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.
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.
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.
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.
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 great platform to host our fundraiser.
Our fundraiser played a crucial role in this project, by contributing over $50.000 and allowing the team to generate enough data to apply for larger research grants. We are very happy to hear that those larger grants have now been approved and that the DIPG single cell sequencing project will continue.
Once again we would like to thank all of you who contributed! It is truly amazing what this fundraising campaign has accomplished. We will keep you updated on the progress of this project.