Cellular Reduction Research Articles

Effect of the nature of the chelated metal on the photodynamic activity of metalloporphyrins

Coordination of metal ions by the tetrapyrrolic macrocyclic ring of porphyrin-based photosensitizers (PSs) affects their photophysical properties and consequently, their photodynamic activity. Diamagnetic metals increase the singlet oxygen quantum yield while paramagnetic metals have the opposite effect. Since singlet oxygen is considered the main cell-damaging species in photodynamic therapy (PDT), the nature of the chelated cation would directly affect PDT efficacy. This expectation, however, is not always supported by experimental results and numerous exceptions have been reported. Understanding the effect of the chelated metal is hindered because different chelators were used. The aim of this work was to investigate the effect of the nature of chelated cation on the photophysical and photodynamic properties of metalloporphyrins, using the same tetrapyrrole core as a chelator of Ag(II), Cu(II), Fe(III), In(III), Mn(III), or Zn(II). Results demonstrated that with the exception of Ag(II), all paramagnetic metalloporphyrins were inefficient as generators of singlet oxygen and did not act as PSs. In contrast, the coordination of diamagnetic ions produced highly efficient PSs. The unexpected photodynamic activity of the Ag(II)-containing porphyrin was attributed to reduction of the chelated Ag(II) to Ag(I) or to demetallation of the complex, caused by cellular reductants and/or by exposure to light. Our results indicate that in biological systems, where PSs localize to various organelles and are subjected to the action of enzymes, reactive metabolites, and reducing or oxidizing agents, their physicochemical and photosensitizing properties change. Consequently, the photophysical properties alone cannot predict the anticancer efficacy of a PS.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p < 0.0001) and MEP (p < 0.0001), but skewing towards GMP (p<0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p < 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

IFN-γ lowers tumor growth by increasing glycolysis and lactate production in a nitric oxide-dependent manner: implications for cancer immunotherapy.

Interferon-gamma (IFN-γ), the sole member of the type-II interferon family, is well known to protect the host from infectious diseases as well as mount anti-tumor responses. The amounts of IFN-γ in the tumor microenvironment determine the host responses against tumors; however, several tumors employ evasive strategies by responding to low IFN-γ signaling. In this study, the response of various tumor cell lines to IFN-γ was studied in vitro. IFN-γ-activation increases glycolytic flux and reduces mitochondrial function in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner in the H6 hepatoma tumor cell line. The higher glycolysis further fueled NO and ROS production, indicating a reciprocal regulation. These processes are accompanied by Hypoxia inducing factor (HIF)-1α stabilization and HIF-1α-dependent augmentation of the glycolytic flux. The IFN-γ enhancement of lactate production also occurred in other NO-producing cell lines: RAW 264.7 monocyte/macrophage and Renca renal adenocarcinoma. However, two other tumor cell lines, CT26 colon carcinoma and B16F10 melanoma, did not produce NO and lactate upon IFN-γ-activation. HIF-1α stabilization upon IFN-γ-activation led to lower cell growth of B16F10 but not CT26 cells. Importantly, the IFN-γ-activation of both CT26 and B16F10 cells demonstrated significant cellular growth reduction upon metabolic rewiring by exogenous administration of potassium lactate. Clinical studies have shown the crucial roles of IFN-γ for successful cancer immunotherapies involving checkpoint inhibitors and chimeric antigen receptor T cells. The positive implications of this study on the metabolic modulation of IFN-γ activation on heterogeneous tumor cells are discussed.

Amelioration of Tumor-promoting Microenvironment via Vascular Remodeling and CAF Suppression Using E7130: Biomarker Analysis by Multimodal Imaging Modalities.

E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFβ1 production, a precursor of TGFβ1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFβ1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFβ1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.

Reversible rearrangement of the cellular cytoskeleton: A key to the broad-spectrum antiviral activity of novel amphiphilic polymers.

