Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer–associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.

BackgroundIn Escherichia coli the mean and cell-to-cell diversity in RNA numbers of different genes vary widely. This is likely due to different kinetics of transcription initiation, a complex process with multiple rate-limiting steps that affect RNA production.ResultsWe measured the in vivo kinetics of production of individual RNA molecules under the control of the lar promoter in E. coli. From the analysis of the distributions of intervals between transcription events in the regimes of weak and medium induction, we find that the process of transcription initiation of this promoter involves a sequential mechanism with two main rate-limiting steps, each lasting hundreds of seconds. Both steps become faster with increasing induction by IPTG and Arabinose.ConclusionsThe two rate-limiting steps in initiation are found to be important regulators of the dynamics of RNA production under the control of the lar promoter in the regimes of weak and medium induction. Variability in the intervals between consecutive RNA productions is much lower than if there was only one rate-limiting step with a duration following an exponential distribution. The methodology proposed here to analyze the in vivo dynamics of transcription may be applicable at a genome-wide scale and provide valuable insight into the dynamics of prokaryotic genetic networks.

Mutual information between the time series of two dynamical elements measures how well their activities are coordinated. In a network of interacting elements, the average mutual information over all pairs of elements I is a global measure of the correlation between the elements' dynamics. Local topological features in the network have been shown to affect I . Here we define a generalized clustering coefficient C_{p} and show that this quantity captures the effects of local structures on the global dynamics of networks. Using random Boolean networks (RBNs) as models of networks of interacting elements, we show that the variation of I ( I averaged over an ensemble of RBNs with the number of nodes N and average connectivity k ) with N and k is caused by the variation of C_{p} . Also, the variability of I between RBNs with equal N and k is due to their distinct values of C_{p} . Consequently, we propose a rewiring method to generate ensembles of BNs, from ordinary RBNs, with fixed values of C_{p} up to order 5, while maintaining in- and out-degree distributions. Using this methodology, the dependency of C_{p} on N and k and the variability of I for RBNs with equal N and k are shown to disappear in RBNs with C_{p} set to zero. The I of ensembles of RBNs with fixed, nonzero C_{p} values, also becomes almost independent of N and k . In addition, it is shown that C_{p} exhibits a power-law dependence on N in ordinary RBNs, suggesting that the C_{p} affects even relatively large networks. The method of generating networks with fixed C_{p} values is useful to generate networks with small N whose dynamics have the same properties as those of large scale networks, or to generate ensembles of networks with the same C_{p} as some specific network, and thus comparable dynamics. These results show how a system's dynamics is constrained by its local structure, suggesting that the local topology of biological networks might be shaped by selection, for example, towards optimizing the coordination between its components.

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

We measured the in vivo production of RNA molecules tagged with MS2d-GFP in Escherichia coli, driven by the lar promoter, under weak and medium induction. The distributions of intervals between consecutive productions of RNAs are found to be sub-exponential, and the process of RNA production is found to be sub-Poissonian. We discuss possible models of transcription initiation and, based on our results and previous in vitro measurements, find that a sequential two-step model of transcription initiation at the promoter region explains well the results.