We are grateful to the Alex's Lemonade Stand Foundation for funding to enable the translation of a basic science discovery to preclinical trials so that Children's Oncology Group phase I and II trials might be possible. The discovery of EphB4 as a therapeutic target in alveolar rhabdomyosarcoma was work of former HHMI medical student fellow, Imran Aslam (now a JHMI internal medicine resident). Working in collaboration with the Druker laboratory, Imran uncovered the EphB4-EphrinB2 as the potentially highest value target in rhabdomyosarcomas. This ALSF REACH award allows us to investigate an anti-EphB4 antibody in embryonal rhabdomyosarcoma, as well as an EphB4-nanoparticle sump in alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and osteosarcoma. This work will be carried on by our star muscle- and mouse models expert, Megan. Check back regularly for updates on Megan's progress!
project updates:
01/14/2014: Project Funded!
02/06/2014: The immunocompromised mice we will be using to test the efficacy of an anti-EphB4 antibody have arrived. These mice will be used to create pilot orthotopic xenograft models of eRMS in the coming weeks.
[ picture: at center is Megan. ]
02/20/2014: This week, the first set of immunocompromised mice were injected with Rh18 cells (a human derived eRMS cell line). This begins a pilot study to demonstrate the latency with which a xenograft from this cell line will successfully create tumors in the strain of mice that we will use for future Alex’s Lemonade Stand experiments. In other strains of immunocompromised mice, Rh18 cells have generated tumors in 6-8 weeks- we’ll have to wait and see if these mice follow suit!
02/27/2014: Currently we are growing a new cell line to inject into the mice which are part of our pilot experiment. To be consistent between experiments, we need the same amount of cells for each mouse we use. This new cell line is a little slow growing; as it is with many other research experiments, the hardest part is waiting!
03/14/2014: more watchful waiting (nothing new).
03/20/2014: A second round of cells have been injected into immunocompromised mice this week to continue our pilot experiments for this Alex’s Lemonade Stand project. Our next steps will be to observe these mice over the next few weeks and take note of when the tumor cells engraft and begin to grow.
03/27/2014: no new news. still in watchful waiting mode.
04/04/2014: still awaiting tumor development before initiating drug trials.
04/10/2014: Our pilot study is progressing nicely this week, with 3 of our orthotopic xenograft mice developing tumors. We will continue to observe our other injected mice for tumor development over the next several weeks.
04/14/2014: See the blog post http://kellerlabblog.blogspot.com/2014/04/imrans-paper-published-in-pnas.html for a link to today's published PNAS paper on this topic. The studies in Imran's paper were the basis of this "next steps" project.
04/17/2014: The growth of the current tumors is progressing as expected. Our second group of mice have not grown any tumors yet, but they are still within the expected latency time for development. We continue to monitor them daily.
04/24/2014: New batch of EphinB2-neutralizing biological agent received from pharma partner, facilitating the next series of therapeutic studies.
05/08/2014: mouse studies about to start for EphinB2-neutralizing biological agent.
05/15/2014: This week our official investigation on the efficacy of an EphrinB2-neutralizing agent has begun. The study mice arrived on Wednesday and were injected with Rh18 cells, a well characterized human eRMS cell line. Using our pilot data, we expect tumors to form within the next 2-3 months. Additionally, our second pilot study is still ongoing; we are continually monitoring the mice for development of tumors.
05/21/2014: Rh18 xenograft studies still ongoing.
05/28/2014: not unexpectedly, still waiting on tumors to develop before treatments can begin...
06/05/14: No new news to report this week. We do not expect treatment on our experimental mice to begin for a few more weeks, and we are continually monitoring both our initial pilot mice and current experimental for tumor development.
06/12/2014: Still patiently waiting this week for mice in both experimental groups to develop tumors.
06/26/2014: Tissue microarrays of EphB4 and EphrinB2 on embryonal rhabdomyosarcoma are complete (it will take 1-2 weeks to analyze these with our pathologist collaborator). We are also still patiently monitoring our immunocompromised mice for development of tumors. We are checking them 3 times a week so as soon as tumors develop we can begin treatment. Stay tuned!
