Physical Health United States Project #19809

Pursuing new treatments for Neuroblastoma children

by Cure First

Project Report | Oct 26, 2016
New drug combinations for neuroblastoma: in vitro and in vivo validation

By Rachele Rosati | Research Fellow

The work flow

Dear Global Giving Donors and Followers,

During the summer, I had the amazing opportunity to attend the Advances in Neuroblastoma Research Congress, a biannual international conference, where the most cutting-edge neuroblastoma (NBL) research is presented. I was excited to be surrounded by leading experts in the field.

I was thrilled to present my recent research in the Translational Medicine section of the conference. I discussed using functional genomics to identify novel therapeutic targets for retinoic acid combinations in NBL cell lines. We identified new genetic targets, and corresponding drugs, that could increase the efficacy of retinoic acid. The combination of these targeted drugs and retinoic acid could improve the overall survival of NBL patients by decreasing recurrences.

Our next step will be to test these new drug combinations in patient derived NBL cells to prove that they can reduce tumor growth. However, acquiring NBL cells from patients can be an invasive procedure. This is often a bottleneck in testing drugs in culture. At the conference, I learned about potential strategies to address this problem. We could use less invasive procedures, such as “liquid biopsies”, to derive circulating cancer cells from blood samples.

We also plan to test the drug combinations in mice carrying NBL tumors. We will use the mice to track the potential toxicity of these drugs in a living organism. All of the experiments will be done at an institute accredited with the Institutional Animal Care and Use Committees (IACUCs), an organization that is centrally important in applying laws about animal research in the United States.

Overall, the validation of new combination treatments for NBL in vivo will help us build the case to move our genomic targets into clinical trials.

Your donation will make these steps toward a cure for neuroblastoma possible!

Thank you for your continued support,

Your Cure First Research Fellow

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Jul 1, 2016
Neuroblastoma PDTXs and high-throughput drug screen

By Rachele Rosati | Cure First Research Fellow

The ANR2016 conference

Dear Global Giving Donors and Followers,

Sorry to be late to report on the progress of this project, but I have been in Australia for the Advances in Neuroblastoma Research Congress (ANR2016). While in Cairns, I was able to share a poster displaying my studies on novel genes as future targets for combination therapies with retinoic acid. I also met with many scientists working on neuroblastoma. It was particularly inspiring to hear talks from Dr. Julie Park from Seattle Children’s Hospital and from Dr. John Maris, from Children’s Hospital of Philadelphia, both of whom are collaborators with Cure First. Now I am back at the bench and computer, and it is my pleasure to give you an update of the past three months on the Neuroblastoma Project.

After the success in establishing the first Patient-Derived Tumor Xenografts (PDTXs) ex-vivo, growing as tumorspheres in 3D, I was able to proceed with designing a high-throughput drug screen experiment with a library of 345 drugs compiled by the (Cancer Target Discovery and Development) CTD2 network. This drug library has 120 FDA-approved drugs and 225 targeted agents that are in clinical trials, phase I or phase II. The main goal of using this drug library was to interrogate as many genes and molecular pathways as possible in order to have a broader and more complete view of the spectrum of the responses of each individual tumor. The first general result is shown here, illustrated by a heat map graph that recorded the AUC (area under the curve) of each drug obtained by high-throughput screeen (HTS) in two patient derived neuroblastoma cell lines. The smaller the AUC (intense blue color), the more the sample shows response to a respective drug, while resistance, i.e. larger AUC, is shown in red. Producing a functional map for every sample, each from a different neuroblastoma sample from a different child, demonstrates both individual and common sensitivities. Many good results are coming from new potential drugs. More work is needed to validate these results and extend them to more samples, but we are extremely hopeful we can identify future new therapies for high-risk neuroblastoma.

Thank you for your support!

Heatmap of AUC obtained by HTS testing 345 drugs

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Mar 18, 2016
Neuroblastoma Organoids from Patient Derived Cells

By Rachele Rosati | Cure First Research Fellow

NB-SD, small tumor spheres at 20X magnification

Dear Supporter,
I would like to share the progress that has been made on the Neuroblastoma Project:
As I previously announced our Director, Dr. Carla Grandori, initiated a collaboration with one of the most prominent Neuroblastoma expert in the country, Dr. John Maris from the Children’s Hospital of Philadelphia (CHOP). Dr. Maris has provided Cure First with ten Patient Derived Tumor Xenograft (PDTX), which I have been working with for the past few months. Each sample came from a patient diagnosed with Neuroblastoma and each had distinct alterations and amplifications. At Dr. Maris’s lab, the patients’ cells had been injected in immunodeficiency mouse and expanded. Then each tumor was chopped out and frozen. Despite the difficulty of retrieving tumor cells from the frozen pieces of tissue where the interstitial mouse cells may have spread, I was able to isolate and expand neuroblastoma tumor cells in vitro.

I started with the following PDTXs: NB-SD, which has a MYCN amplification, TP53, ALK and FLT3/4 mutations and SKNAS, which is an NRAS and TP53 mutant. I have optimized their growth, after several different experiments, with the appropriate culturing media and the appropriate dishes. The results are different for each samples and it took almost a month to achieve a perfect growth in non-adherent tumor spheres. However this is a great achievement as you can see from these photos.

Recently I have received other 2 fresh Neuroblastoma Xenograft samples from CHOP, I processed them and for one of these, NB-Ebc1, which has KRAS and EZH2 mutations, I obtained tumor organoids as well.

Thanks to these results I can announced that we are ready to screen NB-SD and NB-Ebc1 samples, testing a library of 320 compounds compelled by CTD2 (Cancer Target Discovery and Development) group, in order to see which drugs have an impact on the mutations associated with neuroblastoma.

I am looking forward to start these new challenge!

We also want to thank the Soupy for Loopy Foundation that is our main supporter in this project with their donation, and all the people that supports us through Global Giving allowing us to cover the cost of all the laboratory materials needed to carry on the Neuroblastoma project.

Our efforts to discover new drug targets for Neuroblastoma still need your help. Thank you in advance for your help!

NB-SD, tumor organoids at 20X after 10 days

NB-Ebc1, several tumor organoids at 10x

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