To highlight the work being done by our SarcomaStrong recipient we recently interviewed Dr. Lindsey from West Virginia University, Director of their Musculoskeletal Research Laboratory. His team has been doing ground breaking work with Immunotherapy and sarcoma care. We are all optimistic that Dr. Lindsey’s work will lead to a deeper understanding of the immunology of cancer, specifically as it pertains to sarcoma, and to better, less toxic treatments in the future.
How has the research grant from Sarcoma Strong helped to advance your research?
Our lab focuses on immunotherapy and investigating how the immune system responds to cancer and treatments. The grant money was crucial for immunophenotyping, where we identified specific proteins on the surface of many types of white blood cells, including Myeloid derived suppressor cells (MDSCs). This is a tedious and expensive process and we were extremely grateful to have received the grant to help fund this part of the project.
What research studies have you performed over the last few years?
Much of our work has focused on isolating human myeloid derived suppressor cells (MDSCs) through a negative selection process. Specifically, we are trying to create a protocol where we can quickly and consistently remove all other cells from a blood or a tissue sample and leave only the MDSCs. This is uniquely challenging because when cells are manipulated outside of the human body, some properties may be altered. In addition, the longer these cells are held the more they change. Once we isolated the MDSCs from sarcoma patients we ran in vitro experiments based on different immunotherapies for sarcoma.
Our lab also is investigating how nanoparticle-delivered interleukin-12 affects an orthotopic osteosarcoma mouse model. We know MDSCs are directly involved in this process and are testing the effects in vitro and in vivo.
What new questions remain unanswered in that research?
A few important questions remain. We need to identify what dose-specific adjustments that need to be made before applying this research to humans. Although our interleukin-12 nanoparticle treatment was effective, we did not cure every mouse. I think it is likely due a dosage issue. Interleukin-12 has a long history of treating cancer going back to the mid 1990’s and historically it has failed because of difficulty with dosage and delivery of the drug. We need to figure out how to dose-adjust our immunotherapy based on what the immune system is doing in response to both cancer and the treatment. The ability to pivot treatment based on the immune system’s response is the linchpin of personalized immunotherapy.
How close are we to eventually applying this to humans?
This kind of research requires adequate funding and resources, but I think within the next 2-5 years personalized immunotherapy is possible in humans. In fact, the research community is partly doing this already with PD-L1 therapy.
How has the COVID-19 pandemic affected your research progress and goals?
Initially our research progress slowed because the university was shut down and our personnel had to quarantine constantly. Our in vivo mouse model project took 3 months to complete and intermittent shutdowns made this extremely difficult. I was happy with our lab’s ability to acclimate as we held virtual meetings and only brought in personnel sparingly in order to prioritize safety.
In general, COVID affected our perception of how important the immune response is to combating disease as almost all side effects of COVID are immunological in nature. The ability to assess the immune response to disease extends beyond cancer and I hope this sparks interest in immunology research.
Why are you interested in orthopedic oncology and sarcoma?
During my third year of residency, I was introduced to Dr. Mark Goodman an orthopaedic oncologist. He showed me the types of long-term and truthful relationships I could form with patients, the amount of impact orthopedic oncologists have on patients, and the ability to operate all over the body. Every day and every patient is new and unique.
Why should people donate to Sarcoma Strong?
Obtaining resources for an orphan disease like sarcoma is extremely difficult. The more avenues researchers have to obtain funds improves the ability to treat this disease. Institutions like Sarcoma Strong are very important in progressing this research as there are very few other organizations as active and as successful.
Any last comments?
Sarcoma Strong has and continues to do amazing work. I am honored to be a recipient of this grant and am extremely thankful to the Musculoskeletal Tumor Society as well.
