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stimulated or, using antisense targeting strategies, specifically   death or changes in protein expression were successfully
            blocked.  In an analogous study it was demonstrated that   analyzed. Thus, Chadwick et al.  used the OBSC system
                                                                                         [79]
                  [71]
            (1) the invasive behavior of the astrocytoma cells in the brain   as a qualitative and quantitative assay to calculate the fold
            slice co-culture is not always identical to the results obtained   change  in  the number  of  cells  during  the  period  of  slice
            from 2D migration studies, (2) the tumor cells spread out   culture. Furthermore, they investigated either the whole brain
            multidirectionally, (3) frozen human normal brain tissue   or specific regions within the brain, to assess environmental
            can be used for the organotypic culture, (4) there were no   impact on primary brain tumor cell growth.
            obvious signs of necrosis, and (5) the brain cytoarchitecture
            and viability was preserved for at least 14 days. [72]  Organotypic  brain slice  culture  to study the
                                                               microenvironmental impact
            Although the human origin of the biopsies used as the host   Malignant astrocytoma/GBM cause mortality by local tumor
            tissue  in  these  studies  excludes  species-specific  effects  in   growth and brain invasion rather than systemic metastasis.
            the co-culture, slices from newborn rat or mouse brains are   GBM tumor cells diffusely infiltrate the brain parenchyma
            excellent alternatives. They offer several advantages: brain   within and along the white matter tracts or around cerebral
            regions corresponding to the in vivo tumor localization can be   blood vessels,  and rarely penetrate basal lamina structures
                                                                         [53]
            chosen, developmental stage of the brain slice can be adjusted,   at the glial limitans externa.  Analogously, malignant
            multiple replicas from same brain region can be generated,   medulloblastoma  must  also  infiltrate  cerebellar  tissue  for
            and  the  use  of  transgenic  animals  allows  modification  of   distal dissemination. Moreover, resection of MB tumors is
            the cellular microenvironment. Ohnishi et al.  established   inevitably followed by relapse if the patients are not treated
                                                [75]
            OBSCs from 2-day-old neonatal rat brains, which were   with cranio-spinal radiotherapy and chemotherapy, suggesting
            transferred on double-layered membranes consisting of two   the occurrence of local dissemination of tumor cells from the
            different membrane types and maintained at an interface   primary medulloblastoma. In vitro studies aiming at better
            between the air and the culture medium.  The slices were   understanding the local invasion process have been hampered
            then co-cultured with C6 glioma cells labeled with PKH2   by the lack of identification of the brain ECM macromolecules
            fluorescent dye. After 2 days of co-culture, the exogenous   involved and the only poorly understood implication of the
            application of the chemotactic stimulator neural cell adhesion   cellular microenvironment. In vivo approaches on the other
            molecule L1 triggered tumor cell migration from the upper to   hand, offer too little spatial and temporal resolution to
            the bottom membrane through the brain slice.  Since this   monitor tumor-microenvironment interactions appropriately.
                                                 [75]
            study lacked CF-LSM analysis, OBSCs were subsequently   Thus, OBSCs could provide an important platform to study
            performed by the slightly modified Stoppini method, which   the cross-talk between the tumor cells and normal cells in
            allowed quantifying glioma cell invasion using confocal   a physiologically relevant environment. OBSCs can be
            microscopy.  This study revealed that the migrating cells   used for investigating the microenvironment and its impact
                     [76]
            showed a strong increase in immunoreactivity for matrix   on the growth and spread of primary brain tumors, and for
            metalloproteinase 2 and 9.  Analogous OBSC technology   testing the measures that could be taken to prevent or treat it
                                 [76]
            was later used for mouse brain slices to quantify the   effectively.  Although, there is a lack of vascular supply to
                                                                       [79]
            invasiveness of glioma [77]  and to correlate it with histological   the tissue in the slices, capillaries do survive in these sections
            type.  Both studies used human, DiI-stained glioma biopsy   without any circulation.  Despite of the fact that there is
                [78]
                                                                                  [80]
            tumor fragments and GFP-expressing spheroids directly   no blood flow and that the capillaries are not functional, it is
            implanted in the cortex of brain slices derived from 7 day   likely that they are still capable of expressing and secreting
            old mice.  This intraslice implantation system could be   various molecules, [81]  which could affect other cell types in
            maintained  in  culture  for  2  to  4  weeks.  Quantification  of   the slice culture including the tumor cells. In addition, the
            the distance and density of the tumor cell invasion revealed   intriguing exchange between tumor cells and astrocytes and
            that GBMs were 2-4 times more invasive than the lower   the suspected tumor promoting functions of astrocytes [41-43]
            grade glioma cells (LGGs).  Within the different groups   urges for novel studies addressing the therapeutic potential of
            and grades of GBMs and LGGs, heterogeneity in terms of   the astrocyte-tumor interaction, for which organotypic slice
            invasion was seen. It was also observed that the spheroids   culture would be an ideal system.
            were less invasive in comparison to the directly grafted
            fragments. Overall using this system, Palfi et al. [77,78]  and de   Along  with  their  use for  monitoring  tumor dissemination,
            Bouard et al.  could successfully recapitulate, monitor and   OBSCs  have  also  been  used  for  high  resolution  imaging
                      [77]
            quantify the invasion of single cells and the dissemination   of  cytoskeletal structures  in living  glioblastoma  cells.  For
            of  glioma  ex  vivo.  Recently,  Chadwick  et  al. [79]  developed   this, glioblastoma cells were transfected with GFP-actin and
            OBSCs from postnatal day 6 mice and cultured the whole   placed onto murine brain slices and spinal cord explants.
            brain slices on membrane inserts coated with laminin. Tumor   Using live-cell imaging to visualize the cytoskeleton of the
            cells (astrocytoma and medulloblastoma) were stained with   tumor cells, a major change in the gross morphology from a
            Cm-DiI for monitoring, and dispensed on the center of the   solid, two dimensional state to a three dimensional substrate
            slice. This co-culture system remained viable for one week   was  noted.  This morphological  change was characterized
            and effects of drug therapies on tumor cell proliferation, cell   by long, dendritic-like processes that displayed regions


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