07/10/2014: Exciting news from the Keller Lab for our Alex’s Lemonade Stand project this week! The immunocompromised mice have started growing small tumors for the Rh18 eRMS cell line. In the coming weeks, most will begin receiving either an EphrinB2-neutralizing agent or a control drug. Each mouse will be monitored daily and tumors measured three times per week. This experiment is progressing on schedule and the next few months should be full of interesting data.
07/18/2014: The Alex’s Lemonade Stand experiment is progressing nicely this week. All mice are tolerating the dosage of the drug well, and are not exhibiting any side effects/toxicity. Expression of EphB4 and EphrinB2 in a small set of human osteosarcoma samples verified (request for TMA sent to COG); early studies of neuroblastoma are promising.
07/31/2014: This week, our experimental mice continue to be monitored daily. We will be wrapping up the in vivo testing in the coming weeks, then moving on to the data analysis phase.
08/08/2014: EphB4 and EphrinB2 immunohistochemistry optimized for canine osteosarcoma (for pets with spontaneous osteosarcoma a potential preclinical model to help the dogs, and plan for a human osteosarcoma trial.) Request sent to NCI comparative oncology program for canine TMA slides. Meanwhile, the Rh18 eRMS mouse studies with the EphrinB2-neutralizing agent are wrapping up.
08/21/2014: The in vivo portion of our Alex's Lemonade Stand project is slowly coming to a close. This week some of the raw data analysis and number crunching was started, although it will be several weeks until that is finished. Additionally, there are various supporting experiments that still need to be executed to fully determine the efficacy of the EphrinB2 blocking agent.
09/11/2014: This week our supplemental experiments continue. We are now using RT-PCR to examine the amount of metastasis in the harvested lungs of our treated and control mice. These experiments are time consuming, but will help us decide if the EphrinB2 neutralizing agent administered to the mice was effective in diminishing the metastatic capability of RH18 cells.
10/02/2014: Canine osteosarcoma TMA's for staining received; COG neuroblastoma TMA slides approved but MTA still to perform; COG osteosarcoma TMA proposal still in review (since July). rt-pcr strategy for above EphrinB2 blocking agent mouse studies (completed) is in revision. Some delays occurred as Megan spent the last 3 weeks in a collaborator's lab at Cold Spring Harbor Laboratory for a new strategic collaboration.
10/16/2014: A few setbacks this week owing to the particularly specific requirements of some molecular biology techniques. We have ordered a few new reagents in an attempt to complete the RT-PCR experiments, and should be back at the bench soon.
10/30/2014: Our new reagents for the revised experiments have just arrived, and soon we should have a clear picture about the efficacy of the EphrinB2 blocking agent on lung metastasis.
12/04/2014: Preclinical trial of sEphB4-HSA in Rh18 orthotopic xenografts is now complete. The next step is preclinical testing in a patient-derived xenograft (see the description of this autopsy-derived model).
12/31/2014: Project on short term hold pending transfer of grant from OHSU to our new, bold adventure at cc-TDI.
01/06/2014: New lab at cc-TDI started!
01/07/2014: Collaborators Ayeza Bajwa and Atiya Mansoor have completed analysis of human eRMS tissue microarrays with unexpected results that point to key differences in aRMS and eRMS with respect to EphB4 and EphrinB2 biology.
01/08/2015: ALSF REACH grant year 2 funding received... this is the first grant funding for cc-TDI ... thank you, ALSF!
01/15/2015: Strategic meeting (teleconference) held including pharma and CSU partners to consider a companion animal trial for osteosarcoma using EphB4/EphrinB2 therapeutics.
01/27/2015: Integration of animal study underway by Matthew, including ongoing tissue analysis from these studies.
02/06/2015: We have begun considering approaches to separating cell-autonomous and tumor microenvironment contributions of EphB4-EphrinB2 signaling in eRMS.
02/13/2015: analysis of tumor cell vs stroma cell expression of EphB4 and EphrinB2 reveals distinct differences in alveolar and embryonal rhabdomyosarcomas.
02/20/2015: For Rh18 xenograft studies (completed), lung metastasis counts are in progress and pharmacodynamic studies are planned.
02/27/2015: COG TMA requests for osteosarcoma and neuroblastoma slides are approved, but MTAs are still in progress.
03/04/2015: Rh18 xenograft pharmacodynamics studies in progress.
