William J. Goldberg

C6 glioma cell invasion and migration of rat brain after neural homografting : ultrastructure

C6 tumor cells (10(6] were grafted as suspensions into freshly made implantation pockets in rat host cerebral cortex. Specimens were prepared for transmission and scanning electron microscopy 1 to 7 days postimplantation (DPI). By 3 DPI vacuolated C6 cells had migrated on or invaded the host brain. C6 cells were observed on the glia limitans on the surface of the brain, in the corpus callosum, subependymal space, and perivascular space and had invaded the cortex under the implantation pocket. In addition to the tumor mass that was observed under the implantation pocket, by 7 DPI individual C6 cells had migrated into the corpus callosum and internal capsule. Migrated C6 cells were observed in a perineuronal position in the hippocampus and other gray matter structures inferior to the corpus callosum. Micropockets were found around each C6 cell and the processes of these cells had replaced host parenchyma. The preferred routes of migration were on basal lamina and parallel and intersecting nerve fiber bundles. Invasion occurred through gray and white matter. The movement of homografted C6 cells in the brain suggests that these cells actively migrate as individual cells in addition to invading as a mass.

Migration of human malignant astrocytoma cells in the mammalian brain: Scherer revisited

Fresh suspensions of human glioblastoma multiforme were preincubated in the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and implanted into cortical pockets in adult rat brain. Brains were investigated periodically over 30 postoperative days and the migration of the human glioblastoma cells was traced with anti‐PHAL immunofluorescence or the overexpression of human specific p185c‐neu a specific marker of a class of human malignant astrocytoma cells. The principal pathway of migration of the implanted human cells in the rat brain was ventrally through cortical gray matter and into the corpus callosum, with rapid lateral distribution in this and other parallel and intersecting white matter fascicles. Human glioblastoma cells also migrated on basement membrane lined blood vessels, pia‐glia membrane and spaces of Virchow‐Robin, as well as the subependymal space of the ventricles. These paths of migration of human glioblastoma cells in the rat brain are consistent with the pathways of spread of glioblastoma in the human brain as described by Scherer over 50 years ago, indicating that multifocal malignant astrocytomas have common migratory pathways in mature mammalian brain.

C6 glioma-astrocytoma cell and fetal astrocyte migration into artificial basement membrane: a permissive substrate for neural tumors but not fetal astrocytes.

Cortically homografted C6 glioma-astrocytoma cells both invade the rat host brain as a mass and migrate as individual cells. In contrast, fetal astrocytes derived from homografted whole pieces of fetal cortex migrate only as individual cells throughout the brain of the rat but are not capable of invasion. Our experiment explored the migratory capacity (over 7 days) of cultured purified fetal astrocytes and C6 cells after seeding 10(6) cells on a hydrated artificial basement membrane wafer (Matrigel). The artificial basement membrane wafer was not a suitable substrate for the growth of cultured fetal astrocytes. In contrast, C6 cells migrated as individual cells from the surface of the wafer into the substrate. Individual C6 cells migrated 1.8 mm in the first 4 days and then ceased migration. The C6 cells were observed at the base of a digestion tube that extended from and was open to the surface of the wafer. At 3 days, micropockets were observed to form around each C6 cell at the base of each tube. By 7 days, the majority of pockets observed were large and contained several C6 cells. These multiple cell groups appeared to be progenitors of tumor masses. These data indicate that C6 glioma-astrocytoma cells, which in vivo appear to be a model for glioblastoma multiforme, primarily migrate as individual cells through artificial basement membrane and secondarily form tumor masses. Progenitor tumor masses form by coalescence of individual C6 cell micropockets or the division of a single cell in an individual micropocket.

Rapid migration of grafted cortical astrocytes from suspension grafts placed in host thoracic spinal cord

The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and homografted as a cell suspension into host thoracic spinal cord. Animals were sacrificed at 7, 14, 21 and 30 days postimplantation (DPI). Paraffin sections of cervical, thoracic and lumbar spinal cord were double-labeled for the presence of glial fibrillary acidic protein (GFAP) a specific marker for astrocytes, and PHAL, utilized as a marker for graft-derived cells. PHAL-GFAP positive cells were found throughout the thoracic spinal cord at all time periods, indicating that grafted astrocytes migrated along all 3 axes of the host spinal cord (rostral-caudal, dorsal-ventral and left-right). At 30 DPI, graft-derived astrocytes were found in host cervical and lumbar spinal cord. There appeared to be a minimal delay in the onset of migration.

Individual C6 glioma cells migrate in adult rat brain after neural homografting

Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and grafted as a cell suspension (106 cells in 5.0 μl) into freshly made cortical implantation pockets in adult host rats. Animals were killed 1–21 days post‐implantation (DPI). The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 μm. Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes. Cells positive for both PHAL and GFAP were graft‐derived C6 cells. By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma. This mass increased in size over the next 14 days. The invading tumor mass contained double labeled cells at all time periods examined. In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells). Individual graft‐derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.

