101% to completion
$7,550 / $7,500 goal
"I am the father of Nathalie Traller who fought against ASPS for 3 1/2 years until finishing her race October of 2015. She chose to push against the boundaries of what is known about the disease, pioneering new treatment paths and reducing barriers for teens to participate in clinical trials..."

Nathan Traller

Dad & Teacher
Children's Cancer Therapy Development Institute

I am the father of Nathalie Traller who fought against ASPS for 3 1/2 years until finishing her race October of 2015. She chose to push against the boundaries of what is known about the disease, pioneering new treatment paths and reducing barriers for teens to participate in clinical trials which are increasingly the best option for treatment of rare diseases. She frankly was in contempt of the limited state of ASPS research and began raising money to support projects we found. We are all connected.

I am by profession a teacher. I was plunged into the world of oncology and joined my wife in an unwelcome crash course into research and our "systems" which can move painfully slow. 

I am incredibly grateful to partner with Dr. Charles Keller in this endeavor. His vision for Children's Cancer Therapy Development Institute (ccTDI) allow for this type of work. He is the scientific "brains" behind the project. Dr. Noah Berlow is the scientific lead for this project at cc-TDI.  Nathalie has supplied the rest of us with more than enough heart.

Charles Keller, M.D.

Scientific Director
Children's Cancer Therapy Development Institute

Summary

Alveolar Soft Part Sarcoma is a rare sarcoma that has no known systemic treatment. It is a devastating disease that most commonly impacts children and young adults who typically are diagnosed with an advanced metastatic stage. 

This pilot project is designed to develop pediatric Alveolar Soft Part Sarcoma (ASPS) cell lines from different years of a disease progression for a female teenage patient. This resource could be very helpful as a preclinical research tool to investigate new potential therapies for different stages of progression for ASPS.

AIM 1. To develop 3 cell lines from independent primary tumor cell cultures currently in the laboratory.The lines will be made publicly available once validated, even before publication.

AIM 2. To test the potential of each culture to develop engraft as a xenograft in vivo. For this aim, we will mix the culture with Matrigel and engraft it onto the quail chorioallantoic membrane (CAM assay), as we have previously described [PMID 21447712].

AIM 3. To define the therapeutic space for these cell line. We will screen each cell line on a 60-agent chemical screen composed of clinical investigational agents for adult, adolescent and pediatric cancers. 

Results of all the above studies would be moved to publication, and all resources would be shared with other researchers openly.  This proposal that seeks to create new understanding and treatment options for adolescents whose lives are touched by ASPS.

Who will benefit?

By creating this shared research resource for ASPS  (as well as glimpsing therapeutic opportunities), patients with ASPS can know that their disease is not forgotten and is being actively studied for therapeutic options as there is currently NO FDA Indicated course of treatment. A trip of a thousand miles begins with a single step. This is the type of step neccessary towards the long journey towards a cure.

Budget

Nathalie Traller Memorial Fund - via The Rutledge Foundation, Sunset High School Class of 2016 & the Family of Nathalie Traller have provided 75% of the base funding for this project with the final 25% to be raised on Consano.

Updates

ASPS-1

Our sincere thanks to all the supporters!

In addition to the original goals to create a specific patient's tumor into cell lines, we're trying to gather (all) other existing ASPS cell lines. Here is the ASPS-1 cell lines that Noah is growing up now, and about go into a drug screen. Thought you'd enjoy the picture!

Charles & Noah

Our Progress to Date

Our thanks to the early supporters of this project. Below is our progress to date. Sincerely, Noah Berlow PhD and Charles Keller MD, cc-tdi.org

AIM 1. To develop 3 cell lines from independent primary tumor cell cultures currently in the laboratory. To convert a primary tumor cell culture, one serially passages the primary cultures until they move past a telomere-shortening ‘crisis’ in defiance of the ‘Hayflick limit’ -- after which a clone or subpopulation becomes immortalized. One definition of a cell line is that the culture has gone through 30 or more ~weekly passages from petri dish to petri dish. Thus, for this aim we will passage each of the 3 cell cultures serially for approximately 30 weeks. We will do genetic STR analysis to confirm that contamination has not occurred every 7-8 passages. At the time the culture becomes a cell line, we will perform DNA exome sequencing and RNA deep sequencing to compare it to a reference for the patient’s tumor. Genetic STR markers and DNA/RNA reference results will be taken from a recent publication [PMID 26994145]. The lines will be made publicly available once validated, even before publication.

We have in culture the non-immortalized cell cultures PCB999B, PCB999C and CF-99.

