Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions.

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The advanced or recurrent endometrial cancer (EC) has a poor prognosis because of chemoresistance. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is overexpressed in a variety of human cancers and plays important roles in promoting tumor cell growth. Here, we showed that high expression of PFKFB3 in EC cell lines is associated with chemoresistance. Pharmacological inhibition of PFKFB3 with PFK158 and or genetic downregulation of PFKFB3 dramatically suppressed cell proliferation and enhanced the sensitivity of EC cells to carboplatin (CBPt) and cisplatin (Cis). Moreover, PFKFB3 inhibition resulted in reduced glucose uptake, ATP production, and lactate release. Notably, we found that PFK158 with CBPt or Cis exerted strong synergistic antitumor activity in chemoresistant EC cell lines, HEC-1B and ARK-2 cells. We also found that the combination of PFK158 and CBPt/Cis induced apoptosis- and autophagy-mediated cell death through inhibition of the Akt/mTOR signaling pathway. Mechanistically, we found that PFK158 downregulated the CBPt/Cis-induced upregulation of RAD51 expression and enhanced CBPt/Cis-induced DNA damage as demonstrated by an increase in γ-H2AX levels in HEC-1B and ARK-2 cells, potentially revealing a means to enhance PFK158-induced chemosensitivity. More importantly, PFK158 treatment, either as monotherapy or in combination with CBPt, led to a marked reduction in tumor growth in two chemoresistant EC mouse xenograft models. These data suggest that PFKFB3 inhibition alone or in combination with standard chemotherapy may be used as a novel therapeutic strategy for improved therapeutic efficacy and outcomes of advanced and recurrent EC patients.