Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Rural Versus Urban Health Disparities in the COVID-19 Era Among Veterans With COPD.

Individuals living in rural areas in the United States experienced disparities in COVID-19 incidence and mortality rates, and people with chronic obstructive pulmonary disease (COPD) are at high risk of poor outcomes. We sought to determine whether veterans with COPD living in rural areas experienced different perceptions and practices of COVID-19 mitigation strategies, access to care, and health disparities during the COVID-19 pandemic, compared to their urban-living counterparts. We performed a one-time survey of veterans with COPD, collecting COVID-19-related information including individual perceptions and practice of mitigation strategies, COVID-19 vaccination status, access to care, and respiratory symptoms stratified by rural-urban status. A total of 100 participants completed the survey with 47 living in rural areas and 53 living in urban areas. There were no significant differences in perceptions and practices related to COVID-19 mitigation strategies (including vaccination), access to care, or respiratory and mental health outcomes. This lack of disparity between rural and urban veterans with COPD might be explained by the strength of the Veterans Health Administration in telemedicine or by an increased uptake of mitigation practices in people with chronic respiratory illness.

Design of a photoswitchable cadherin.

There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell-cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca(2+) binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca(2+) binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin's biological function in cell-cell adhesion and downstream signaling.

Intracycle power distribution in a heterogeneous multi-compartmental mathematical model: possible links to strain and VILI.

BACKGROUND: Repeated expenditure of energy and its generation of damaging strain are required to injure the lung by ventilation (VILI). Mathematical modeling of passively inflated, single-compartment lungs with uniform parameters for resistance and compliance indicates that standard clinical modes (flow patterns) differ impressively with respect to the timing and intensity of energy delivery-the intracycle power (ICP) that determines parenchymal stress and strain. Although measures of elastic ICP may accurately characterize instantaneous rates of global energy delivery, how the ICP component delivered to a compartment affects the VILI-linked variable of strain is determined by compartmental mechanics, compartmental size and mode of gas delivery. We extended our one-compartment model of ICP to a multi-compartment setting that varied those characteristics. MAIN FINDINGS: The primary findings of this model/simulation indicate that: (1) the strain and strain rate experienced within a modeled compartment are nonlinear functions of delivered energy and power, respectively; (2) for a given combination of flow profile and tidal volume, resting compartmental volumes influence their resulting maximal strains in response to breath delivery; (3) flow profile is a key determinant of the maximal strain as well as maximal strain rate experienced within a multi-compartment lung. By implication, different clinician-selected flow profiles not only influence the timing of power delivery, but also spatially distribute the attendant strains of expansion among compartments with diverse mechanical properties. Importantly, the contours and magnitudes of the compartmental ICP, strain, and strain rate curves are not congruent; strain and strain rate do not necessarily follow the compartmental ICP, and the hierarchy of amplitudes among compartments for these variables may not coincide. CONCLUSIONS: Different flow patterns impact how strain and strain rate develop as compartmental volume crests to its final value. Notably, as inflation proceeds, strain rate may rise or fall even as total strain, a monotonic function of volume, steadily (and predictably) rises. Which flow pattern serves best to minimize the maximal strain rate and VILI risk experienced within any sector, therefore, may strongly depend on the nature and heterogeneity of the mechanical properties of the injured lung.

Macrophage-Mediated Antibody Dependent Effector Function in Aggressive B-Cell Lymphoma Treatment is Enhanced by Ibrutinib via Inhibition of JAK2.

Targeted inhibition of Bruton's Tyrosine Kinase (BTK) with ibrutinib and other agents has become important treatment options in chronic lymphocytic leukemia, Waldenström's Macroglobulinemia, Mantle cell lymphoma, and non-GCB DLBCL. Clinical trials combining small molecule inhibitors with monoclonal antibodies have been initiated at rapid pace, with the biological understanding between their synergistic interactions lagging behind. Here, we have evaluated the synergy between BTK inhibitors and monoclonal antibody therapy via macrophage mediated antibody dependent cellular phagocytosis (ADCP). Initially, we observed increased ADCP with ibrutinib, whilst second generation BTK inhibitors failed to synergistically interact with monoclonal antibody treatment. Kinase activity profiling under BTK inhibition identified significant loss of Janus Kinase 2 (JAK2) only under ibrutinib treatment. We validated this potential off-target effect via JAK inhibition in vitro as well as with CRISPR/Cas9 JAK2-/- experiments in vivo, showing increased ADCP and prolonged survival, respectively. This data supports inhibition of the JAK-STAT (Signal Transducers and Activators of Transcription) signaling pathway in B-cell malignancies in combination with monoclonal antibody therapy to increase macrophage-mediated immune responses.

Assessment of 213Bi-anti-EGFR MAb treatment efficacy in malignant cancer cells with [1-13C]pyruvate and [18F]FDG.

