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Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

All-trans-retinoic acid (ATRA), a pan-agonist for retinoic acid receptors (RARs), regulates diverse cellular functions including growth, differentiation and immune function. We report here that ATRA-treatment represses tumor growth in syngeneic, immunocompetent but not in immunodeficient mice. Tumor immune microenvironment was implicated since depletion of cytotoxic T lymphocytes antagonized these effects in syngeneic mice. Combining ATRA with immune checkpoint blockade did not inhibit lung cancer growth in mice. We sought to augment retinoid anti-tumor effects without affecting its pro-tumorigenicity. We previously reported that CD38 mediated resistance to checkpoint blockade in murine 344SQ lung cancer cells via RARα transcriptional activation of CD38 expression. Yet, combining the RARα antagonist (IRX6696) with anti-PD-L1 did not augment anti-tumorigenicity in transplanted 344SQ cells in syngeneic mice. Prior work implicated RARγ in regulating T cell response. Combining the novel RARγ agonist (IRX4647) with anti-PD-L1 statistically-significantly repressed 344SQ lung cancer cell growth in syngeneic mice. This line is relatively resistant to checkpoint blockade. Immunofluorescent analysis of these treated tumors revealed that combined IRX4647 and anti-PD-L1 treatments reduced CD38 expression in the tumor stroma relative to IRX4647 or anti-PD-L1 treatment alone. Statistically-significantly elevated helper (CD4+) T cells were detected in treated tumors along with increased IL-5 and IL-13 expression observed in plasma and tumors. These cytokines can activate helper T cells, altering lung cancer growth. These microenvironment effects were associated with in vivo anti-tumorigenicity. IRX4647-treatment did not appreciably alter in vitro growth of lung cancer cells although retinoid receptors expression profiles were affected. Pharmacokinetic study of IRX4647 found its plasma half-life was 6 hours. Combining an RARγ agonist with immune checkpoint blockade exerted superior anti-neoplastic efficacy against lung cancer versus an ATRA-based regimen. Given these findings we propose exploring activity of this RARγ agonist with an optimal checkpoint inhibitor in a lung cancer clinical trial. Citation Format: Cheng-Hsin Wei, Lu Huang, Blair Kreh, Xiuxia Liu, Liliya Tyutyunyk-Massey, Zibo Chen, Mi Shi, Vidyasagar Vuligonda, Martin Sanders, Serguei Kozlov, King Chan, Amir Horowitz, Mary Carrington, Patrick Hwu, Weiyi Peng, Ethan Dmitrovsky, Xi Liu. Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1836.

Background Iron deficiency can result in hyporetinolemia and hepatic vitamin A (VA) sequestration. Objectives We used model-based compartmental analysis to determine the impact of iron repletion on VA metabolism and kinetics in iron-deficient rats. Methods At weaning, Sprague-Dawley rats were assigned to either a VA-marginal diet (0.35 mg retinol equivalent/kg) with adequate iron (35 ppm, control group [CN]) or reduced iron (3 ppm, iron-deficient group [ID-]), with an equivalent average body weight for each group. After 5 wk, n = 4 rats from each group were euthanized for baseline measurements of VA and iron indices, and the remaining rats (n = 6 CN, n = 10 ID-) received an intravenous injection of 3H-labeled retinol in an emulsion as tracer to initiate the kinetic study. On day 21 after dosing, half of the ID- rats were switched to the CN diet to initiate iron repletion, referred to as the iron-repletion group (ID+). From the time of dosing, 34 serial blood samples were collected from each rat over a 92-d time course. Plasma tracer and tissue tracee data were fitted to 6- and 4-compartment models, respectively, to analyze the kinetic behavior of VA in all groups. Results Our mathematical model indicated that ID- rats exhibited a nearly 6-fold decrease in liver VA secretion and >4-fold reduction in whole-body VA utilization, compared with CN rats, whereas these perturbed kinetic behaviors were notably corrected in ID+ rats, close to those from the CN group. Conclusions Iron repletion can remove the inhibitory effect that iron deficiency exerts on hepatic mobilization of VA and restore retinol kinetic parameters to values similar to that of never-deficient CN rats. Together with improvements in iron and VA indices, our results suggest that restoration of an iron-adequate diet is sufficient to improve VA kinetics after a previous state of iron deficiency.

