PROJECT SUMMARY/ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 24-029. Metastatic colorectal cancer (mCRC) is the major cause of death from colon cancer and is unresponsive to current approved immune checkpoint therapy. We have previously identified that tumor associated macrophages (TAMs) in the tumor microenvironment (TME) of mCRCs are defined by high expression of the marker gene SPP1. These TAMs interact with fibroblasts and immune cells in the TME and generate a pro-tumorigenic niche that allows cancer to flourish at distant metastatic sites. However, our understanding of the functional mechanisms by which SPP1+ TAMs promote colorectal cancer (CRC) metastasis is limited. Our study focuses on understanding the influence of SPP1+ TAMs in migration and invasion in mCRC. Leveraging sophisticated three-dimensional multi-cellular microfluidic devices, we will investigate TAM functions in physiological model systems that maintain the complexity of the metastatic TME. Our hypothesis is that SPP1+ TAMs can migrate into metastatic niches and further promote tumor cell invasion through physiological barriers such as the blood-brain barrier (BBB). In Aim 1, we will compare the differences in migration potential of SPP1 +/+ and CRISPR knockout SPP1 -/- macrophages in the presence of tumor cells. We will utilize advanced imaging techniques and time- lapse microscopy in microfluidic models in the presence of extracellular matrix. We will also test if fibroblasts have an additive role in promoting TAM migration. Aim 2 investigates the contribution of SPP1+ TAMs to mCRC invasion through the BBB in brain metastasis. We will investigate this in a microfluidic model that mimics the in vivo human BBB. We will also test if this process can be inhibited by therapeutic interventions such as pirfenidone, an anti-fibrotic small molecule, and an SPP1 neutralizing antibody. Together, we will identify the mechanisms by which TAMs promote CRC metastasis and develop strategies to inhibit them.