(J-377) Phagocytosis and Polarization

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row J - Poster # 377

Presenting Author(s)

Simeon Williams, II, n/a

Undergraduate Student (Research)
Center for Engineering MechanoBiology at The University of Pennsylvania
St.Louis, Missouri, United States

Last Author(s)

SP

Steven Phan

PhD Student
University of Pennsylvania, United States

Primary Investigator(s)

Dennis E. Discher, PhD (he/him/his)

Robert D Bent Professor
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Introduction:: Macrophages play an essential role in the future of immunology and cancer treatment. These white blood cells are responsible for removing dead cells, destroying cancer tumor cells, and combating other harmful microorganisms. Additionally, they have the ability to stimulate other immune system cells, making them essential in the fight against cancer. To study macrophage activity and behavior accurately, we use a phagocytosis assay to quantify their engulfment capabilities. Another observed process is polarization, where macrophages respond to environmental stimuli by adopting specific phenotypes. To better understand macrophage behavior, a conditionally immortalized macrophage progenitor (CIM) line will be utilized. These CIMs share characteristics and markers with primary bone marrow-derived macrophages (BMDM) but are more genetically manipulable. In this experiment, macrophages will phagocytose the B16F10 melanoma cell line, which allows us to determine their phagocytic capabilities and polarity, which is whether they adopt the M1 phenotype (pro-inflammatory), or M2 phenotype (wound-healing) based on specific perturbations. In essence, the aim of this research is to elucidate the optimal conditions that enhance macrophage efficiency. By achieving this, we hope to develop better immunology methods and more effective strategies to combat cancer, a leading cause of death that impacts so many patients and their loved ones. Moreover, this research is something that I am confident will help scientists and engineers curate efficient ways to take cancer head-on.

Materials and Methods:: The CIMs are cultured in a modified RPMI medium, and supplemented with fetal bovine serum (FBS), pen-strep (P/S), beta-estradiol, and granulocyte-macrophage colony-stimulating factor (GM-CSF), then diluted. Similarly, the melanoma cells are cultured in RPMI medium supplemented with FBS and P/S, and after being detached using Trypsin-EDTA, they are subcultured at the correct ratio. After washing the CIMs twice with PBS, they were supplemented with FBS and added to Iscove's Modified Dulbecco's Medium (IMDM) supplemented with FBS, P/S, and M-CSF. Following differentiation, fresh medium was added to the plate on the third day of it. After a week of differentiation, the melanoma cells are added to plates containing macrophages at a ratio of 2:1, where phagocytosis is allowed to occur over two hours and the amount of engulfment is quantified using a fluorescent microscope. Following exposure to certain stimuli or specific perturbations in their environment, the cells can polarize into either M1, pro-inflammatory eaters, or M2, anti-inflammatory, wound healing. They can be quantified using flow cytometry by staining for common M1 macrophage markers and M2 markers.

Results, Conclusions, and Discussions::

Figure 1 above shows the results of the phagocytosis assay performed with DMSO being the control and the first condition, along with nocodazole, which was the second condition.

Figure 2 above shows the results of the phagocytosis assay performed with the first condition which was the B16 media, and the second condition which was nocodazole.

It is extremely significant to highlight the function and relationship between these various drugs and both the macrophages and B16s. The first drug that was used and that acted as the control was DMSO or dimethyl sulfoxide. The second drug was nocodazole, which is a microtubule inhibitor. Nocodazole prevents the assembly of microtubules, and subsequently, the process of cell division which is vital to the B16s. Furthermore, these lower percentages of eating done by the macrophages of the B16s may be attributed to this toxic condition that nocodazole is for phagocytosis. In addition to these two drugs, B16 media served as another condition within this assay. This condition specifically yielded the most amount of eating, which made sense given the likelihood of the B16s dividing, therefore increasing the opportunity for the macrophages to engulf more of them.

Acknowledgements (Optional): : I’d like to acknowledge that this project was funded by the NSF-funded Science and Technology Center, Center for Engineering Mechanobiology (CEMB) at the University of Pennsylvania. Along those same lines, I want to thank Dr.Dennis Discher for providing me with the space and resources to do this research as well as my project, along with my mentor Steven Phan who guided me every step of the way throughout this project.

References (Optional): :