Calcium signalling in glioblastoma multiforme: Combining oncology and neurogenesis with synthetic biology and optical imaging to relate calcium signalling with neural stemness. – calciumGlioStem

we have now established, for GiCs, a reliable system of culture which allows the study of the passage from self proliferation to quiescence. Differentiation can be obtained by substituting growth factors EGF and FGF with 0.5% serum in the culture medium of self renewal cells. This was the pre-requisite to tackle the task 2
Calcium movements in TG01 cells were performed by photometry using the calcium luminescent probe aequorin. We have designed and tested the functionality of different aequorins; the 1st is targeted to the cytoplasm, the 2nd targeted to the mitochondria and the 3rd targeted to the golgi. Ca2+ homeostasis is measured via SOCE stimulation.
We have achieved a lot of the essential preparation work for the project. This includes preparing some of the first transgenic fish lines that we require for the project as well as developing the techniques to prepare neurospheres from zebrafish brain. We are also in the earliest stages of developing transgenic lines of fish that develop brain tumors.
In order to build a model able to describe the calcium signal of the glioblastoma cancer stem cell, we have followed the following steps:
• Defining a set of genes coding for proteins specifically involved in the calcium signal of the cancer stem cells. The calcium toolkit genes are coding for calcium pumps/calcium exchangers, for calcium channels and for calcium binding proteins involved in the regulation of calcium entry and calcium outflow. Moreover, we are optimizing a workflow to detect specific complexes of those proteins.
• Developing the software parts needed to model our system.
• Finally, describing our model in the language SBML and simulating our model.

Task2
we have established, for GiCs, a reliable system of culture which allows the study of the passage from self proliferation to quiescence. we have shown that Ca2+ homeostasis is different in self renewal and quiescent TG01 cells. In quiescent cells the Ca2+ homeostasis is controlled by an increase of the mitochondria Ca2+ uptake.
IP3 receptors may play a role in the calcium homeostasis in the two states of TG01 cells
When self renewal TG01 cells are cultured in acidic medium (pH6.5) they adopt a quiescent state after 5 days of culture. The same experiment done in presence of 2-APB, prevents the passage in quiescence. This important result shows that Ca2+ homeostasis can control the different states of TG01
We have established acorrelation between the states of TG01 cells and the morphology of mitochondria. This is an important result since it has been already shown in other model that the changes in mitochondrial shape is linked to crucial cellular functions such as Ca2+ signalling
Preliminary experiments indicate that the Ca2+ toolkit is different in self renewal and quiescent cells
We have achieved a lot of the essential preparation work for the project. This includes preparing some of the first transgenic fish lines that we require for the project as well as developing the techniques to prepare neurospheres from zebrafish brain. We are also in the earliest stages of developing transgenic lines of fish that develop brain tumors.
Task4
we have a specific calcium gene profile for each subset.
In order to characterize what are the calcium toolbox expressed genes in a given subset, we have developed an algorithm to detect the genes expressed specifically in a subset and the genes expressed everywhere.
We show that i) the mechanism of SOCE is strongly perturbed in cancer cells ii) the process of calcium reuptake into mitochondria is more important in cancer cells than in their normal counterparts, i

Task2 1st part
This part of the work is in total accordance with what was planned on the calendar for the application.
1st deliverable: characterization of protein expression for different Ca2+ channels (STIM and ORAI, TRPs) and IP3 receptors (January 2016)
2nd deliverable: deciphering the role of Ca2+ in the transition self renewal/quiescence. 2-APB gives an interesting result by inhibiting the quiescence state. The target(s) of 2-APB will be précised (March 2016).
3rd deliverable: writing an article (March-May 2016)
4th deliverable: Functional implication of Ca2+ signalling in the transition self renewal/quiescence. Inhibition of chosen targets by ShRNA. Relations with cell cycle (October 2016).
Writing an article (1st semester 2016).
Task2 2nd part
We have demonstrated that cells isolated from the brain and maintained with growth factors in suspension culture will form neurospheres as early as 3 days in culture. We aim to determine the presence and localization of the ER-based Ca2+ toolkit. We already have the appropriate antibodies in order to conduct these experiments.
nduction of tumors in the fish brain
KRasG12Vconstructs will be microinjected into early zebrafish embryos in order to induce brain tumors .
Task 4
UNDERSTANDING THE PROPERTIES OF Ca2+ SIGNALLING NETWORKS IN CANCER STEM CELLS
This first deliverable is expected to be completed in September.
?Our second deliverable will be the building of models that simulate the observed Ca2+ signals in the Toulouse laboratory. We are expecting to get a first model in January.
Our third deliverable will be to pinpoint the critical steps of our model in order to design experiments to perturb such steps. When perturbed, if the cell responds as they do in our in silico model, . More precisely, pharmacological molecules are used in Toulouse to perturb the calcium signal. We will test our model by looking if our model simulates the Toulouse experimental observations.