We have performed STR genetic fingerprinting on cultures PCB999B and CF-99 and found that they share identical genetic fingerprints with a snap frozen piece of the index patient's tumor, indicating the cultures are correctly identified as originating from her.

We have not yet sent cultures for DNA exome and RNA deep sequencing, but will once significant cell populations have been grown.

In addition to the above cultures, we have requested other human ASPS cell lines from the Kanazawa Cancer Center Research Institute in Japan (ASPS-KY), the National Cancer Institute (ASPS-1 and ASPS tumor fragments), and the University of Illinois Hospital (FU-UR-1). We are also establishing MTAs to access materials generated from transgenic mice bearing ASPS tumors from the University of Utah.

AIM 2. To test the potential of each culture to develop engraft as a xenograft in vivo. For this aim, we will mix the culture with Matrigel and engraft it onto the quail chorioallantoic membrane (CAM assay), as we have previously described [PMID 21447712].

We are in parallel optimizing the CAM assay; currently, our CAM assay optimization is focused on the optimization of a high-throughput egg cracking mechanism capable of drastically simplifying the time consuming and highly technical process of extracting the quail embryo. This process ordinarily requires several minutes and extreme delicacy to open a single quail egg, and is not highly reproducible from scientist to scientist. The mechanism developed here can extract 6 quail embryos simultaneously in under a minute. We are finalizing additional improvements to improve quail viability over the course of the 8 days of experimental viability, and presently have around 50% long term viability, which is the present goal for the mechanism.

We will next test newly acquired imaging equipment to enable better visualization of cell and tumor engraftment onto the quail CAM assay to move the quail-based PDX modeling approach to experimental usability.

AIM 3. To define the therapeutic space for these cell line. We will screen each cell line on a 60-agent chemical screen composed of clinical investigational agents for adult, adolescent and pediatric cancers. A similar approach coordinated by our laboratory, albeit with a larger number of cell lines, was recently published in Nature Medicine [PMID 25939062] and resulted in a clinical trial.

We have printed master plates of the drug screen.

We have not yet printed ‘production’ plates for screening ASPS cultures. We have acquired a high-throughput plate printing robot to perform this task in-house, which will be performed once the robot is calibrated and tested for reproducible plate printing.

Results of all the above studies would be moved to publication, and all resources would be shared with other researchers openly. Thank you for considering this proposal that seeks to create new understanding and treatment options for adolescents whose lives are touched by ASPS.

(update 08/06/16):

We will present results of our studies at a University of Miami/Manny Alvarez Foundation Patient Meeting/Research Workshop on Sept 22-24.

Our Progress (12/08/16)

Nathalie’s cells from 3 origins have been cultured: PCBxyzB, PCBxyzC, and CF-xy. PCB-xyzB and PCB-xyzC were digested in collagenase and cultured in the lab. Unfortunately, cells from PCB-xyzC did not adhere to the tissue culture plate and no cells appeared viable after a reasonable amount of time. PCB-xyzB did grow adherent cells and has been developing as a cell culture in the lab; we are expanding it and attempting to develop it into a cell line. We also procured CF-xy (our internal notation) as a culture from Lara Davis, Nathalie’s oncologist, and we cultured it alongside PCB-xyzB.

We also began a collaboration with Dr. Kevin Jones at University of Utah, who has been developing transgenic mouse models of ASPS. His group sent us several tumor pieces from transgenic mice, which were digested and cultured as well.

However, a recent incubator failure during a weekend unfortunately caused us to lose these cultures. The PCB-xyzB culture has been restarted and is growing well, and we are working to replace the other lost cultures.

In addition, we have expanded the initial goal of this project to also study existing the existing ASPS cell lines (ASPS-1 and ASPS-KY) as well as other cell lines from different diseases with one key commonality; like ASPS, these diseases all have translocations involving the gene TFE3; our goal is to explore the commonalities and differences in these diseases, and to see what we can learn about one from the others.

The process of developing collaborations and collecting cell lines is always slow (but steady). Thus far we have procured ASPS-1 (which we have performed a 48 hour and 72 hour drug screen on, and we will soon be testing it as a quail xenograft) and S-TFE (which has just arrived). The screening results for ASPS-1 are available to share on request.

Nathalie’s cells are growing in culture ( stretched-out appearing cells), and once they have reached a high enough passage we will perform ploidy analysis to determine if these are indeed cancer cells; there is always the risk that other connective tissue cells will grow instead of tumor cells.