BACKGROUND: Although iron deficiency is known to interrupt vitamin A (VA) metabolism, the ability of iron repletion to restore VA metabolism and kinetics in iron-deficient rats is not well understood. OBJECTIVES: In the present study, we examined the effects of dietary iron repletion on VA status in rats with pre-existing iron deficiency. METHODS: Weanling Sprague-Dawley rats were fed a VA-marginal diet (0.35 mg retinol/kg diet) containing either a normal concentration of iron [35 ppm, control group (CN)] or reduced iron (3 ppm, iron-deficient group, ID−); after 5 wk, 4 rats/group were killed for baseline measurements. A (3)H-labeled retinol emulsion was administered intravenously to the remaining rats (n = 6, CN; n = 10, ID−) as tracer to initiate the kinetic study. On day 21 after dosing, n = 5 ID− rats were switched to the CN diet, generating an iron-repletion group (ID+). Blood samples were collected at 34 time points ≤92 d after dose administration, when all rats were killed and iron and VA status were determined. RESULTS: At baseline, ID− rats had developed iron deficiency, with a reduced plasma VA concentration (0.67 compared with 1.20 μmol/L in ID− and CN rats, respectively; P

Whether vitamin A (VA) has a role in the development of metabolic abnormalities associated with intake of a high-fat diet (HFD) is unclear. Sprague–Dawley rats after weaning were fed an isocaloric VA sufficient HFD (VAS-HFD) or a VA deficient HFD (VAD-HFD) for 8 weeks. Body mass, food intake, liver and adipose tissue mass, and the hepatic expression levels of key proteins for metabolism were determined. VAD-HFD rats had lower body, liver, and epididymal fat mass than VAS-HFD rats. VAD-HFD rats had lower hepatic protein expression levels of cytochrome P450 26A1, glucokinase, and phosphoenolpyruvate carboxykinase than VAS-HFD rats. VAD-HFD rats had higher protein levels of glycogen synthase kinase (GSK)-3α and lower levels of GSK-3β, but not glycogen synthase, than VAS-HFD rats. VAD-HFD rats had higher hepatic levels of insulin receptor substrate-1 (IRS-1), insulin receptor β-subunit, mitogen-activated protein kinase proteins, and peroxisome proliferator-activated receptor-gamma coactivator 1α mRNA, and lower level of IRS-2 protein than VAS-HFD rats. These results indicate that in a HFD setting, VA deficiency attenuated HFD-induced obesity, and VA status altered the expression levels of proteins required for glucose metabolism and insulin signaling. We conclude that VA status contributes to the regulation of hepatic glucose and lipid metabolism in a HFD setting, and may regulate hepatic carbohydrate metabolism.

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.