The battle against emerging viral infections has been uneven, as there is currently no broad-spectrum drug available to contain the spread of novel pathogens throughout the population. Consequently, the pandemic outbreak that occurred in early 2020 laid bare the almost empty state of the pandemic box. Therefore, the development of novel treatments with broad specificity has become a paramount concern in this post-pandemic era. Here, we propose copolymers of poly (sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and poly (sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn) and random (P(AMPSm-co-AaUn)) that show efficacy against a broad range of alpha and betacoronaviruses. Owing to their intricate architecture, these polymers exhibit a highly distinctive mode of action, modulating nano-mechanical properties of cells and thereby influencing viral replication. Through the employment of confocal and atomic force microscopy techniques, we discerned perturbations in actin and vimentin filaments, which correlated with modification of cellular elasticity and reduction of glycocalyx layer. Intriguingly, this process was reversible upon polymer removal from the cells. To ascertain the applicability of our findings, we assessed the efficacy and underlying mechanism of the inhibitors using fully differentiated human airway epithelial cultures, wherein near-complete abrogation of viral replication was documented. Given their mode of action, these polymers can be classified as biologically active nanomaterials that exploit a highly conserved molecular target-cellular plasticity-proffering the potential for truly broad-spectrum activity while concurrently for drug resistance development is minimal.

Cellular reductive stress: Is plasma membrane electron transport an evolutionarily-conserved safety valve?

Cellular respiration is highly regulated, changes dynamically in response to the microenvironment of individual cells and during differentiation and differs between cell and tissue types. Too little cell respiration can cause an accumulation of reductants, leading to reductive stress, while inefficient respiration, that causes a build-up of reactive oxygen species (ROS), can result in oxidative stress. Most of the discussion of this central redox dichotomy has centred around oxidative stress because the damaging effects of cellular oxidants on DNA, lipids and proteins are well-established, and have been shown to contribute to health issues including, mitochondrial and cardiovascular diseases, tumorigenesis, and to the effects of ageing. Much less attention has been paid to cellular reductive stress. Nevertheless, excessive levels of key cellular reductants including NADH, NADPH and glutathione, as well as an imbalance in protein thiols, and insufficient levels of ROS to maintain cell signalling pathways, can be harmful to cells and result in poor health outcomes. Recently, cellular mechanisms that sense and regulate cellular reductive stress associated with low ROS levels have been identified. In addition, plasma membrane electron transport has been shown to be a key player in cellular redox homeostasis involving NAD(P)H/NAD(P)+ ratios. It is now well-established that the plasma membrane contains coenzyme Q-mediated electron transport pathways capable of oxidizing intracellular NAD(P)H and reducing extracellular electron acceptors such as molecular oxygen. A better understanding of the origins, cellular and subcellular compartmentalization and regulation of cellular reductants could lead to the development of new anticancer strategies.

Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Serum from patients with asthma potentiates macrophage phagocytosis and human mesenchymal stromal cell therapy in experimental allergic asthma

Background/AimsAlthough several studies have demonstrated that mesenchymal stromal cells (MSCs) exhibit beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been controversial. Recent evidence has shown that MSCs modify their in vivo immunomodulatory actions depending on the specific inflammatory environment encountered. Accordingly, we assessed whether the therapeutic properties of human mesenchymal stromal cells (hMSCs) could be potentiated by conditioning these cells with serum (hMSC-serum) obtained from patients with asthma and then transplanted in an experimental model of house dust mite (HDM)-induced allergic asthma. MethodshMSC and hMSC-serum were administered intratracheally 24 h after the final HDM challenge. hMSC viability and inflammatory mediator production, lung mechanics and histology, bronchoalveolar lavage fluid (BALF) cellularity and biomarker levels, mitochondrial structure and function as well as macrophage polarization and phagocytic capacity were assessed. ResultsSerum preconditioning led to: (i) increased hMSC apoptosis and expression of transforming growth factor-β, interleukin (IL)-10, tumor necrosis factor-α–stimulated gene 6 protein and indoleamine 2,3-dioxygenase-1; (ii) fission and reduction of the intrinsic respiratory capacity of mitochondria; and (iii) polarization of macrophages to M2 phenotype, which may be associated with a greater percentage of hMSCs phagocytosed by macrophages. Compared with mice receiving hMSCs, administration of hMSC-serum led to further reduction of collagen fiber content, eotaxin levels, total and differential cellularity and increased IL-10 levels in BALF, improving lung mechanics. hMSC-serum promoted greater M2 macrophage polarization as well as macrophage phagocytosis, mainly of apoptotic hMSCs. ConclusionsSerum from patients with asthma led to a greater percentage of hMSCs phagocytosed by macrophages and triggered immunomodulatory responses, resulting in further reductions in both inflammation and remodeling compared with non-preconditioned hMSCs.

YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis

Metabolic reprogramming is one of the key features of tumors facilitating their rapid proliferation and adaptation to harsh microenvironments. Yin Yang 2 (YY2) has recently been reported as a tumor suppressor downregulated in various types of tumors; however, the molecular mechanisms underlying its tumor-suppressive activity remain poorly understood. Furthermore, the involvement of YY2 in tumor cell metabolic reprogramming remains unclear. Herein, we aimed to elucidate the novel regulatory mechanism of YY2 in the suppression of tumorigenesis. Using transcriptomic analysis, we uncovered an unprecedented link between YY2 and tumor cell serine metabolism. YY2 alteration could negatively regulate the expression level of phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthesis pathway, and consequently, tumor cell de novo serine biosynthesis. Mechanistically, we revealed that YY2 binds to the PHGDH promoter and suppresses its transcriptional activity. This, in turn, leads to decreased production of serine, nucleotides, and cellular reductants NADH and NADPH, which subsequently suppresses tumorigenic potential. These findings reveal a novel function of YY2 as a regulator of the serine metabolic pathway in tumor cells and provide new insights into its tumor suppressor activity. Furthermore, our findings suggest the potential of YY2 as a target for metabolic-based antitumor therapeutic strategies.

RINGSIDE phase 2/3 trial of AL102 for treatment of desmoid tumors (DT): Phase 2 results.

11515 Background: For patients with desmoid tumors (DT, aggressive fibromatosis), systemic therapy that results in tumor regression, symptom improvement and durable tolerability is needed. Gamma secretase inhibitors (GSIs) have demonstrated antitumor activity against DT. AL102 is a potent, orally available, selective GSI under investigation for treatment of DT. Methods: RINGSIDE (AL-DES-01) is a Phase 2/3 study for patients with progressing DT. In the open-label Phase 2 study (Part A), adults with progressing DT (≥10% unidimensional growth within 18 months or DT-related pain requiring non-opioid medication) were randomized to three dosing regimens: 1.2 mg QD, 2 mg intermittent BIW (2 days on 5 days off), or 4 mg intermittent BIW. Patients who complete Phase 2 roll over into an open-label extension (OLE). RINGSIDE Phase 3 (Part B) is a double-blind, placebo-controlled study evaluating the chosen dose regimen from Phase 2 (1.2 mg once daily) utilizing PFS as the primary endpoint. We report updated efficacy and safety results from RINGSIDE Phase 2. Results: Enrollment of all 42 patients into Phase 2 was completed as of March 2022. As of January 3, 2023, median time on study was 10.5 months (range 0.8 – 14.7) and 30 patients (71.4%) were still on study, 10 (23.8%) of whom rolled over to the OLE. Mean age was 39.9 years, 73.8% were women and 69% had received prior desmoid cancer therapy. The best response in the evaluable population as assessed by blinded independent central review (BICR) was partial response (PR) in 6/12 patients (50%) for 1.2 mg QD, 3/13 patients (23.1%) for 4 mg BIW, and 5/11 patients (45.5%) for 2 mg BIW. Disease control rate was 100%, 91%, and 97% in these groups, respectively. A consistent pattern of deeper, more rapid and maintained response was observed with 1.2 mg QD. Median volume change (BICR) from baseline was -51.9% for 1.2 mg QD, -9.5% for 4 mg BIW, and -15.2% for 2 mg BIW at Week 16 and -76.4%, -35.5%, and -51.2%, respectively, at Week 28. Similar patterns were observed for % changes from baseline in T2 signal intensity, suggesting reduction of tumor cellularity. Consistent with the mechanism of action of GSIs, the five most common Grade 1-2 treatment-emergent adverse events (TEAEs) were diarrhea, nausea, fatigue, alopecia, and dry skin. Grade 3 drug-related TEAEs were reported in 26.2% of patients across all tested doses. There were no Grade 4, Grade 5, or serious TEAEs related to AL102 per investigator assessment. There were no new safety signals. Conclusions: In this Phase 2 study, AL102 was safe and generally well tolerated across all tested doses. The safety profile was consistent with the GSI class of drugs. Tumor response, volume reduction and T2 signal reduction were observed earlier in the 1.2 mg QD group, with deeper and maintained treatment responses. This dose was selected for study in RINGSIDE Phase 3, which is currently enrolling in multiple countries. Clinical trial information: NCT04871282 .

