Relevance of gene mutations and methylation to the growth of pancreatic intraductal papillary mucinous neoplasms based on pyrosequencing

We aimed to assess some of the potential genetic pathways for cancer development from non-malignant intraductal papillary mucinous neoplasm (IPMN) by evaluating genetic mutations and methylation. In total, 46 dissected regions in 33 IPMN cases were analyzed and compared between malignant-potential and benign cases, or between malignant-potential and benign tissue dissected regions including low-grade IPMN dissected regions accompanied by malignant-potential regions. Several gene mutations, gene methylations, and proteins were assessed by pyrosequencing and immunohistochemical analysis. RASSF1A methylation was more frequent in malignant-potential dissected regions (p = 0.0329). LINE-1 methylation was inversely correlated with GNAS mutation (r = − 0.3739, p = 0.0105). In cases with malignant-potential dissected regions, GNAS mutation was associated with less frequent perivascular invasion (p = 0.0128), perineural invasion (p = 0.0377), and lymph node metastasis (p = 0.0377) but significantly longer overall survival, compared to malignant-potential cases without GNAS mutation (p = 0.0419). The presence of concordant KRAS and GNAS mutations in the malignant-potential and benign dissected regions were more frequent among branch-duct IPMN cases than among the other types (p = 0.0319). Methylation of RASSF1A, CDKN2A, and LINE-1 and GNAS mutation may be relevant to cancer development, IPMN subtypes, and cancer prognosis.

Brain Invasion along Perivascular Spaces by Glioma Cells: Relationship with Blood–Brain Barrier

The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood–brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.

A 3D Topographical Model of Parenchymal Infiltration and Perivascular Invasion in Glioblastoma

Glioblastoma (GBM) is the most common and invasive primary brain cancer. GBM tumors are characterized by diffuse infiltration, with tumor cells invading slowly through the hyaluronic acid (HA)-rich parenchyma toward vascular beds and then migrating rapidly along microvasculature. Progress in understanding local infiltration, vascular homing, and perivascular invasion is limited by an absence of culture models that recapitulate these hallmark processes. Here we introduce a platform for GBM invasion consisting of a tumor-like cell reservoir and a parallel open channel “vessel” embedded in 3D HA-RGD matrix. We show that this simple paradigm is sufficient to capture multi-step invasion and transitions in cell morphology and speed reminiscent of those seen in GBM. Specifically, seeded tumor cells grow into multicellular masses that expand and invade the surrounding HA-RGD matrices while extending long (10-100 µm), thin protrusions resembling those observed for GBM in vivo. Upon encountering the channel, cells orient along the channel wall, adopt a 2D-like morphology, and migrate rapidly along the channel. Structured illumination microscopy reveals distinct cytoskeletal architectures for cells invading through the HA matrix versus those migrating along the vascular channel. Substitution of collagen I in place of HA-RGD supports the same sequence of events but with faster local invasion and a more mesenchymal morphology. These results indicate that topographical effects are generalizable across matrix formulations, but that mechanisms underlying invasion are matrix-dependent. We anticipate that our reductionist paradigm should speed the development of mechanistic hypotheses that could be tested in more complex tumor models.

EphrinB2 drives perivascular invasion and proliferation of glioblastoma stem-like cells

Glioblastomas (GBM) are aggressive and therapy-resistant brain tumours, which contain a subpopulation of tumour-propagating glioblastoma stem-like cells (GSC) thought to drive progression and recurrence. Diffuse invasion of the brain parenchyma, including along preexisting blood vessels, is a leading cause of therapeutic resistance, but the mechanisms remain unclear. Here, we show that ephrin-B2 mediates GSC perivascular invasion. Intravital imaging, coupled with mechanistic studies in murine GBM models and patient-derived GSC, revealed that endothelial ephrin-B2 compartmentalises non-tumourigenic cells. In contrast, upregulation of the same ephrin-B2 ligand in GSC enabled perivascular migration through homotypic forward signalling. Surprisingly, ephrin-B2 reverse signalling also promoted tumourigenesis cell-autonomously, by mediating anchorage-independent cytokinesis via RhoA. In human GSC-derived orthotopic xenografts, EFNB2 knock-down blocked tumour initiation and treatment of established tumours with ephrin-B2-blocking antibodies suppressed progression. Thus, our results indicate that targeting ephrin-B2 may be an effective strategy for the simultaneous inhibition of invasion and proliferation in GBM.