Publications
Noémie Robil, Fabien Petel, Marie-Claude Kilhoffer and Jacques Haiech (2015) Glioblastoma and Calcium signalling. Analysis of the calcium toolbox expression. Int. J. Dev. Biol (in press)

Active participation in scientific meetings
Jacques Haiech : Calcium signaling in normal and cancer stem cells : different languages for different fates (invited speaker) 13th International meeting of the European Calcium Society, Aix en Provence, September 13-17, 2014

Francisco Aulestia : Cancer stem cells: Ca2+ influx through store operated Ca2+ channels. 13th International meeting of the European Calcium Society, Aix en Provence, September 13-17, 2014

Catherine Leclerc : Characterization of the Ca2+ signatures in cancer stem cells from glioblastoma multiforme (invited speaker) 6th European Calcium Society workshop Calcium and Cell Fate, Seillac France June 21--24 2015

Marc Moreau, Catherine Leclerc and Jacques Haiech are three of the co-organizers of the 6th European Worskhop Calcium and Cell Fate , 21-24 June 2015, Seillac (France)

Glioblastoma multiforme (GBM) is the most frequent aggressive malignant primary brain tumor with a median survival of 15 months. A host of signaling molecules activates GBM cells to affect proliferation, motility, and invasiveness and modulate downstream effectors of intracellular signaling pathways. These aggressive tumors display a heterogeneous cellular composition. Some tumor cells bear significant migratory capacity rendering therapies ineffective. This results in invasive spread and recurrent growth. Despite recent advances in the understanding of GBM pathogenesis, no ideal outcomes have appeared. This necessitates a new therapeutic approach based on a broader understanding of GBM pathogenesis.
The cellular origin of GBM is still a matter of debate but a subpopulation of glioma cells share many properties with adult neural stem cells (NSCs), leading to call them Glioblastoma initiating Cells (GiCs) with distinct self renewal “embryonic characteristics” and local metastatic ability capable of initiating tumor growth. These characteristics can be compared with adult neurogenesis. The general consensus identifies astrocyte-like cells as NSCs. An important aspect of adult vs embryonic neurogenesis is the transition to a slow proliferative mode. We hypothesize that GiCs in GBM originate from quiescent astrocyte-like cells and that deregulation of this proliferative transition mode contributes to the generation of brain tumors.
What is the mechanism of the transformation of NSCs into GiCs? The current dogma proposes a two-hit mechanism: a 1st event in stem cells allowing proliferating and accumulating mutations, and a 2nd mutation in tumor mass cells to acquire properties of GiCs, each event might be genetic or epigenetic.
We propose that these alterations perturb regulatory Ca2+ signalling pathways. Aberrant ciliogenesis is associated with GBM and this deficiency likely contributes to malignancy. Primary cilia are implicated in signal transduction, cell cycle progression and regulation of Ca2+ signaling pathways. Genome-wide analysis of GBM has implicated many elements of the Ca2+ toolkit, including Ca2+ channels and calmodulins (CaM). Interestingly, CaMkinase II is probably the direct target of a TRPP2-Ca2+ channel located to the primary cilia. Our team and others have shown that Ca2+-dependant signalling pathways are important triggers during neurogenesis.
We propose that the 1st hit induces aberrant ciliogenesis with a consequence on Ca2+ signaling and that the 2nd hit targets the balance of proliferation/differentiation via alterations of CaMKinases.
The following questions will be investigated in 3 Tasks:
Question 1: How and by which structures does Ca2+ signaling maintain the state of GiCs?
Question 2: How and by which targets does Ca2+ signaling regulate the proliferating state in GBM?
1: We will use Ca2+ imaging to compare Ca2+ homeostasis in NSCs and in proliferating GBM cells. We will take advantage of lower vertebrate models (Xenopus and zebrafish) where we have already published important results concerning Ca2+ signalling during early neurogenesis.
2: We will determine the elements of the Ca2+ signalling pathways that are potentially modified in GiCs. The perturbation of CaMKinases activities, the hub of Ca2+ signaling, will be performed using optogenetic techniques. With this tool we will attempt to orientate the GiCs toward a particular proliferating fate.
3: We will utilize the specific characteristics (frequency, amplitude, shape, and mode of generation) of the Ca2+ signals to speculate on the mechanisms responsible for both their encoding and decoding. We will establish and test a hypothetical predictive model using specific tools developed by J. Haiech and we will develop an in silico model to identify other possible signalling hubs and networks that drive GiCs towards a proliferative state. This will hopefully help us to explore and develop novel therapies that may be applied GiCs.