Oncogenic signalling by kinases presents a significant opportunity for cancer therapy. The B-Raf selective inhibitor, Vemurafenib, has shown great effect against B-Raf mutant melanoma patients. However, drug resistance emerges in nearly every case. Various mechanisms underlying this resistance are described, however these approaches usually overlook the heterogeneity of the tumour microenvironment, and do not provide information about the temporal aspects of drug response and eventual emergence of resistance. Therefore, uncovering the spatio-temporal heterogeneity, i.e., when, where, and how melanoma cells respond to drug and acquire resistance in a complex tumour microenvironment is the key step toward the comprehensive understanding of resistance mechanisms. To tackle these problems, we will investigate the tolerance and resistance to B-Raf inhibitors with single cell resolution in vivo. We have established melanoma cell lines stably expressing EKAR-EV biosensor that reports ERK activity in living cells. In vitro, the melanoma cells show homogenous responses to BRaf inhibitors, whereas intravital analysis indicate that there is considerable heterogeneity in ERK activity within tumours. This cannot be attributed simply B-Raf mutation as all cells contain the oncogene, and we hypothesize that additional signals from the microenvironment activate ERK, possibly putting the neighbouring melanoma cells in drug-tolerant state. Similar mechanisms are possible at metastatic locations. The tumour microenvironment of CNS metastases is quite different from those of other organs, it is particularly enriched in RTK ligands. In the longer term, we will study how metastatic melanoma cells may respond to and tolerate the drugs in the CNS. In summary we aim to identify the micro-environmental signals that activate ERK, and explore how these signals may confer tolerant to BRaf inhibition and ultimately aid the emergence of drug resistant clones.