Imaging Single Cells in the Breast Tumor Microenvironment

Multi-photon microscopy (MPM) allows the observation of tumor cell behavior at single cell resolution in vivo and has demonstrated that invasive and locomotory tumor cells form migratory streams and intravasate only when associated with macrophages. These behaviors have been reconstituted in vitro with well characterized cell lines and with primary tumor cells from cancer patients. This analysis demonstrates that tumor cell streaming and intravasation are cell autonomous behaviors that are the direct result of the tumor cell -macrophage interaction. The in vivo invasion assay was developed to collect the streaming and intravasation competent tumor cells exhibiting these autonomous behaviors from primary tumors in vivo. Collection of these cells proved of particular value when the in vivo invasion assay was coupled to expression profiling to reveal the genes expressed by tumor cells during streaming and intravasation in rat, mouse and human breast tumors (the Invasion Signature). The invasion, adhesion and motility pathways identified in the Invasion Signature converge on the RhoC/Cofilin/Mena pathway identifying it as a master regulator of chemotaxis, streaming and intravasation of breast tumor cells in vivo. Biosensors were developed to observe the mechanisms by which the RhoC/Cofilin/Mena pathway contributes to complex cancer cell behavior in vivo. The findings indicate that RhoC determines the shape and polarity of locomotory and invasive tumor cell protrusions by regulating the spatial and temporal extent of Cofilin activity that is stimulated by Mena. Mena sensitizes the EGFR to EGF supporting stream formation and intravasation. Using prognostic markers derived from the RhoC/Cofilin/Mena pathway, anatomical landmarks have been developed for use with breast cancer patients. One of these, composed of the tumor cell-macrophage intravasation complex, is called TMEM (Tumor MicroEnvironment for Metastasis) in human breast tumors. Another is Cofilin x P-Cofilin intensity, a marker of amplitude of activation of the Cofilin/Mena pathway in tumor cells. TMEM density has been found to predict metastatic risk, while Cofilin x P-Cofilin intensity predicts disease free survival in human invasive ductal carcinomas of the breast as shown in two separate retrospective clinical trials. In summary, multi-photon imaging in deep tissue locations in live animals in real time has revealed molecular mechanisms associated with complex cancer cell behavior leading directly to new markers of metastatic risk and survival.