05/06/2015: Rh18 xenograft pharmacodynamics studies done; Rh18 lung metastasis count in progress.
05/20/2015: COG osteosarcoma TMA slides arrived May 8; working to verify EphrinB2 expression in PDX models.
06/03/2015: Rh18 xenograft lung metastasis count done.
07/29/2015: CTEP approved neuroblastoma TMA slides protocol July 26.
08/07/2015: westerns on PDXs still ongoing; neuroblastoma TMA slides received.
02/06/2014: The immunocompromised mice we will be using to test the efficacy of an anti-EphB4 antibody have arrived. These mice will be used to create pilot orthotopic xenograft models of eRMS in the coming weeks.
[ picture: at center is Megan. ]
02/20/2014: This week, the first set of immunocompromised mice were injected with Rh18 cells (a human derived eRMS cell line). This begins a pilot study to demonstrate the latency with which a xenograft from this cell line will successfully create tumors in the strain of mice that we will use for future Alex’s Lemonade Stand experiments. In other strains of immunocompromised mice, Rh18 cells have generated tumors in 6-8 weeks- we’ll have to wait and see if these mice follow suit!
02/27/2014: Currently we are growing a new cell line to inject into the mice which are part of our pilot experiment. To be consistent between experiments, we need the same amount of cells for each mouse we use. This new cell line is a little slow growing; as it is with many other research experiments, the hardest part is waiting!
03/14/2014: more watchful waiting (nothing new).
03/20/2014: A second round of cells have been injected into immunocompromised mice this week to continue our pilot experiments for this Alex’s Lemonade Stand project. Our next steps will be to observe these mice over the next few weeks and take note of when the tumor cells engraft and begin to grow.
03/27/2014: no new news. still in watchful waiting mode.
04/04/2014: still awaiting tumor development before initiating drug trials.
04/10/2014: Our pilot study is progressing nicely this week, with 3 of our orthotopic xenograft mice developing tumors. We will continue to observe our other injected mice for tumor development over the next several weeks.
04/14/2014: See the blog post http://kellerlabblog.blogspot.com/2014/04/imrans-paper-published-in-pnas.html for a link to today's published PNAS paper on this topic. The studies in Imran's paper were the basis of this "next steps" project.
04/17/2014: The growth of the current tumors is progressing as expected. Our second group of mice have not grown any tumors yet, but they are still within the expected latency time for development. We continue to monitor them daily.
04/24/2014: New batch of EphinB2-neutralizing biological agent received from pharma partner, facilitating the next series of therapeutic studies.
05/08/2014: mouse studies about to start for EphinB2-neutralizing biological agent.
05/15/2014: This week our official investigation on the efficacy of an EphrinB2-neutralizing agent has begun. The study mice arrived on Wednesday and were injected with Rh18 cells, a well characterized human eRMS cell line. Using our pilot data, we expect tumors to form within the next 2-3 months. Additionally, our second pilot study is still ongoing; we are continually monitoring the mice for development of tumors.
05/21/2014: Rh18 xenograft studies still ongoing.
05/28/2014: not unexpectedly, still waiting on tumors to develop before treatments can begin...
06/05/14: No new news to report this week. We do not expect treatment on our experimental mice to begin for a few more weeks, and we are continually monitoring both our initial pilot mice and current experimental for tumor development.
06/12/2014: Still patiently waiting this week for mice in both experimental groups to develop tumors.
06/26/2014: Tissue microarrays of EphB4 and EphrinB2 on embryonal rhabdomyosarcoma are complete (it will take 1-2 weeks to analyze these with our pathologist collaborator). We are also still patiently monitoring our immunocompromised mice for development of tumors. We are checking them 3 times a week so as soon as tumors develop we can begin treatment. Stay tuned!
07/10/2014: Exciting news from the Keller Lab for our Alex’s Lemonade Stand project this week! The immunocompromised mice have started growing small tumors for the Rh18 eRMS cell line. In the coming weeks, most will begin receiving either an EphrinB2-neutralizing agent or a control drug. Each mouse will be monitored daily and tumors measured three times per week. This experiment is progressing on schedule and the next few months should be full of interesting data.
07/18/2014: The Alex’s Lemonade Stand experiment is progressing nicely this week. All mice are tolerating the dosage of the drug well, and are not exhibiting any side effects/toxicity. Expression of EphB4 and EphrinB2 in a small set of human osteosarcoma samples verified (request for TMA sent to COG); early studies of neuroblastoma are promising.
