Gene context drift identifies drug targets to mitigate cancer treatment resistance

Cancer treatment often fails because combinations of different therapies evoke complex resistance mechanisms that are hard to predict. We introduce REsistance through COntext DRift (RECODR): a computational pipeline that combines co-expression graph networks of single-cell RNA sequencing profiles with a graph-embedding approach to measure changes in gene co-expression context during cancer treatment. RECODR is based on the idea that gene co-expression context, rather than expression level alone, reveals important information about treatment resistance. Analysis of tumours treated in preclinical and clinical trials using RECODR unmasked resistance mechanisms–invisible to existing computational approaches–enabling the design of highly effective combination treatments for mice with choroid plexus carcinoma, and the prediction of potential new treatments for patients with medulloblastoma and triple negative breast cancer. Thus, RECODR may unravel the complexity of cancer treatment resistance by detecting context-specific changes in gene interactions that determine the resistant phenotype. CPC cells (mc300) tagged with luciferase were grown in tissue culture in complete neurobasal media (with 20ng FGF, 20ng EGF and 0.1% BSA ). When neurospheres formed, 5000 cells were implanted in to CD1 nude mice. After 3 days, Bioluminescence (BLI) signals were taken using the IVIS to look for tumor growth. Mice were then randomised in to non-treatment and treatment arms to an average BLI signal of 1-5*10^5 p/s/cm2/sr. Tumour growth was monitored by IVIS imaging. When mice reached an endpoint (either through clinical signs or a BLI of 1-9*10^8 p/s/cm2/sr) mice were killed by exposure to carbon dioxide. Brains were harvested and tumors removed before undergoing enzymatic dissociation with 100µg/ml of DNAseI and 20U/ml Papain for one hour at 37°C. After an hour, samples were triturated 5-6 times before being passed through a 40μm filter. The original tube was washed with 5ml HBSS which was then passed through the same filter. Samples were centrifuged for 5 mins at 4°C and 300g. The supernatant was discarded and cells re-suspended in 10ml HBSS before being passed through a second 40μm filter. The cell suspension was then centrifuged for 5 mins at 4°C and 300g. Finally, the supernatant was discarded and cells were re-suspended and transferred to FACS tubes before being being sorted using a BD Aria II (BD Biosciences) with the following gating strategy, forward and side scatter, singlets and dual RFP (561nm-585/29) and YFP(488nm-530/40) positive cells. Dead cells were excluded with DAPI, UV (355nm-450/50).