Metabolic reprogramming is a general feature of cancer cells. Regrettably, the comprehensive quantification of metabolites in biological specimens does not promptly translate into knowledge on the utilization of metabolic pathways. By estimating fluxes across metabolic pathways, computational models hold the promise to bridge this gap between data and biological functionality. These models currently portray the average behavior of cell populations however, masking the inherent heterogeneity that is part and parcel of tumorigenesis as much as drug resistance. To remove this limitation, we propose single-cell Flux Balance Analysis (scFBA) as a computational framework to translate single-cell transcriptomes into single-cell fluxomes. We show that the integration of single-cell RNA-seq profiles of cells derived from lung adenocarcinoma and breast cancer patients into a multi-scale stoichiometric model of a cancer cell population: significantly 1) reduces the space of feasible single-cell fluxomes; 2) allows to identify clusters of cells with different growth rates within the population; 3) points out the possible metabolic interactions among cells via exchange of metabolites. The scFBA suite of MATLAB functions is available at https://github.com/BIMIB-DISCo/scFBA, as well as the case study datasets. Cytotoxicity of chemotherapeutic agents and resistance to targeted treatments are the main reasons why cancer is still one of the top causes of death. As tumor cells are intrinsically resistant to therapies that target signaling pathways, targeting the metabolic hallmarks of cancer holds promise for more incisive treatments. Regrettably, the heterogeneity of cancer metabolism hinders the identification of effective treatments. To fully uncover the metabolic heterogeneity within tumors, characterization of metabolic programs (metabolic flux distributions) at the single-cell level is required. To fill the gap between current technologies for genomics and future technologies for fluxomics, both at the single-cell and the genome-wide scale, we propose to integrate cancer data from: 1) single-cell transcriptomics and 2) bulk metabolomics, into a multi-scale stoichiometric model, to deliver for the first time metabolic fluxomes at the single-cell level. To this end, we introduce a new paradigm for flux balance analysis and data integration in cancer metabolism to: 1) characterize metabolic heterogeneity, not only at the inter-, but also at the intra-tumor level 2) identify the metabolic interactions between cancer populations, whose role in resistance to metabolic treatments has been recently recognized 3) predict the collective response to drug targeting of metabolism.

Metabolism is directly and indirectly fine-tuned by a complex web of interacting regulatory mechanisms that fall into two major classes. On the one hand, the expression level of the catalyzing enzyme sets the maximal theoretical flux level (i.e., the net rate of the reaction) for each enzyme-controlled reaction. On the other hand, metabolic regulation controls the metabolic flux through the interactions of metabolites (substrates, cofactors, allosteric modulators) with the responsible enzyme. High-throughput data, such as metabolomics and transcriptomics data, if analyzed separately, do not accurately characterize the hierarchical regulation of metabolism outlined above. They must be integrated to disassemble the interdependence between different regulatory layers controlling metabolism. To this aim, we propose INTEGRATE, a computational pipeline that integrates metabolomics and transcriptomics data, using constraint-based stoichiometric metabolic models as a scaffold. We compute differential reaction expression from transcriptomics data and use constraint-based modeling to predict if the differential expression of metabolic enzymes directly originates differences in metabolic fluxes. In parallel, we use metabolomics to predict how differences in substrate availability translate into differences in metabolic fluxes. We discriminate fluxes regulated at the metabolic and/or gene expression level by intersecting these two output datasets. We demonstrate the pipeline using a set of immortalized normal and cancer breast cell lines. In a clinical setting, knowing the regulatory level at which a given metabolic reaction is controlled will be valuable to inform targeted, truly personalized therapies in cancer patients.

Metabolomics is a rapidly expanding technology that finds increasing application in a variety of fields, form metabolic disorders to cancer, from nutrition and wellness to design and optimization of cell factories. The integration of metabolic snapshots with metabolic fluxes, physiological readouts, metabolic models, and knowledge-informed Artificial Intelligence tools, is required to obtain a system-level understanding of metabolism. The emerging power of multi-omic approaches and the development of integrated experimental and computational tools, able to dissect metabolic features at cellular and subcellular resolution, provide unprecedented opportunities for understanding design principles of metabolic (dis)regulation and for the development of precision therapies in multifactorial diseases, such as cancer and neurodegenerative diseases.

Classical random Boolean networks (RBN) are not well suited to describe experimental data from time-course microarray, mainly because of the strict assumptions about the synchronicity of the regulatory mechanisms. In order to overcome this setback, a generalization of the RBN model is described and analyzed. Gene products (e.g., regulatory proteins) are introduced, with each one characterized by a specific decay time, thereby introducing a form of memory in the system. The dynamics of these networks is analyzed, and it is shown that the distribution of the decay times has a strong effect that can be adequately described and understood. The implications for the dynamical criticality of the networks are also discussed.

Cell metabolism is the biochemical machinery that provides energy and building blocks to sustain life. Understanding its fine regulation is of pivotal relevance in several fields, from metabolic engineering applications to the treatment of metabolic disorders and cancer. Sophisticated computational approaches are needed to unravel the complexity of metabolism. To this aim, a plethora of methods have been developed, yet it is generally hard to identify which computational strategy is most suited for the investigation of a specific aspect of metabolism. This review provides an up-to-date description of the computational methods available for the analysis of metabolic pathways, discussing their main advantages and drawbacks. In particular, attention is devoted to the identification of the appropriate scale and level of accuracy in the reconstruction of metabolic networks, and to the inference of model structure and parameters, especially when dealing with a shortage of experimental measurements. The choice of the proper computational methods to derive in silico data is then addressed, including topological analyses, constraint-based modeling and simulation of the system dynamics. A description of some computational approaches to gain new biological knowledge or to formulate hypotheses is finally provided.

Despite myriads of possible gene expression profiles, cells tend to be found in a confined number of expression patterns. The dynamics of Boolean models of gene regulatory networks has proven to be a likely candidate for the description of such self-organisation phenomena. Because cells do not live in isolation, but they constantly shape their functions to adapt to signals from other cells, this raises the question of whether the cooperation among cells entails an expansion or a reduction of their possible steady states. Multi random Boolean networks are introduced here as a model for interaction among cells that might be suitable for the investigation of some generic properties regarding the influence of communication on the diversity of cell behaviours. In spite of its simplicity, the model exhibits a non-obvious phenomenon according to which a moderate exchange of products among adjacent cells fosters the variety of their possible behaviours, which on the other hand are more similar to one another. On the contrary, a more invasive coupling would lead cells towards homogeneity.