07/31/2014: This week, our experimental mice continue to be monitored daily. We will be wrapping up the in vivo testing in the coming weeks, then moving on to the data analysis phase.
08/08/2014: EphB4 and EphrinB2 immunohistochemistry optimized for canine osteosarcoma (for pets with spontaneous osteosarcoma a potential preclinical model to help the dogs, and plan for a human osteosarcoma trial.) Request sent to NCI comparative oncology program for canine TMA slides. Meanwhile, the Rh18 eRMS mouse studies with the EphrinB2-neutralizing agent are wrapping up.
08/21/2014: The in vivo portion of our Alex's Lemonade Stand project is slowly coming to a close. This week some of the raw data analysis and number crunching was started, although it will be several weeks until that is finished. Additionally, there are various supporting experiments that still need to be executed to fully determine the efficacy of the EphrinB2 blocking agent.
09/11/2014: This week our supplemental experiments continue. We are now using RT-PCR to examine the amount of metastasis in the harvested lungs of our treated and control mice. These experiments are time consuming, but will help us decide if the EphrinB2 neutralizing agent administered to the mice was effective in diminishing the metastatic capability of RH18 cells.
10/02/2014: Canine osteosarcoma TMA's for staining received; COG neuroblastoma TMA slides approved but MTA still to perform; COG osteosarcoma TMA proposal still in review (since July). rt-pcr strategy for above EphrinB2 blocking agent mouse studies (completed) is in revision. Some delays occurred as Megan spent the last 3 weeks in a collaborator's lab at Cold Spring Harbor Laboratory for a new strategic collaboration.
10/16/2014: A few setbacks this week owing to the particularly specific requirements of some molecular biology techniques. We have ordered a few new reagents in an attempt to complete the RT-PCR experiments, and should be back at the bench soon.
10/30/2014: Our new reagents for the revised experiments have just arrived, and soon we should have a clear picture about the efficacy of the EphrinB2 blocking agent on lung metastasis.
12/04/2014: Preclinical trial of sEphB4-HSA in Rh18 orthotopic xenografts is now complete. The next step is preclinical testing in a patient-derived xenograft (see the description of this autopsy-derived model).
12/31/2014: Project on short term hold pending transfer of grant from OHSU to our new, bold adventure at cc-TDI.
01/06/2014: New lab at cc-TDI started!
01/07/2014: Collaborators Ayeza Bajwa and Atiya Mansoor have completed analysis of human eRMS tissue microarrays with unexpected results that point to key differences in aRMS and eRMS with respect to EphB4 and EphrinB2 biology.
01/08/2015: ALSF REACH grant year 2 funding received... this is the first grant funding for cc-TDI ... thank you, ALSF!
01/15/2015: Strategic meeting (teleconference) held including pharma and CSU partners to consider a companion animal trial for osteosarcoma using EphB4/EphrinB2 therapeutics.
01/27/2015: Integration of animal study underway by Matthew, including ongoing tissue analysis from these studies.
02/06/2015: We have begun considering approaches to separating cell-autonomous and tumor microenvironment contributions of EphB4-EphrinB2 signaling in eRMS.
02/13/2015: analysis of tumor cell vs stroma cell expression of EphB4 and EphrinB2 reveals distinct differences in alveolar and embryonal rhabdomyosarcomas.
02/20/2015: For Rh18 xenograft studies (completed), lung metastasis counts are in progress and pharmacodynamic studies are planned.
02/27/2015: COG TMA requests for osteosarcoma and neuroblastoma slides are approved, but MTAs are still in progress.
03/04/2015: Rh18 xenograft pharmacodynamics studies in progress.
05/06/2015: Rh18 xenograft pharmacodynamics studies done; Rh18 lung metastasis count in progress.
05/20/2015: COG osteosarcoma TMA slides arrived May 8; working to verify EphrinB2 expression in PDX models.
06/03/2015: Rh18 xenograft lung metastasis count done.
07/29/2015: CTEP approved neuroblastoma TMA slides protocol July 26.
08/07/2015: westerns on PDXs still ongoing; neuroblastoma TMA slides received.
