, there exists at the synapse a pool of surface cadherins that is untethered to the actin cytoskeleton and capable of a rapid and reversible dispersion along the plasmalemma under conditions of strong activity. These observations suggest that synaptic activity dynamically regulates both the strength and the localization of cadherin-cadherin bonds across the synaptic junctional interface, changes that may be crucial for regulating synaptic plasticity.

Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP.

Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively.

Consumo de carne por niños preescolares adscritos a guardería / Meat consumption by preschoolers assisting to a daycare center

Se determina el consumo de carne en niños sanos adscritos a guardería por medio de un estudio tipo encuesta. Se pesaron las raciones de carne que se incluyen en la dieta habitual de 76 niños preescolares, clínicamente sanos, adscritos a la guardería 201 del IMSS en Culiacán, Sinaloa. El grupo estudiado se clasificó en niños de peso normal, subnormal o con sobrepeso, de acuerdo al índice de masa corporal (IMC). Al término del lapso usual para toma de alimentos, se pesó la carne no consumida. El consumo de carne fue 59 por ciento del total de las raciones otorgadas; dicho consumo no mostró diferencias estadísticamente significativas entre los niños según su IMC (p=0.06). Concluimos que existe baja predilección por la carne en niños preescolares que se encuentran al cuidado de los servicios de guardería y que su consumo es aparentemente independiente de sus condiciones ponderales