Elucidating heterogeneity of cancer associated immune cells - functions and roles of neutrophils and arginase/arginine in breast cancer.
No new change in experiment summary, only procedure is refined.
Tumor associated immune cells (TAIs) form a significant part of the tumor and are highly dynamic. The distinct TAI subsets demonstrate divergent activities (protumor and antitumor) that play critical roles in disease progression and metastasis. Research from various laboratories, as well as our work (on the 4T1 mouse breast cancer model) shows that metastasis-able and metastasis-unable tumors are associated with different immune subsets, and in our work, we find tumor associated neutrophils (TANs) particularly interesting. The metastasis-able tumors are associated with a significantly higher number of neutrophils. Additionally, our preliminary work indicates systemic changes in neutrophil levels in mice bearing metastasis-able tumors. Work from our lab indicates that TANs have a high arginase activity, and we hypothesize that arginase activity of TANs further drives an influx of protumor immune cells which enable metastasis.
We aim to investigate and establish the role of arginase in suppression of the anti-tumor immune cells to enable disease progression. We will use the established 4T1 mouse breast cancer model in immunocompetent BALB/c mice in this application. 66cl4 (metastasis-able) and 67NR (metastasis-unable) 4T1 derived tumors will be grown in female balb/c mice. The role of arginase in 4T1 (highly metastatic) tumors is unclear and we will study the arginase activity in this model. The total number of mice in this experiment will be: 151). We will use a variety of ex vivo assays and measures to study the role of arginase in cancer immune suppression. Additionally, manipulation of tumor microenvironment (TME) by using arginase inhibitors is expected to shed light on its role in tumor immune responses. In our approach, arginase inhibition serves two purposes:
1. A tool to assess pro-tumor functions of arginase
2. Arginase inhibition as a potential therapeutic approach
Nanoparticles (liposomes) are a widely used tool for drug delivery, and we have liposomes that accumulate at very high levels in 66cl4 tumor associated neutrophils (published data). We want to investigate the biodistribution, pharmacokinetics and cellular uptake of these liposome formulations in healthy mice before they can be used as vehicles for delivering drugs. We will then use these nanoparticles with nor-NOHA (arginase inhibitor) and we hypothesize that this will result in stronger arginase inhibition compared to an identical dose of nor-NOHA in solution. The plan is to inject over a longer period (2 weeks), which would help mouse recovery between procedures. This will also enable us to study the systemic immune response with weekly blood sampling.
Tumors will be allowed to grow up to 15 mm in diameter (about 3 to 4 weeks). The well-being of mice will be monitored closely and upon appearance of significant discomfort (using the attached score sheet), they will be sacrificed. The experiment is expected to cause the mice low to moderate discomfort. Majority of our work will be done ex vivo and if scientific merit is not compromised, we will reduce the number of animals used in the experiments.
Tumor associated immune cells (TAIs) form a significant part of the tumor and are highly dynamic. The distinct TAI subsets demonstrate divergent activities (protumor and antitumor) that play critical roles in disease progression and metastasis. Research from various laboratories, as well as our work (on the 4T1 mouse breast cancer model) shows that metastasis-able and metastasis-unable tumors are associated with different immune subsets, and in our work, we find tumor associated neutrophils (TANs) particularly interesting. The metastasis-able tumors are associated with a significantly higher number of neutrophils. Additionally, our preliminary work indicates systemic changes in neutrophil levels in mice bearing metastasis-able tumors. Work from our lab indicates that TANs have a high arginase activity, and we hypothesize that arginase activity of TANs further drives an influx of protumor immune cells which enable metastasis.
We aim to investigate and establish the role of arginase in suppression of the anti-tumor immune cells to enable disease progression. We will use the established 4T1 mouse breast cancer model in immunocompetent BALB/c mice in this application. 66cl4 (metastasis-able) and 67NR (metastasis-unable) 4T1 derived tumors will be grown in female balb/c mice. The role of arginase in 4T1 (highly metastatic) tumors is unclear and we will study the arginase activity in this model. The total number of mice in this experiment will be: 151). We will use a variety of ex vivo assays and measures to study the role of arginase in cancer immune suppression. Additionally, manipulation of tumor microenvironment (TME) by using arginase inhibitors is expected to shed light on its role in tumor immune responses. In our approach, arginase inhibition serves two purposes:
1. A tool to assess pro-tumor functions of arginase
2. Arginase inhibition as a potential therapeutic approach
Nanoparticles (liposomes) are a widely used tool for drug delivery, and we have liposomes that accumulate at very high levels in 66cl4 tumor associated neutrophils (published data). We want to investigate the biodistribution, pharmacokinetics and cellular uptake of these liposome formulations in healthy mice before they can be used as vehicles for delivering drugs. We will then use these nanoparticles with nor-NOHA (arginase inhibitor) and we hypothesize that this will result in stronger arginase inhibition compared to an identical dose of nor-NOHA in solution. The plan is to inject over a longer period (2 weeks), which would help mouse recovery between procedures. This will also enable us to study the systemic immune response with weekly blood sampling.
Tumors will be allowed to grow up to 15 mm in diameter (about 3 to 4 weeks). The well-being of mice will be monitored closely and upon appearance of significant discomfort (using the attached score sheet), they will be sacrificed. The experiment is expected to cause the mice low to moderate discomfort. Majority of our work will be done ex vivo and if scientific merit is not compromised, we will reduce the number of animals used in the experiments.