Vitamin A (VA) deficiency remains prevalent in resource limited countries, affecting over 250 million preschool aged children. Vitamin A deficiency is associated with reduced intestinal barrier function and increased risk of mortality due to mucosal infection. Using Citrobacter rodentium (C. rodentium) infection in mice as a model for diarrheal diseases in humans, previous reports showed reduced pathogen clearance and survival in vitamin A deficient (VAD) mice compared to their vitamin A sufficient (VAS) counterparts.ObjectivesTo characterize and compare the impact of preexisting VA deficiency on gene expression patterns in the small intestine (SI) and the colon, and to discover novel target genes in VA-related biological pathways.MethodsVAD mice were generated by feeding VAD diet to pregnant C57/BL6 dams and their post-weaning offspring. RNAseq were performed using the total mRNAs extracted from SI and colon. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression and co-expression patterns.ResultsDEGs compared between VAS and VAD groups detected 49 SI and 94 colon genes. By GO information, SI DEGs were significantly enriched in categories relevant to retinoid metabolic process, molecule binding, and immune function. Immunity related pathways, including “humoral immune response” and “complement activation” were positively associated with VA in SI. Three co-expression modules showed significant correlation with VA status in SI; these modules contained four known retinoic acid targets. In addition, other SI genes of interest (e.g. Mbl2, Cxcl14, and Nr0b2) in these modules were suggested as new candidate genes regulated by VA. Furthermore, our analysis showed that markers of two cell types in SI, mast cells and Tuft cells, were significantly altered by VA status. In colon, “cell division” was the only enriched category and was negatively associated with VA. Thus, comparison of co-expression modules between SI and colon indicated distinct networks under the regulation of dietary VA and suggest that preexisting VAD could have a significant impact on the host response to a variety of disease conditions.

OBJECTIVES: We investigated the impact of iron repletion on vitamin A (VA) metabolism and kinetics in iron-deficient rats, hypothesizing that iron repletion can remove the inhibitory effect of iron deficiency on hepatic mobilization of VA and improve the hyporetinolemia of iron deficiency. METHODS: At weaning, rats were divided into two groups and fed either a VA-marginal diet (control group, CN) with normal levels of iron (37 ppm) or the same diet with reduced iron (5 ppm, iron deficient group, ID-). After 5 weeks, n = 4 rats from each group were euthanized for baseline measurements, and the remaining rats (n = 6 CN, n = 10 ID-) received an i.v. dose of (3)H-labeled retinol in an emulsion as tracer to initiate the kinetic study. On day 21 after dosing, half of the ID- rats were switched to the CN diet, thus creating an iron repletion group (ID+). Serial blood samples were collected from each rat at 34 preselected time points after dosing. On day 92, all rats were euthanized. Iron and VA status were determined, and model-based compartmental analysis was applied to the plasma tracer data to develop VA kinetic models for rats from all groups. RESULTS: Rats fed the iron-deficient diet developed iron deficiency, and exhibited reduced plasma retinol (0.67 vs. 1.19 µmol/L), with higher liver VA (265 vs. 187 nmol) at the start of the kinetic study, suggesting impaired hepatic VA mobilization. At the study conclusion, ID+ rats had recovered from iron deficiency, as indicated by hematological indices including hemoglobin, hematocrit, and serum iron. While liver VA storage was more than doubled in ID- rats (456 nmol) vs. CN rats (190 nmol), hyporetinolemia was maintained in ID- rats. In contrast, VA in the liver of ID+ rats was mobilized, reflected by the reduced liver VA mass (276 nmol) and increased hepatic output of VA into plasma based on predictions of the compartmental model, and plasma retinol in ID + rats returned to normal. CONCLUSIONS: Iron repletion restored plasma retinol concentrations and increased liver vitamin A mobilization in iron deficient rats. FUNDING SOURCES: National Institutes of Health.

The ability to generate stable, spatiotemporally controllable concentration gradients is critical for resolving the dynamics of cellular response to a chemical microenvironment. Here we demonstrate an acoustofluidic gradient generator based on acoustically oscillating sharp-edge structures, which facilitates in a step-wise fashion the rapid mixing of fluids to generate tunable, dynamic chemical gradients. By controlling the driving voltage of a piezoelectric transducer, we demonstrated that the chemical gradient profiles can be conveniently altered (spatially controllable). By adjusting the actuation time of the piezoelectric transducer, moreover, we generated pulsatile chemical gradients (temporally controllable). With these two characteristics combined, we have developed a spatiotemporally controllable gradient generator. The applicability and biocompatibility of our acoustofluidic gradient generator are validated by demonstrating the migration of human dermal microvascular endothelial cells (HMVEC-d) in response to a generated vascular endothelial growth factor (VEGF) gradient, and by preserving the viability of HMVEC-d cells after long-term exposure to an acoustic field. Our device features advantages such as simple fabrication and operation, compact and biocompatible device, and generation of spatiotemporally tunable gradients.

