Abstract Dysregulated dynamics and trafficking of receptor tyrosine kinases (RTK) have been linked to oncogenesis and metastasis. The mechanisms for disfunction of RTK dynamics are emerging and still not clear. Using single-particle tracking (SPT) techniques, we studied the dynamics and trafficking patterns of epidermal growth factor receptor (EGFR) and underlying mechanisms using prostate cancer cells (PCa) (LNCaP, DU145 and PC3) exhibiting variant metastatic capability. Through SPT and super-resolution imaging, it was revealed that cortical actin disorganization modulates increased dynamics of EGFRs, including high EGFR diffusivity, enlarged EGFR confinement size on the plasma membrane and faster EGFR internalization in advanced invasive cells. The elevated EGFR dynamics were associated with advanced aggressive behaviors, and highly upregulated EGFR, EPHB and SRC signaling as identified using single cell RNA-seq. Strong EGFR activation with relatively low EGFR expression in PC3 implicated some other mechanisms, likely biophysical, beyond EGFR quantity. Moreover, the upregulated EPHB and SRC pathways have been shown to regulate actin organization and metastasis and our in silico analysis indicated that genes in the two pathways predict poor prognosis for disease free and survival statuses in The Cancer Genome Atlas prostate cancer patient cohort. To interrogate the roles of EPHB and SRC pathways in actin organization and EGFR dynamics, a series of drug inhibitions and siRNA knockdown were applied to PCa cells followed by mRNA profiling, evaluations of EGFR dynamics, and cell behaviors. Functional knockdown of EPHB2 and SRC led to decreased EGFR dynamics, cell proliferation, migration and invasion. The loss-of-function effects were more profound in advanced invasive PCa. In this study, we discovered the roles of EPHB and SRC pathways in modulating actin organization and EGFR dynamics and leading to aggressive metastatic phenotypes. Additionally, EGFR dynamics were potential biophysical parameters to differentiate the highly-invasive from the non- and less-invasive PCa. Thus, we believe that the SPT-based EGFR dynamics can serve as a new biophysical assay to probe the metastatic malignancy of cancer cells and to monitor their response to anti-cancer drug treatment. Citation Format: Yen-Liang Liu, Aaron M. Horning, Che-Kuang Lin, Brandon Lieberman, Chia-Nung Hung, Chih-Wei Chou, Chiou-Miin Wang, Michael A. Liss, Mirae Kim, Rohan Vasisht, Evan P. Perillo, Katherine Blocher, Hannah Horng, Xi Tian, Chun-Lin Lin, Andrew K. Dunn, Tim H.-M. Huang, Hsin-Chih Yeh, Chun-Liang Chen. Upregulated EPHB2 and SRC pathways modulate spatial EGFR dynamics and malignant phenotypes and predict poor prognosis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 173.