Reprogramming the immunosuppressive tumor microenvironment results in successful clearance of tumors resistant to radiation therapy and anti-PD-1/PD-L1.

Despite breakthroughs in immune checkpoint inhibitors (ICI), the majority of tumors, including those poorly infiltrated by CD8+ T cells or heavily infiltrated by immunosuppressive immune effector cells, are unlikely to result in clinically meaningful tumor responses. Radiation therapy (RT) has been combined with ICI to potentially overcome this resistance and improve response rates but reported clinical trial results have thus far been disappointing. Novel approaches are required to overcome this resistance and reprogram the immunosuppressive tumor microenvironment (TME) and address this major unmet clinical need. Using diverse preclinical tumor models of prostate and bladder cancer, including an autochthonous prostate tumor (Pten-/-/trp53-/-) that respond poorly to radiation therapy (RT) and anti-PD-L1 combinations, the key drivers of this resistance within the TME were profiled and used to develop rationalized combination therapies that simultaneously enhance activation of anti-cancer T cell responses and reprogram the immunosuppressive TME. The addition of anti-CD40mAb to RT resulted in an increase in IFN-y signaling, activation of Th-1 pathways with an increased infiltration of CD8+ T-cells and regulatory T-cells with associated activation of the CTLA-4 signaling pathway in the TME. Anti-CTLA-4mAb in combination with RT further reprogrammed the immunosuppressive TME, resulting in durable, long-term tumor control. Our data provide novel insights into the underlying mechanisms of the immunosuppressive TME that result in resistance to RT and anti-PD-1 inhibitors and inform therapeutic approaches to reprogramming the immune contexture in the TME to potentially improve tumor responses and clinical outcomes.

Circadian rhythmicity in murine blood: Electrical effects of malaria infection and anemia.

Circadian rhythms are biological adaptations to the day-night cycle, whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. We examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. Blood samples were analyzed at 4-h intervals over 2 days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malaria infected cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities.

Detecting Circadian Rhythms in Human Red Blood Cells by Dielectrophoresis.

Dielectrophoresis (DEP) enables the measurement of population-level electrophysiology in many cell types by examining their interaction with an externally applied electric field. Here we describe the application of DEP to the measurement of circadian rhythms in a non-nucleated cell type, the human red blood cell. Using DEP, population-level electrophysiology of ~20,000 red blood cells can be measured from start to finish in less than 3 min, and can be repeated over several days to reveal cell-autonomous daily regulation of membrane electrophysiology. This method is amenable to the characterization of circadian rhythms by altering entrainment and free-run conditions or through pharmacological perturbation.

Vm-related extracellular potentials observed in red blood cells.

Even in nonexcitable cells, the membrane potential Vm is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. Vm arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that Vm only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; Vm is detectable beyond the cell surface, and modulating Vm produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between Vm and the electrical double layer, rather than molecular transporters. We show that modulating Vm changes the extracellular ion composition, mimicking the behaviour if voltage-gated ion channels in non-excitable channels. We also observed Vm-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease.

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Rhythmic potassium transport regulates the circadian clock in human red blood cells.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.