Mouse ASPS-1 cells look very different (rounded), but we were able to successfully culture them before the incubator failure, and we will attempt to again.

solidly moving forward to Kick ASPS! (06/28/2017)

We have recently focused on expanding and validating the cell culture/cell line for Nathalie’s cells. To validate the culture being cancer, the cultures is being karyotyped at the OHSU Cytogenetics Research Laboratory. We sent a culture to the Cytogenetics lab a short time ago; unfortunately, the culture became contaminated, possibly during transportation from cc-TDI to the Cytogenetics lab. We restarted the culture and transported it again recently. Thus far we have not had reports of contamination, so the culture should be safely in their hands. We are now awaiting data from the Cytogenetics researchers. We have already confirmed that cultures come from Nathalie’s cells (determined by short tandem repeat (STR) analysis of cultured cells vs. original tumor pieces). We have also recently optimized a ploidy analysis on a known cancer cell lines vs. match normal on our Guava Flow Cytometer - . Once the current culture has been expanded, we will perform ploidy analysis to determine if the cells appear to be cancer cells. These three tests will allow us to know with a great degree of certainty whether or not Nathalie’s cultures are indeed ASPS cancer cells.

We are also now culturing the ASPS-1 cell line for xenograft development in our quail egg assay to test our current methods for adding human cells to quail eggs, focusing on ASPS cells. Unfortunately, we have still not been able to obtain the ASPS-KY cell line from Japan, they have not responded to any of the previous emails.  We remain hopeful.  

Moving ahead (11/13/17)

To determine if cultured ASPS-origin cells were indeed cancer, we sent cultures of both sites for Fluorescent In Situ Hybridization (FISH) analysis at the University of Washington cytogenetics core. Unfortunately, both probes were negative for the expected ASPL-TFE3 gene fusion. This means the cells growing are likely not cancer. Nonetheless, we are pursuing one additional validation method: we will inject cardiotoxin-injured immunodeficient SHO mice with large cell populations to attempt engraftment of the cells into the damaged mouse leg. Engraftment of cells and development of a mass on the mouse leg would indicate the cells are indeed cancerous, in spite of FISH probe results.

 

Even if the engraftment does not occur, this will not be the end of the project. The cells are confirmed to originate from Nathalie, meaning the cells bear much of her biology. As tumorigenesis is associated with the ASPL-TFE3 gene fusion and potentially additional mutations, we will “reverse engineer” Nathalie’s ASPS by systematically inducing new mutations into her cell cultures until they exhibit behaviors consistent with cancer cells. This reverse engineering process is significantly more time consuming, but it should allow us to build a new ASPS cell model for research use, and to better understand the biology of ASPS by tracing the genetic lineage of induced mutations.

 

We have requested an ASPL-TFE3 fusion plasmid from a collaborator in Japan and we are finalizing logistics to have it brought to cc-TDI. We will also be able to isolate ASPL-TFE3 cDNA from Nathalie’s frozen tumor tissue, though having an established plasmid will be a dependable transfection system. We also have the ASPS-1 cell line with which to explore ASPS biology and drug sensitivity.

 

Due to the slow growth rate of Nathalie’s cells we expect engraftment will take time, but we will monitor them daily for development of tumors. Nonetheless, we will be updating as soon as possible with results from the ASPS mouse engraftment experiment.

Kick ASPS Update (1/30/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. Unfortunately, none have yet grown any visible tumors. They will continue to be monitored regularly for development of tumors. We have also accessed another preserved ASPS sample for sequencing experiments, which will be performed shortly.

We have progressed on the secondary model generation plans initiated late in 2017. We have received the ASPL-TFE3 fusion plasmid from our collaborator in Japan, and we are also purchasing a lentiviral ASPL-TFE3 fusion system which will allow us to easily transfect cells in a stable manner, i.e. the cells will permanently express the ASPL-TFE3 gene found in ASPS. This lentivirus system is expected February 9th at the latest. We have purchased two versions of the lentivirus system: 1) Myc-DDK tagged ASPL-TFE3 which should enable ease of use in protein-based studies due to the well characterized antibodies available for Myc and DDK, 2) GFP-tagged ASPL-TFE3 which enables easy visualization due to the green fluorescent protein expressed in conjunction with ASPL-TFE3. Once we have the lentivirus system, we will be able to transfect Nathalie’s cells to express ASPL-TFE3 and push them towards the tumor cell genotype to help in our studies of ASPS. We will also be able to transfect other normal cell models with ASPL-TFE3 and explore changes in drug response in ASPS-naïve cells. The reverse engineering ASPS model development should be expediated by the lentiviral system, and is the first step in serial addition of variations to determine the necessary genetic milieu needed to push cells towards an ASPS-like state.