Mechanisms of C6 glioma cell and fetal astrocyte migration into hydrated collagen I gels

Fetal basal ganglia astrocytes and C6 glioma cells were plated on the surface of 1.5 cm thick hydrated collagen I wafers. Both cell types migrated through the entire thickness of the wafer within 1 day after plating. The collagen in the wafer was digested and the fine collagen I fibrils were clumped into large strands. By 2-3 days, the collagen strands were digested from the wafers and replaced by a mass of fetal astrocytes or C6 cells joined by their processes. The collagen I digestion and cell migration suggested protease production. In a second series of experiments, cultured C6 cells and E14 fetal astrocytes were immunohistochemically stained for the presence of plasminogen activators as an index of protease production. Both tissue (tPA) and urokinase (uPA) types were observed. Fetal astrocytes and C6 cells were also positive for guanidinobenzoatase, a serine protease associated with migrating cells. These data demonstrate that rapid migration of the cells on and through collagen I fibrils is concomitant with expression of plasminogen activators and proteases which can either activate or function as collagenases and release the cells from the substrate.

Injury-related spinal cord astrocytes are immunoglobulin-positive (IgM and/or IgG) at different time periods in the regenerative process

IgG-positive astrocytes have been reported in scrapie-induced and Alzheimers cortical plaques, multiple sclerosis, and CNS tissue around abscesses, metastatic tumors and primary tumors of glial origin. The present experiments ascertain if this immunoglobulin positivity is specific for these cases or a function of astrocytes around any site of injury in the CNS. The spinal cords of 30, 300-g Sprague-Dawley male rats were lesioned by passing a 26 gauge needle through the cord at T6. After periods as long as 9 months, the spinal cords were processed for paraffin immunohistochemistry with antisera to IgM, IgG or double labeled for these immunoglobulins and GFAP (glial fibrillary acidic protein) a specific cytoplasmic marker for astrocytes. From 1 through 7 days after lesioning double labeled astrocytes in and around the site of injury are both IgM- and IgG-positive. From 14 days through 9 months postlesion, double labeled astrocytes surrounding the lesion are only positive for IgG. These data indicate a relationship between immunoglobulin availability, continued blood-brain barrier perturbation to immunoglobulins and the ability of reactive astrocytes to sequestor immunoglobulins. IgM is an early determinant for reactive astrocytes and IgG positivity is determinant for reactive astrocytes at any time period.

Immunohistochemistry of Human Malignant Astrocytoma Cells Xenografted to Rat Brain: Apolipoprotein E

Fresh xenografted human malignant astrocytoma cells migrate throughout the host rat brain. Cells from three Grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutinin (PHAL) and then xenografted into implantation pockets in rat host cerebral cortex. The human malignant astrocytoma cells in the host brain were immunocytochemically double-labeled for the presence of PHAL, which is used as a marker for graft derived cells, and either glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and astrocytoma cells, or apolipoprotein E (APOE) 7 days, 14 days, 21 days, and 1 month later. Fresh human malignant astrocytoma cells (Grade 3 and 4) contained APOE and GFAP. The xenografted cells preserved APOE and GFAP in the host. PHAL double-labeled human malignant astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the space of Virchow-Robin, migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. APOE was a consistent marker for the migrating human malignant astrocytoma cells, but not an exclusive marker of the xenografted cells, since host rat reactive astrocytes also expressed APOE.

Migration of fresh human malignant astrocytoma cells into hydrated gel wafers in vitro

Individual astrocytoma cells expressing a cytoplasmic form of pl85 c-neu migrated along basement membrane lined surfaces after xenografing fresh low or high grade human malignant astrocytomas into host rat brain. We now study the migratory capacity of fresh human malignant astrocytoma cells seeded on hydrated gel wafers composed of artificial basement membrane or collagen I, a normal and lesion-related CNS extracellular matrix component. Approximately 107 mechanically disrupted cells (with small clumps) of 3 fresh low grade and 6 fresh high grade astrocytomas were seeded on the surface of artificial basement membrane and collagen I wafers (11 × 16 mm). The wafers were then prepared for scanning electron microscopy and immunohistochemstry at 1, 3, 5, and 7 days after seeding. Regardless of tumor grade, a morphologically similar class of cells was observed to migrate through collagen I gels in 24 hours and 0.5–1.5 mm into artificial basement membrane gels in 7 days. Immunohistochemistry revealed that the migrated cells from low and high grade astrocytomas were positive for glial fibrillary acidic protein (GFAP)) and expressed cytoplasmic human-specific pl85 c-neu . These data indicate that fresh human malignant astrocytoma cells that contain GFAP and express cytoplasmic pl85 c-neu have a high degree of migratory capacity and could be the cell in the tumor involved in intraparenchymal metastasis and poor patient survival in high grade astrocytomas of the human brain.

Transplantation of cultured fetal spinal cord grafts, grown on a histocompatible substrate, into adult spinal cord

Cell suspensions from 14-day gestation rat spinal cord can be successfully cultured on collagen gels containing laminin. After 7 and 14 days in culture the gels were removed from the dish and slices of gel with cultured cells were transplanted into the dorsal column of adult rats. Thirty days later there was no evidence of the gel. However, grafted neurons with well-developed organelles, axons, dendrites, axosomatic and axodendritic synapses were observed in the white matter of the host dorsal columns. Oligodendrocytes and astrocytes were also observed in the graft. This histocompatible substrate for culturing fetal CNS is a mechanism for transplanting cultured CNS cells as grafts of known maturity and synaptic organization into adult host CNS.