Mariella Filbin, MD, PhD
I first met a young girl with DIPG during my early years of medical training. I followed her all through her therapies and had to learn the hard way that we did not have many treatments to offer. I remember the desperation I felt: we could fly to the moon, send data across the planet in seconds, but still have not made any advances in this terrible disease over decades. I decided then to dedicate my career to patients with highly aggressive brain tumors and have since focused all of my research efforts on finding new therapeutic approaches for these devastating tumors.
Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most lethal brain tumors afflicting children. Most succumb to their disease within a year of diagnosis. Pediatric patients are currently treated with radiation therapy and often chemotherapy; however, tumors exhibit rapid resistance to these therapies and commonly start to grow again within months of treatment completion. Understanding the biology of DIPG is critical in order to identify new effective treatments for this disease.
While considerable progress has been made in the identification of molecular subtypes of many cancers, the transfer of these findings into improved therapies for children with DIPG has not yet been realized. Genetic studies of DIPG have recently shed light on their mutations, but much remains to be learned about how this tumor starts growing and maintains itself. One key limitation to current analyses is that they are performed on bulk tumor pieces and do not inform at the single-cell level or on the functional states of individual cells.
Our hypothesis is that DIPG is a heterogeneous disease containing many diverse cell types. Only a small percentage of cells have the potential to cause the tumor to grow by aberrantly reactivating stem-cell genes that were turned off after normal brain development.
Our proposed research leverages single-cell RNA sequencing technologies and applies them directly to patient samples. These novel methods are revolutionizing our understanding of cancer, as they allow for the first time to look comprehensively (up to 10,000 active genes in any given single cell) at all cell types present in a tumor, including rare sub-populations. This will enable us to identify all cell types in patient-derived DIPG biopsies in an unbiased fashion, identify their genetic mutations, and characterize the programs that drive them at the single-cell level for the first time. Uncovering these aspects of DIPG tumor biology will effectively fill a very large gap of knowledge and allow the design of therapies aimed at targeting the entire spectrum of DIPG cancer cell subpopulations that exist in patients.
Why is this important?
Single-cell RNA-sequencing methods applied to rare and precious patient-derived DIPG samples will shed unprecedented light on tumor cellular architecture and composition that are not identifiable by bulk tumor analyses. We will exploit this knowledge to define and target the transcriptional networks underlying DIPG growth and reveal novel tumor vulnerabilities that can rapidly enter pre-clinical and clinical trials.
Who will benefit?
The proposed study is the first ever to study systematically the transcriptional programs of a pediatric cancer at the single-cell level. Using pioneering single-cell technologies and computational analyses available at Dana-Farber Cancer Institute, Massachusetts General Hospital and the Broad Institute of MIT and Harvard will provide a novel view of the molecular pathways driving DIPG, with the lessons learned widely applicable across all areas of pediatric oncology.
BudgetThe project is divided in five identical steps. Each step involves the sequencing of 1,000-2,000 single cells from a DIPG biopsy and costs about $20,000 including all reagents, materials, technician salaries and bioinformatic analysis. We have learned from adult glioma that a total of five tumors is needed to get a comprehensive picture of the tumor architecture.
You did something great!!!
To our dear supporters,
We have some great news regarding our fundraiser. Even though we did not reach our initial goal of $100.000, we raised over $50.000 for DIPG research. This money has already been put to use. Marielle Filbin, Mario Suvá and their team were able to perform single cell RNA sequencing on five different DIPG samples.
This generated enough preliminary data to successfully apply for additional funding to continue this project. So we hope more DIPG samples will be sequenced soon. We are confident the data from this project will be published in the near future and will quickly translate into new approaches on how to treat DIPG.
Thank you all for making this groundbreaking project possible! Also a big thank you to the people at consano for providing us with this greatplatform to host our fundraiser.
For more information and future updates go to:
Kate's Birthday and news from the lab!
