Tumor cells form networks to penetrate the brain

Researchers from MD Anderson have revealed how glioma cells move and infiltrate the brain, providing a new potential treatment pathway to explore against these deadly brain tumors.

“Glioma is not usually resectable or curable because it spreads diffusely throughout the brain, like mold in bread. We found that glioma cells form a moving network, and we’ve identified a novel mechanism that could be targeted to halt that cellular movement,” says Peter Friedl, M.D., Ph.D., professor of Genitourinary Medical Oncology and senior author of the article published in Nature Cell Biology.

The research group first mapped the three-dimensional (3D) microanatomy of glioma clinical samples. Conventional single-slice (i.e., two-dimensional) pathological analysis of these samples showed that cells were spread far apart, with little evidence of intercellular connections.

However, Friedl’s research group used 3D image reconstruction of 30 to 50 slices of tumor tissue stained to differentiate glioma cells from other brain cells. This 3D method revealed extensive multicellular glioma cell networks in both the center of the tumor and at the infiltrating margins. These cellular networks were found in both low-grade astrocytomas and glioblastomas. The 3D imaging showed two types of connections between glioma cells: long filaments and compact, epithelial-like junctions.

Glioma cells move like a fungal colony to progress rapidly

To characterize these connections, the researchers used PDX models (samples of human glioma tissue xenografted into a mouse brain). The PDX models showed that the type of connection depends upon cellular density: each branched filament connected four to six neighboring but dispersed glioma cells, and compact linear junctions formed between directly adjacent cells.

Using complementary 3D in vitro models to mimic brain infiltration, the researchers found that glioma cells maintained contact with three to five neighboring cells while moving at 120–140 micrometers per day. This collective invasion shows mechano-chemical cooperation among cells. In fact, 3D time-lapse microscopy showed that glioma cells can dynamically convert between the two types of connections as cell density changes.

“We found that glioma cells move like the mycelium of a fungal colony; these cellular networks allow glioma to cover large volumes and progress rapidly,” says Friedl.

The research team also investigated the biochemical mechanisms of glioma cell connections. Adherens junctions act like Velcro for cells, forming attachments mediated by E-cadherin and N-cadhedrin. The research team found that downregulating N-cadhedrin, b-catenin and p120-catenin disrupted these cell connections.

Stifling p120-catenin inhibits brain tumor growth

p120-catenin is essential for cell-cell interactions throughout the body. Downregulation of p120-cateninin in the PDX models impaired filament connections that help diffuse glioma cells join together to move outward and severely inhibited growth. This, in turn, compromised the glioma networks.

Results from a 3D astrocyte scaffolding assay also showed that downregulating p120-catenin disrupted the filament connections between glioma cells. Although the glioma cells were still mobile, they were no longer able to migrate away from the center of the tumor. However, expression of p120-catenin rescued multicellular network formation.

The researchers also investigated the effect of downregulating p120-catenin as tumors develop in vivo by implanting fluorescent glioma cells in mice. After 4 weeks of tumor growth, p120-catenin-deficient cells did not form large or multifocal lesions and lacked diffuse brain infiltration. These tumors were 90%-98% smaller than those of control cells.

“This is the biggest effect in reducing the aggressiveness in glioma that we have seen so far in a mouse model,” says Friedl.

For these experiments, the research team used a novel method that allows whole-brain reconstruction and volumetric analysis through 3D confocal microscopy of 200-micron-thick slices of the entire brain (15-17 slices for a mouse brain). “This method allows us to see every filament in the entire brain,” Friedl says.

Next: search for a drug that will work in human brain tumors

The team analyzed p120-catenin expression in two independent databases of glioma patients and found significantly prolonged survival among patients with low p120-catenin levels in their brain tumor cells.

Future research will focus on finding an inhibitor for p120-catenin or related molecules that will work in humans. “We are trying to target something of general relevance for the body, in the same way that chemotherapy inhibits mitosis. We need to find the sweet spot where glioma cells are more vulnerable than normal cells,” Friedl says.

This novel strategy will likely have to be used in combination therapy. “Targeting p120-catenin-dependent adherens junctions will sensitize glioma cells to traditional therapies, such as radiation. This will help reduce side effects for patients and could help reduce therapy resistance,” Friedl says.

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