Once we have established a cell model that recapitulates some characteristics of Nathalie’s tumor, we will perform drug screening, sequencing, and quail engraftment to characterize the ASPS cell model. The reverse engineering experiment will likely be the current endpoint for the ASPS model development project. However, ASPS research will continue at cc-TDI, with a new look at the disease once model systems are in place.

Kick ASPS Update (3/21/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop, so we remain optimistic. They will continue to be monitored regularly for development of tumors and the time frame on development is still wide open.

We have ordered the Myc-DDK tagged ASPL-TFE3 and GFP-tagged ASPL-TFE3 lentivirus systems through OriGene. We are in frequent contact with our customer support representative Tiffany. Unfortunately, the OriGene lab has had difficulty creating the custom ASPL-TFE3 plasmid, which means a delay in the arrival of the plasmid. Having worked with OriGene on several projects, they have earned our trust in their ability to create even complicated lentiviruses such as gene fusion products. We will continue contacting OriGene regularly to ensure delivery of the lentivirus as quickly as possible. Once it does, we will be able to immediately add the ASPL-TFE3 fusion into Nathalie’s cells, recapitulating the driving mutation behind the ASPS. Additionally, with recent experience in transfection of plasmids and lentivirus systems, establishing and confirming stable ASPL-TFE3 expression is a technique well in hand.

Kick ASPS Update 03/28/2018

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop, so we remain optimistic. They will continue to be monitored regularly for development of tumors and the time frame on development is still wide open.

Working closely with our sales representative at OriGene, we have identified the cause of the plasmid delay. Due to the method of sequence delivery, the lab was having difficulty synthesizing the fusion gene. Working with Tiffany, I have re-transcribed the data into a readable format and resubmitted to the lab. We should have updates soon!

Kick ASPS Update (4/4/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop, so we remain optimistic. They will continue to be monitored regularly for development of tumors and the time frame on development is still wide open.

The new transcript of the ASPL-TFE3 gene is at the lab, and we should be hearing back soon. Tiffany has not sent me an update yet, but once she has I will post it!

Thank you very much, Kick ASPS supporters!

Kick ASPS Update (4/11/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop, so we remain optimistic. They will continue to be monitored regularly for development of tumors and the time frame on development is still wide open.

Per Tiffany, the lab at OriGene has said the new ASPL-TFE3 gene plasmids should be ready in the next two weeks. I believe we will be receiving them soon!

Thank you very much, Kick ASPS supporters!

Noah

Kick ASPS Update (4/18/2018)

-- still watching/waiting for tumors in mice --

Per Tiffany at OriGene all ASPL-TFE3 plasmids should be ready in the next week or two. This week we received two of the four plasmids we ordered (for a parallel project), so the ASPL-TFE3 plasmids should be arriving shortly.

Thank you very much, Kick ASPS supporters!

Kick ASPS Update (4/25/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop. They will continue to be monitored regularly for development of tumors and the time frame on development is still wide open.

Per Tiffany, the lab at OriGene is *still* working on the ASPL-TFE3 plasmids, hopefully we should be receiving them next week. I will follow up with more information as soon as possible.

Kick ASPS Update (5/2/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, at least not yet.

 

There have been no updates from Tiffany on our vectors, so I will be following up with her again today. Hopefully we will receive an update from the OriGene lab soon.

Kick ASPS Update (5/10/2018)

We have continued monitoring the mice engrafted with cells from Nathalie’s cultures. The mice have not yet developed tumors, though patient-derived xenograft models routinely take multiple months to develop. They will continue to be monitored regularly for development of tumors, but we are in Plan B mode to study Nathalie's tumor by adding ASPL-TFE3 to her normal cells.  On that front, unfortunately have been no further updates on the APSL-TFE3 fusion gene construct. I expect updates soon, and I will ask daily for them.

 

Kick ASPS Update (5/17/2018)

The mice injected with Nathalie’s cells have been observed for approximately 6 months, and have yet shown no tumors. This likely means that her cells will not engraft (recreate a tumor in the mouse). Nonetheless, we still have the reverse-engineering plan B to move Nathalie’s cells closer to a tumor state. This past week, we received the ASPL-TFE3 fusion protein lentivirus. I will be able to transfect Nathalie’s cells with the fusion protein associated with ASPS.

More updates soon!

Update 6/6/2018

By popular demand, we are switching to less-frequent, more content-rich monthly posts.  These will appear as new blog posts on our website.  For exclusive! week to week updates of late-breaking results, email molly@cc-tdi.org to be subscribed.  Thank you for supporting this ASPS research!