Thread by @VarunVenkatara2

How do glioblastoma invade and colonize the entire brain ?
Overlaying molecular, structural and functional data helped us to uncover three layers of neuronal mechanisms driving glioblastoma invasion (authors.elsevier.com/c/1fVaEL7PXipIV). A thread 🧵: (1/24)

Previously, we saw a subpopulation of glioblastoma cells was connected with each other via gap junctions while other tumor cells appeared unconnected . The tumor-tumor network was resistant toward therapy. But what is the role of the seemingly unconnected tumor cells ? (2/24)

To characterize molecular and functional glioblastoma cell states we used a combination of in vivo imaging and single cell RNA-sequencing with the dye SR101 that could distinguish tumor-connected and and tumor-unconnected glioblastoma cells. (3/24)

Surprisingly, we found that some tumor cells not only talk to each other but also with astrocytes via gap junctions while the unconnected cells were neither connected to tumor cells nor astrocytes. (4/24)

Using in vivo timelapse imaging in xenograft models, we could identify tumor cells not connected to astrocytes and other tumor cells as drivers of glioblastoma invasion. (5/24)

Next, we wanted to understand whether these connectivity states can be distinguished and how they evolve over time. scRNAseq showed that they can be molecularly separated and potentially transition from unconnected to connected tumor cells over time. (6/24)

Leveraging in vivo two photon microscopy with SR101 as connectivity marker we could demonstrate that tumor cell/astrocyte-unconnected glioblastoma cells evolve to tumor cell/astrocyte-connected glioblastoma cells over time. (7/24)

What molecular cell states are now associated with invasion ? Using different classifications, we see that neuronal-like and neural progenitor-like/oligodendrocyte-precursor are enriched in the glioblastoma subpopulation driving invasion. (8/24)

Does the molecular neuronal-like cell state translate to neuronal cellular patterns of invasion ? We adapted restoration microscopy algorithms together with in-vivo two-photon imaging to follow small neurite-like processes called tumor microtubes. (9/24)

Using this approach, we found three mechanisms govering tumor microtube dynamics similar to neurites during path-finding: branching, protrusion and retraction. (10/24)

But we also find highly dynamic, small processes that are smaller than tumor microtubes that contribute much to the scanning behaviour of glioblastoma cells. (11/24)

We analyzed the scanning behaviour of tumor microtubes in more depth and found that this could be described a Levy-like movement pattern, a search-efficient mechanism that could previously also be seen in animal predators looking for scarce sources of food. (12/24)

Observation with in vivo imaging revealed that three overall movement patterns could describe the invasion pattern, reminiscent of neuronal migration patterns during development: branching migration, locomotion and translocation. (13/24)

Lastly, we wanted to understand whether neuron-glioma synapses that we previously described (nature.com/articles/s4158…)also influence tumor microtube genesis and dynamics.And indeed ! Neuronal activity increased tumor microtube genesis and dynamics as well as invasion speed.(14/24)

Investigating the mechanisms with 3D in vivo calcium imaging, we found that glioblastoma cells not only show calcium waves and whole-cell calcium transients but also calcium microdomains. (15/ )

Inhibiting calcium transients with a calcium chelator inhibited tumor microtube dynamics and reduced invasion. (16/24)

Genetic perturbation and pharmacological AMPA receptor inhibition led to reduced reduced tumor microtubes per cell, reduced branching points and overall reduced tumor microtube length. (17/24)

Taken together, we find three neuronal layers: a molecular neuronal-like cell state driving glioblastoma invasion, neuronal-like cellular mechanisms and neuron-glioma synapses driving neurite-like sprouting of tumor microtubes. (18/24)

However, there remain open questions: How do these neuronal mechanisms change under therapeutic pressure ? What is the role of synaptic contacts on different cell and connectivity states ? (19/24)

How do single glioblastoma cells evolve over time on a molecular, cellular, connectivity and functional level ? How does the microenvironment change in parallel ? And very importantly: How do these results translate to the clinic ? (20/24)

It is very exciting to see that recent work by @roelverhaak and @fsvarn could confirm that neuronal signaling was indeed associated with a more invasive phenotype of human glioblastoma upon recurrence (sciencedirect.com/science/articl…) (21/24)

Our work can only be a small puzzle piece in a very exciting, emerging area of #CancerNeuroscience research. It is clear that we need to study these incurable brain tumors in a multidisciplinary fashion... (22/24)

...with further preclinical and clinical-translational work to push this field forward. (23/24)

I would like to thank everyone involved in this work which could not have been possible without the joint enormous efforts by everyone involved ! It was a great journey and I am looking forward to the next steps ! (24/24)

@YvoYang @MarcCSchubert
@ekinreyhann
@SvenjaKTetzlaff
@nikwiss
@StellaSoyka
@RPramatarov
@Rb409Wagner
@wick_wolfgang
@Winkler_Lab
@Miriam__Ratliff
@DirkCHoffmann
@sahm_lab
@AnGolebiewska @yahayayabo
@uniklinik_hd
@DKFZ
@HdNeurooncology
@NCT_HD @CancerNeurosci1