Today would have been Kate’s 7th birthday. She battled DIPG for 18 months and inspired so many people with her beautiful and brave spirit. Even after her passing Kate’s fight continues in so many ways. One good example are the kind people at "Unbridled Acts“ who not only set up a fundraiser in Kate’s honor, but pledged to match the total donations $ .50 for every $1 donated, up to $10,000!!!
All the funds will go towards the DIPG research project here on Consano. The fundraiser and the matching program will end next week. So go to http://unbridledacts.org/continuing-kates-fight/ and increase the impact of your contribution by donating within this generous matching system!
Furthermore, we’ve received great news from the lab!
Since the first DIPG biopsy has been successfully sorted for single cell analysis a few weeks ago, the researchers at Dana-Faber and MGH were able to isolate the RNA from the individual tumor cells. The RNA was then reverse transcribed into cDNA in order to be sequenced. After the reverse transcription the cDNA from each cell was tagged with a DNA barcode (a short genetic marker). The code enables bioinformaticians to identify which piece of cDNA came from which cell, later in the process. You can see the workflow depicted in the graphic.
Before the cDNA is sequenced, the scientists need to make sure that the quality of the sample is good enough and that the reverse transcription actually worked. This is done by an electrophoresis of the sample. You can see the "quality check" electropherogram of the cDNA from DIPG cells in the graphic. The curve suggests an almost perfect sample quality and the sample concentration is more than sufficient for sequencing.
In the next few days the first cDNA-pool of DIPG cells will be sequenced! Analysis of the data will take some time, but it will give scientists a better understanding of DIPG biology and is going to help them to identify new vulnerabilities of this disease.
This revolutionary project was only made possible by your generous donations, but could not have been executed without the brilliant scientists at Dana-Farber Cancer Institute and Massachusetts General Hospital.
In particular Mariella Filbin (DFCI), who initiated this project, Mario Suvà (MGH), who oversees this project as a Principle Investigator and single cell sequencing expert and Leah Escalante (MGH), who is a tremendously skilled technician with a lot of single cell sequencing experience.
We will keep you updated on any new progress.
T-shirt sale is now online!!!
In order to boost our fundraising efforts to the next level we started a t-shirt sale on www.booster.com/DIPG. You can buy high quality t-shirts for women, men and children in all sizes. All the profits will go directly to the DIPG research project here on consano. The shirts will only be available for a limited time, so be sure to check them out!
First DIPG biopsy sorted for single cell analysis
Thanks to all the generous donations to our project the first DIPG biopsy was successfully sorted for single cell sequencing!
Dr. Filbin and her team got a small piece of a DIPG biopsy. The biopsy was immediately brought to the lab and was gently dissociated into single cells by an enzymatic dissociation kit. The single cells were then stained with three different markers. Calcein was used for the staining of viable tumor cells (yellow population on the left in the picture), CD45 was used to stain lymphocytes and microglia which infiltrated the tumor (orange population in the middle and purple population on the right) and TOPro was used to stain non viably or dying cells (not shown in picture).
The staining enabled our team to only sort viable tumor cells (Calcein positive cells) into plates. With the help of fluorescence-activated cell sorting (FACS) Dr. Filbin and her team were able to sort one single cell into each well of a 96-well plate. 14 plates could be sorted in total from this biopsy. The plates were frozen down at -80°C and will be prepared for sequencing in the next week. Once the sequencing is done, a bioinformatic analysis of the sequencing data will reveal the identiy of each cell. This will give us a very detailed picture of what a DIPG consists of and can hopefully reveal different subpopulations within the tumor and their unique vulnerabilities.
Due to the heterogeneity between different tumors in patients, the analysis of a single DIPG biopsy will not be enough to characterize these tumors as a whole. Only a larger number of biopsies will reveal the universal and generalizable architecture of DIPGs.
Thanks again to all of our supporters. It is truly amazing what your support made possible here. We will keep you updated on any new progress.