We present a programmable acoustofluidic pump that utilizes the acoustic streaming effects generated by the oscillation of tilted sharp-edge structures. This sharp-edge-based acoustofluidic pump is capable of generating stable flow rates as high as 8 μL min(-1) (~76 Pa of pumping pressure), and it can tune flow rates across a wide range (nanoliters to microliters per minute). Along with its ability to reliably produce stable and tunable flow rates, the acoustofluidic pump is easy to operate and requires minimum hardware, showing great potential for a variety of applications.

OBJECTIVES: To characterize and compare the impact of vitamin A (VA) deficiency on gene expression patterns in the small intestine (SI) and the colon, and to discover novel target genes in VA-related biological pathways. METHODS: vitamin A deficient (VAD) mice were generated by feeding VAD diet to pregnant C57/BL6 dams and their post-weaning offspring. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. RESULTS: The comparison between vitamin A sufficient (VAS) and VAD groups detected 49 and 94 DEGs in SI and colon, respectively. According to GO information, DEGs in the SI demonstrated significant enrichment in categories relevant to retinoid metabolic process, molecule binding, and immune function. Immunity related pathways, such as “humoral immune response” and “complement activation,” were positively associated with VA in SI. On the contrary, in colon, “cell division” was the only enriched category and was negatively associated with VA. WGCNA identified modules significantly correlated with VA status in SI and in colon. One of those modules contained five known retinoic acid targets. Therefore we have prioritized the other module members (e.g., Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) to be investigated as candidate genes regulated by VA. Comparison of co-expression modules between SI and colon indicated distinct VA effects on these two organs. CONCLUSIONS: The results show that VA deficiency alters the gene expression profiles in SI and colon quite differently. Some immune-related genes (Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) may be novel targets under the control of VA in SI. FUNDING SOURCES: NIH training grant and NIH research grant. SUPPORTING TABLES, IMAGES AND/OR GRAPHS

We demonstrate the first microfluidic-based on-chip liquefaction device for human sputum samples. Our device is based on an acoustofluidic micromixer using oscillating sharp edges. This acoustofluidic sputum liquefier can effectively and uniformly liquefy sputum samples at a throughput of 30 μL min(−1). Cell viability and integrity are maintained during the sputum liquefaction process. Our acoustofluidic sputum liquefier can be conveniently integrated with other microfluidic units to enable automated on-chip sputum processing and analysis.

α-galactosylceramide (αGalCer) and all trans-retinoic acid (RA) are both known as potent immune regulators on various steps of the immune responses. Our previous studies have shown that αGalCer and RA could regulate B cell differentiation in vitro and increase antibody production in vivo. To investigate the underlying mechanisms related to the antibody response, animal experiments (in 8-wk old female Balb/c mice) were performed to determine the antigen presentation and B cell response during primary immunization. In a one-day experiment, dextran-FITC or KLH-FITC was injected intravenously with or without αGalCer (2 μg in 100 μl PBS with antigen, iv) or RA (37.5 μg in 10 μl canola oil, oral dose); in a 10-day experiment, KLH was injected (100 μg/100 μl PBS) with or without αGalCer/RA treatment. Spleens were analyzed by flow cytometry and immunofluorescent staining. We observed that Dextran and KLH after 24h of injection were localized at the spleen marginal zone (MZ) area, with only KLH colocalized with the metallophilic macrophages; in the presence of αGalCer and RA, the band of metallophilic macrophages became wider. While dextran moved markedly toward the follicular area after treatment, KLH signal was not seen within the follicle, and also became weaker at the MZ area, suggesting the different presentation and processing mechanisms of the antigens. At day 10, substantially more active germinal centers were formed in the presence of αGalCer and RA, with more focused staining of CD23+GL-7+ cells, markers of germinal center B cells. B cell numbers were increased at both time points. Our study suggests that αGalCer and RA together may promote antigen presentation and induce B cell differentiation that ultimately increases antibody production.

