Project Update 2024:

This project is focused upon understanding how a combination of difluoromethylornithine (DFMO), a modified amino acid, and a diet lacking in arginine affects high-risk neuroblastoma tumors. We have studied DFMO in combination with mouse chow deficient in arginine and proline, an amino acid that typically gets converted into arginine in neuroblastoma. We have shown that this combination, which does not include any chemotherapy or specific anti-tumor drugs, prolongs the overall and tumor-free survival of two types of mice: TH-MYCN+/+ mice (which spontaneously grow neuroblastoma tumors, typically die within 45 days of life, and have an intact immune system) and immunocompromised mice implanted with a human neuroblastoma tumor (IMR5) under their skin. In this past year, we have also found that the dietary removal of arginine alone, when combined with DFMO, can also shrink mouse tumors and enable them to live longer. However, this effect is not quite as pronounced as in mice that had a more profound arginine depletion with the simultaneous removal of proline. We have collected all of the tumors from these mice and are studying their RNA and cytokine/chemokine levels to better understand 1) how the combination of DFMO and removal of arginine prevent neuroblastoma growth and 2) how this treatment affects the local immune system (tumor microenvironment) within each tumor. So far, we have tested a few tumors’ RNA and have found that, interestingly, tumors with DFMO and a proline/arginine deficient diet have upregulated immunosuppressive genes, as well as increased expression of genes affiliated with macrophages, a type of white blood cell that can be programmed by the tumor microenvironment to either promote or inhibit tumor cell growth. Tumors that only had arginine removed, however, uniformly demonstrated decreased expression of immune-stimulating genes. We have also begun to look at the cytokines and chemokines in each tumor and have found that DFMO alone is affiliated with an increase in all three types of TGFß, a context-dependent immune molecule that has both pro-tumor and anti-tumor effects. DFMO-treated tumors also have an increase in macrophage colony stimulating factor (M-CSF), which triggers the growth of macrophages.We are in the process of completing all our RNA and cytokine analyses for each tumor generated in our TH-MYCN+/+ mice, as well as designing and testing a streamlined way to create a tumor “fingerprint” showing how RNA and protein expressions interacts. This year, we will also test a drug that prevents the activity of arginine to see if we can replicate the success of DFMO with the dietary intervention. Ultimately, these efforts will help us to develop this therapeutic strategy for its eventual translation to a treatment for children with high-risk neuroblastoma.

Project Update 2025:

This year, we continued to evaluate the efficacy of treating high-risk neuroblastoma with polyamine deprivation. Polyamines are critical for cancer cell division but also play important roles in immune cell functioning. We previously tested the drug difluoromethylornithine (DFMO), which inhibits polyamine production, with a diet lacking proline and arginine, the critical building blocks for polyamines. This treatment completely cured 1/3 of mice from their tumors without chemotherapy, so we tested whether we could achieve this same success with DFMO and arginine deiminase (ADI-PEG20), a drug that metabolizes arginine. We tested this drug combination in TH-MYCN mice (which have a complete immune system) and in immunocompromised mice injected with human tumors (xenografts), and we found that this drug combination works almost as well as the diet and DFMO strategy. This is important because these two drugs would be much more likely to be tolerable for children with neuroblastoma to take in the future.

We also looked at the tumors from our TH-MYCN mice treated with DFMO with and without the proline/arginine deficient diet. We found that these tumors had increased amounts of macrophages and cancer-associated fibroblasts. When we looked at the RNA expression of the macrophages in untreated and DFMO-treated tumors, we saw that the macrophages in DFMO-treated tumors had different gene expression. One type of pro-inflammatory, anti-tumor macrophage (M1) was increased, but these M1 macrophages were being segregated away from neuroblasts. Another type of macrophage (M2a) normally increases the amount of fibrosis and is pro-tumor; these M2a cells use a lot of arginine and make polyamines normally. We looked at the protein expression of tumors treated with DFMO and DFMO+ProArg diet and saw that both of these treatment groups expressed cytokines/signaling molecules related to both M1 and M2a macrophages, which confirms that these tumors are attracting this type of immune cell. We think that the M2a macrophages could be a tumor resistance mechanism and that something is preventing the M1 macrophages from infiltrating the neuroblastoma and killing the tumor cells. In the next year, we will learn more about these macrophages and investigate ways in which we can increase M1 cell activity and inhibit M2a resistance.