Evaluation of response to therapy is among the key objectives of oncology. A new method to evaluate this response includes magnetic resonance spectroscopy (MRS) with hyperpolarized 13C-labelled metabolites, which holds promise to provide new insights in terms of both therapeutic efficacy and tumor cell metabolism. Human EJ28Luc urothelial carcinoma and LN18 glioma cells were treated with lethal activity concentrations of a 213Bi-anti-EGFR immunoconjugate. Treatment efficacy was controlled via analysis of DNA double-strand breaks (immunofluorescence γH2AX staining) and clonogenic survival of cells. To investigate changes in metabolism of treated cells vs controls we analyzed conversion of hyperpolarized [1-13C]pyruvate to [1-13C]lactate via MRS as well as viability of cells, lactate formation and lactate dehydrogenase activity in the cellular supernatants and [18F]FDG uptake in treated cells vs controls, respectively. Treatment of malignant cancer cells with 213Bi-anti-EGFR-MAb induced intense DNA double-strand breaks, resulting in cell death as monitored via clonogenic survival. Moreover, treatment of EJ28Luc bladder cancer cells resulted in decreased cell viability, [18F]FDG-uptake and an increased lactate export. In both EJ28Luc and LN18 carcinoma cells treatment with 213Bi-anti-EGFR-MAb triggered a significant increase in lactate/pyruvate ratios, as measured with hyperpolarized [1-13C]pyruvate. Treatment with 213Bi-anti-EGFR-MAb resulted in an effective induction of cell death in EJ28Luc and LN18 cells. Lactate/pyruvate ratios of hyperpolarized [1-13C]pyruvate proved to detect early treatment response effects, holding promise for future clinical applications in early therapy monitoring.

Hyperpolarized 13C Diffusion MRS of Co-Polarized Pyruvate and Fumarate to Measure Lactate Export and Necrosis.

Background: Non-invasive tumor characterization and monitoring are among the key goals of medical imaging. Using hyperpolarized 13C-labelled metabolic probes fast metabolic pathways can be probed in real-time, providing new opportunities for tumor characterization. In this in vitro study, we investigated whether measurement of apparent diffusion coefficient (ADC) measurements and magnetic resonance spectroscopy (MRS) of co-polarized 13C-labeled pyruvic acid and fumaric acid can non-invasively detect both necrosis and changes in lactate export, which are parameters indicative of tumor aggressiveness. Methods:13C-labeled pyruvic acid and fumaric acid were co-polarized in a preclinical hyperpolarizer and the dissolved compounds were added to prepared samples of 8932 pancreatic cancer and MCF-7 breast carcinoma cells. Extracellular lactate concentrations and cell viability were measured in separate assays. Results: The mean ratios of the ADC values of lactate and pyruvate (ADClac/ADCpyr) between MCF-7 (0.533 ± 0.015, n = 3) and 8932 pancreatic cancer cells (0.744 ± 0.064, n = 3) showed a statistically significant difference (p = 0.048). 8932 cells had higher extracellular lactate concentrations in the extracellular medium (22.97 ± 2.53 ng/µl) compared with MCF-7 cells (7.52 ± 0.59 ng/µl; p < 0.001). Fumarate-to-malate conversion was only detectable in necrotic cells, thereby allowing clear differentiation between necrotic and viable cells. Conclusion: We provide evidence that MRS of hyperpolarized 13C-labelled pyruvic acid and fumaric acid, with their respective conversions to lactate and malate, are useful for characterization of necrosis and lactate efflux in tumor cells.

Developmental programming of somatic growth, behavior and endocannabinoid metabolism by variation of early postnatal nutrition in a cross-fostering mouse model.

BACKGROUND: Nutrient deprivation during early development has been associated with the predisposition to metabolic disorders in adulthood. Considering its interaction with metabolism, appetite and behavior, the endocannabinoid (eCB) system represents a promising target of developmental programming. METHODS: By cross-fostering and variation of litter size, early postnatal nutrition of CB6F1-hybrid mice was controlled during the lactation period (3, 6, or 10 pups/mother). After weaning and redistribution at P21, all pups received standard chow ad libitum. Gene expression analyses (liver, visceral fat, hypothalamus) were performed at P50, eCB concentrations were determined in liver and visceral fat. Locomotor activity and social behavior were analyzed by means of computer-assisted videotracking. RESULTS: Body growth was permanently altered, with differences for length, weight, body mass index and fat mass persisting beyond P100 (all 3>6>10,p<0.01). This was paralleled by differences in hepatic IGF-I expression (p<0.01). Distinct gene expression patterns for key enzymes of the eCB system were observed in fat (eCB-synthesis: 3>6>10 (DAGLα p<0.05; NAPE-PLD p = 0.05)) and liver (eCB-degradation: 3>6>10 (FAAH p<0.05; MGL p<0.01)). Concentrations of endocannabinoids AEA and 2-AG in liver and visceral fat were largely comparable, except for a borderline significance for higher AEA (liver, p = 0.049) in formerly overfed mice and, vice versa, tendencies (p<0.1) towards lower AEA (fat) and 2-AG (liver) in formerly underfed animals. In the arcuate nucleus, formerly underfed mice tended to express more eCB-receptor transcripts (CB1R p<0.05; CB2R p = 0.08) than their overfed fellows. Open-field social behavior testing revealed significant group differences, with formerly underfed mice turning out to be the most sociable animals (p<0.01). Locomotor activity did not differ. CONCLUSION: Our data indicate a developmental plasticity of somatic growth, behavior and parameters of the eCB system, with long-lasting impact of early postnatal nutrition. Developmental programming of the eCB system in metabolically active tissues, as shown here for liver and fat, may play a role in the formation of the adult cardiometabolic risk profile following perinatal malnutrition in humans.