Signaling dynamics in coexisting monoclonal cell subpopulations unveil mechanisms of resistance to anti-cancer compounds.

BACKGROUND: Tumor heterogeneity is a main contributor of resistance to anti-cancer targeted agents though it has proven difficult to study. Unfortunately, model systems to functionally characterize and mechanistically study dynamic responses to treatment across coexisting subpopulations of cancer cells remain a missing need in oncology. METHODS: Using single cell cloning and expansion techniques, we established monoclonal cell subpopulations (MCPs) from a commercially available epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer cell line. We then used this model sensitivity to the EGFR inhibitor osimertinib across coexisting cell populations within the same tumor. Pathway-centered signaling dynamics associated with response to treatment and morphological characteristics of the MCPs were assessed using Reverse Phase Protein Microarray. Signaling nodes differentially activated in MCPs less sensitive to treatment were then pharmacologically inhibited to identify target signaling proteins putatively implicated in promoting drug resistance. RESULTS: MCPs demonstrated highly heterogeneous sensitivities to osimertinib. Cell viability after treatment increased > 20% compared to the parental line in selected MCPs, whereas viability decreased by 75% in other MCPs. Reduced treatment response was detected in MCPs with higher proliferation rates, EGFR L858R expression, activation of EGFR binding partners and downstream signaling molecules, and expression of epithelial-to-mesenchymal transition markers. Levels of activation of EGFR binding partners and MCPs' proliferation rates were also associated with response to c-MET and IGFR inhibitors. CONCLUSIONS: MCPs represent a suitable model system to characterize heterogeneous biomolecular behaviors in preclinical studies and identify and functionally test biological mechanisms associated with resistance to targeted therapeutics.

Metagenomic Analysis of a Continuous-Flow Aerobic Granulation System for Wastewater Treatment.

Aerobic granulation is an emerging process in wastewater treatment that has the potential to accelerate sedimentation of the microbial biomass during secondary treatment. Aerobic granulation has been difficult to achieve in the continuous flow reactors (CFRs) used in modern wastewater treatment plants. Recent research has demonstrated that the alternation of nutrient-abundant (feast) and nutrient-limiting (famine) conditions is able to promote aerobic granulation in a CFR. In this study, we conducted a metagenomic analysis with the objective of characterizing the bacterial composition of the granular biomass developed in three simulated plug flow reactors (PFRs) with different feast-to-famine ratios. Phylogenetic analyses revealed a clear distinction between the bacterial composition of aerobic granules in the pilot simulated PFRs as compared with conventional activated sludge. Larger and denser granules, showing improved sedimentation properties, were observed in the PFR with the longest famine time and were characterized by a greater proportion of bacteria producing abundant extracellular polymeric substances (EPS). Functional metagenomic analysis based on KEGG pathways indicated that the large and dense aerobic granules in the PFR with the longest famine time showed increased functionalities related to secretion systems and quorum sensing, which are characteristics of bacteria in biofilms and aerobic granules. This study contributes to a further understanding of the relationship between aerobic granule morphology and the bacterial composition of the granular biomass.

Dynamic response of aerobic granular sludge to feast and famine conditions in plug flow reactors fed with real domestic wastewater.

Plug flow reactors (PFRs) approximated by the connection of multiple completely stirred tank reactors (CSTRs) in series were used to achieve continuous flow aerobic granulation in real domestic wastewater. This study revealed, possibly for the first time, that the morphology and characteristics of aerobic granular sludge transformed in the course of a mixed liquor flow through a PFR. The feast zone, located at the front end of the PFR, can quickly develop filamentous structure on the surface of aerobic granular sludge which later disappeared in the famine zone at the back end of the PFR. Detention time from the front to the back end of the PFR was only 6.5 h. During this period the observed sludge morphological change led to sludge settleability fluctuation as much as 66% in zone settling velocity, 16% in specific gravity, and 40% in settled sludge volume. Further analysis revealed these types of sludge morphologies and characteristics were closely related to the specific substrate removal rate profiles of the PFR, i.e., the feast zone might have encouraged filamentous bacteria to extend outward into the bulk solution for soluble substrate, and the famine zone appeared to play an essential role in solidifying the structure of granular sludge structure prior to subjecting it to the gravity selection pressure. It can be inferred from this study that the lack of a famine zone in aerobic granulation reactors can loosen the granule structure and in turn deteriorate granule settleability. For a PFR, a famine zone following the feast zone is essential for maintaining the structural integrity of aerobic granular sludge in a continuous flow wastewater treatment system.