Written by Zach Troiani from interview with Dr. Lindsey 9/2/2021
Congratulations to Dr. Lindsey and his entire team for their ongoing work and progress. He recently presented some of his work at the Fall Meeting of the Musculoskeletal Tumor Society in Baltimore, MD. Paper #33
The Team at West Virginia University School of Medicine Department of Orthopaedic Surgery: Justin Markel BS, Hillary Pratt BS, Amanda Stewart PhD, Alan Mizener BS, Ryan Lacinski BS, Brock Lindsey MD
Systemic Administration of IL-12 Loaded PLGA Nanoparticles is 50% Curative in Murine Osteosarcoma Model
While immunostimulatory cytokine therapy has long been established as an effective treatment in murine osteosarcoma models, dosing issues currently preclude this therapy from entering a clinical setting. Historically, clinical trials administering large bolus doses of Interleukin-12 (IL-12) resulted in numerous study participants developing systemic inflammatory response syndrome followed by an immunocompromised state characterized by T cell exhaustion. This study aimed to establish the efficacy of poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a controlled release delivery vehicle for systemic IL-12 therapy. We hypothesized that providing sustained, low levels of systemic IL-12 would maintain treatment efficacy while simultaneously mitigating the toxic side effects of bolus therapy. We tested this hypothesis in vivo using an orthotopic model of murine osteosarcoma.
Thirty syngeneic BALB/c mice were inoculated with luciferase-transfected K7M2 osteosarcoma cells. The mice were then allowed three weeks to develop palpable tumors. Before treatment began, 18 mice were excluded for various reasons: standardization of study timing, premature death, and failed tumor uptake. The remaining 12 mice were divided evenly into three dosage groups: 4 mg/kg, 40 mg/kg, & 400 mg/kg. Beginning 3 weeks post-inoculation, after significant palpable tumors had formed, the mice received weekly 250 μL intraperitoneal injections of IL-12 loaded PLGA nanoparticles at the prescribed dose. Disease progression was tracked every other week using in vivo fluorescence imaging (IVIS) and cheek bleeds for immunophenotyping of peripheral blood mononuclear cells (PBMCs) via spectral flow cytometry.
One week following the start of treatment, palliative amputation was performed. Treatment regimens were subsequently continued until 11 weeks post-inoculation, at which point all surviving mice were humanely euthanized. Of the 12 mice included in the study, six were considered disease-free at euthanasia. Disease-free mice lacked detectable metastases and recurrent tumors upon terminal IVIS imaging and were reported to be negative for metastases upon blinded histopathological analysis of the lungs by a board-certified pathologist. The 50% cure rate demonstrated in this study is a notable improvement over the 6% cure rate with our labs historical controls in this model following palliative amputation. Additionally, immunophenotypic metrics for these mice returned to healthy status, in stark contrast to the metrics of diseased mice. Dosage affected treatment efficacy with 3 mice from the curative group belonging to the 4 mg/kg group, 2 to the 40 mg/kg group, and 1 to the 400 mg/kg group. This trend did not achieve statistical significance using the Cochran Armitage Trend Test (p = 0.16) due to small groups but may suggest that dosage could be further reduced to improve treatment response. Lastly, neutrophil-to-lymphocyte ratio (NLR) and natural killer cell percentage of total PBMCs (NK%) were discovered as disease tracking and treatment response metrics, respectively. NLR for all mice peaked shortly after amputation when disease burden was greatest.
Beginning four weeks post-amputation, mice that would eventually be euthanized with disease had a statistically significant increase in NLR compared to mice that were eventually cured (p = 0.03, 0.02, & 0.05 at 4, 6, & 8 weeks post-amputation, respectively). In contrast, animals with a curative response to therapy had NLR return to pre-inoculation levels. NK% was also a predictive metric for treatment response, with NK% significantly greater beginning four weeks post-amputation among mice that were eventually cured compared to those that were euthanized with disease (p = 0.03, <0.01, <0.01 at 4, 6, & 8 weeks post-amputation, respectively).
Collectively, these results demonstrate that PLGA nanoparticles allow for systemic IL-12 therapy to be administered in an efficacious manner at logarithmically decreased dosages compared to previous clinical trials. Therefore, PLGA nanoparticles are a promising drug delivery vehicle for translating immunostimulatory cytokine therapy to a clinical setting. Additionally, immunophenotyping results establish NLR and NK% as reliable disease tracking and treatment response metrics that could be leveraged to tailor treatment through dosage modulation. This study makes a significant argument to pursue nanoparticle delivery of immunostimulatory cytokines to the clinical setting.
