Research > Focus A > Work Packages A01

TARGETING TUMOR CELL NETWORKS TO OVERCOME PRIMARY AND ADAPTIVE RESISTANCE IN GLIOBLASTOMA

Summary

The main goal of this project is to gain further insights into the biology and relevance of tumor microtubes (TMs), which are thin membrane connections between single glioma cells and an important cellular mechanism of primary and adaptive tumor resistance in glioma animal models. Ultimately, we aim at the development of innovative strategies to inhibit some of their important functions. The specific aims are (1) to understand whether TMs are biomarkers for response to established therapies and adaptive resistance in human glioblastoma, and (2) to develop anti-TM treatments that break resistance to standard glioblastoma therapies. The visual abstract below shows the aims and planned tasks of the work package.

Task 1

Validation of TMs and their networks as a primary and adaptive mechanism of resistance in human glioblastoma

Steps / Workflow

Quantification of TM length, number and interconnectivity; response to therapy in primary/ recurrent patient glioma tissue
Correlation of TM occurrence and therapy resistance (time to progression; OS) in human gliomas
Validation of new relevant genes (Task2) in human samples

A

Tumor microtube networks in diffuse intrinsic pontine glioma (DIPG) harbouring a typical H3.3 K27M mutation. Autopsy material from a pediatric patient, stained with an anti-nestin antibody that specifically stains tumor cells in this brain region. Tissue provided by M. Monje, Stanford. Image size, 100 x 100 μm

B

C

Task 2

Identification of new genes involved in TM formation

Steps / Workflow

Two combined in vitro/ in vivo screening tools of PDX glioblastoma cell lines
Functional validation of new candidates by gene knockdown, in vivo two photon and survival studies

New screening methodologies
Von der Idee zum Konzept
Different growth cond.(adh. vs. serum-free)

SR101 FACS sorting

Not TM-connected (left), and largely TM-connected cells (right) 10 hours after i.v. injection of SR101. Images: preliminary data, R. Xie, F. Winkler Ingenuity analysis: see left, “Background & Preliminary data”: Analysis of TM-connected vs. unconnected glioma cells.
RNA SEQ + PROTEOMICS
10-15 relevant candidates
Validation in 1p19q intact vs. 1p19q codeleted tumors (TCGA), the UNITE databases and tissue from Task 1
3 new TM-relevant targets
Functional validation of new candidates by shRNA knockdown, in vivo two photon, MRI and survival studies

Task 3

In vitro and in vivo screening: effects of selected brain-penetrant compounds on (a) TM morphology and function, (b) glioma growth and

Steps / Workflow

In vitro screen of new compounds (n=100) in 3D co-culture/ brain organoids (selection instructed by new candidates from Task 2)
In vivo screen by two photon microscopy, MRI and mouse survival
Combination of the most convincing compound and standard therapies (radio- and chemotherapy)

A

Glioma cell networks in a novel brain organoid model. Left, Brain organoid cross section demonstrates the infiltrative tumor growth. Right, Dense tumor cell network formation resembling the network structure found in vivo (unpublished, E. Jung and F. Winkler in cooperation with Ph. Koch, Mannheim).

B

Refined 2D in vitro screen (co-culture with astrocyte components) for drug effects on TMs and their networks. Compounds 5 and 10: anti-TM effects, Compound 8: increase in TM-number. Size of every image, 100 x 100 μm (unpublished, E. Jung and F. Winkler).