The objective of this study was to determine the effects of growth stage (age) and dietary vitamin A (VA) on retinoid levels in plasma and main retinoid storage organs in a mouse model that is commonly used for immunological research. A purified VA-marginal diet was fed to Balb/C dams from pregnancy until pups were weaned. Post-weaning pups (total n=46) were randomized to 4 diet groups and fed VA-deficient, marginal, adequate, and supplement diets, which contains 0, 0.35, 4, and 100 μg retinol as retinyl palmitate/g diet, respectively. Tissues were collected from newborn (0 d), neonatal (14 d), young adult (4-wk), and adult (8-wk) mice and analyzed for total retinol (ROH) by UPLC. A dose-response relationship was observed in liver and lung, but not plasma. Surprisingly, the liver and lung ROH levels in VA-deficient and marginal groups for young adult and adult mice were higher as compared to those in rats previously studied in our laboratory. The results suggested that under VA-adequate and supplemented c...

α-Galactosylceramide (αGalCer) and all-trans-retinoic acid (RA) are each potent immune regulators able to increase immune responses in various conditions. αGalCer is particularly known for its direct activation of invariant NKT cells and B cells. In the present study, we used wildtype (WT) and CD1d knockout (KO) mice to characterize the specificity of αGalCer and its interaction with RA on B cell activation. WT and CD1dKO spleen cells were cultured ± αGalCer (100 ng/ml), or LPS (100 ng/ml) as a direct B-cell stimulus, and RA (20 nM), and B cell proliferation and differentiation were determined by flow cytometry. αGalCer and LPS both markedly increased the CD19+ B cell division. Whereas αGalCer mainly enriched spleen follicular CD23+ B cells, LPS increased the marginal zone CD21+ B cell population. The effects of αGalCer were completely absent in CD1dKO mice. However, although spleen B cells of CD1dKO mice failed to respond to αGalCer, these cells had a higher rate of proliferation compared to cells of WT mice, suggesting that CD1d molecules may have immune tonic effects in addition to reacting to αGalCer. Moreover, RA worked together with αGalCer to increase the proportions of IgG+ and CD138+ cells among CD23+ B cells, suggesting enhanced B-cell differentiation in splenic follicles. An in vivo immunization study using Keyhole limpet hemocyanin (KLH, 50 μg, ip) further confirmed that administration of RA (37 μg, po) and αGalCer (2 μg, sc), given at the time of primary immunization, increased antibody production in both the primary and secondary responses, which was not observed in CD1dKO mice. Thus, immune enhancement by αGalCer requires CD1d, and, when combined with RA promotes follicular B cell differentiation and increased antibody production.

The critical roles of retinoic acid (RA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic immune responses have been documented. However, how VA affects the development of autoimmunity is still unclear. Here we demonstrate that pharmacological doses of VA had adverse effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, RA (6 mg/kg body weight/day) or retinyl palmitate combined with 10% RA (VARA, a total of 6.6 mg/kg/day) to the mice starting from 6 weeks of age. At this age, the mice do not exhibit overt signs of lupus. However, the immunogenic environment preceding disease onset has been established that is evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of RA or VARA treatment, significantly higher pathological scores in kidney, lung, and brain were observed, which were correlated with a marked increase in B-cell responses. In addition, although the number of T cells in the mesenteric lymph node decreased with RA that led to reduced tissue weight, the proportions of T-cell subsets did not change. Importantly, neither RA nor VARA affected regulatory T cells in these mice. Altogether, our results suggest that under the immunogenic environment in autoimmune lupus, VA can generate more severe disease rather than inducing tolerance. Thus, administration of VA in lupus treatment should be approached with caution.