Evolutionary analysis of cellular reduction and anaerobicity in the hyper-prevalent gut microbe Blastocystis

Blastocystis is the most prevalent microbial eukaryote in the human and animal gut, yet its role as commensal or parasite is still under debate. Blastocystis has clearly undergone evolutionary adaptation to the gut environment and possesses minimal cellular compartmentalization, reduced anaerobic mitochondria, no flagella, and no reported peroxisomes. To address this poorly understood evolutionary transition, we have taken a multi-disciplinary approach to characterize Proteromonas lacertae, the closest canonical stramenopile relative of Blastocystis. Genomic data reveal an abundance of unique genes in P.lacertae but also reductive evolution of the genomic complement in Blastocystis. Comparative genomic analysis sheds light on flagellar evolution, including 37 new candidate components implicated with mastigonemes, the stramenopile morphological hallmark. The P.lacertae membrane-trafficking system (MTS) complement is only slightly more canonical than that of Blastocystis, but notably, we identified that both organisms encode the complete enigmatic endocytic TSET complex, a first for the entire stramenopile lineage. Investigation also details the modulation of mitochondrial composition and metabolism in both P.lacertae and Blastocystis. Unexpectedly, we identify in P.lacertae the most reduced peroxisome-derived organelle reported to date, which leads us to speculate on a mechanism of constraint guiding the dynamics of peroxisome-mitochondrion reductive evolution on the path to anaerobiosis. Overall, these analyses provide a launching point to investigate organellar evolution and reveal in detail the evolutionary path that Blastocystis has taken from a canonical flagellated protist to the hyper-divergent and hyper-prevalent animal and human gut microbe.

Evaluation of the effectiveness of non-invasive methods of compressive microvibration and ablative sensory microvibration on local areas with excess fat deposits in volunteers of different age groups

The aim of the study was the apparatus of compressive microvibration “Endospheres Therapy® Body” (Registration certificate RZN 2016/3863 of 29 December 2017) used in women with mild to moderate edematous fibrosclerosis (panniculopathy) of peri- and post-menopausal age. The purpose of the project is to evaluate the effectiveness of the non-invasive technique of compressive microvibration in volunteers of different age groups. The basis for the work is the agreement No. 002/2022-n between the GBUZ MO MONIKI named after M.F. Vladimirsky and ItalConsult, LLC of 01.03.2022. The main tasks were: form 3 cohort groups of volunteers in accordance with the criteria agreed with the Customer (mild or moderate edematous fibrosclerosis, as panniculopathy), aged 30 to 44 years old, 45 to 54 years old and 55-65 years old; ensure that all participants receive a course on procedures (12 procedures every other day) regularly and correctly; evaluate the effectiveness of the non-invasive method of compressive microvibration on local areas of excess fat deposits in volunteers of different age groups through subjective testing (with a questionnaire), objective results of laboratory tests (blood test with hormone levels) and instrumental studies (ultrasound examination of the skin of fat trap zones, in abdomen and thighs) and assessment of the microcirculation system. Hypothesis: compressive microvibration generated by the manipulator-cylinder, consisting of 50 spheres of hypoallergenic silicone that generates low-frequency vibrations, makes it possible to activate microcirculation, stimulate lymphatic drainage, increase muscle tone and improve skin trophism. For all study results, mean values (Mean), standard deviations (Std.Dev.), minimum (Minimum), maximum (Maximum) values, and SD confidence of all results were obtained. The difference between the mean values and the variation coefficients was tested using a two-sample Student’s t-test (considering the distribution’s normality). Our results have convincingly demonstrated the effectiveness of the Endospheres Therapy® procedures for treating cellulite, with the effect maintained for at least 60 days after the course of therapy. Instrumental studies confirmed a pronounced improvement in the skin’s condition, microcirculation, cellular metabolism activation, hypoxia reduction and tissue perfusion improvement. Endospheres Therapy® procedures are safe and effective in treating cellulite in the long term without side effects. Patients noted a high degree of satisfaction with the Endospheres Therapy® course, which indicates the quality of the procedures and the level of specialists.

Reverse-QTY code design of active human serum albumin self-assembled amphiphilic nanoparticles for effective anti-tumor drug doxorubicin release in mice

Human serum albumin (HSA) is a highly water-soluble protein with 67% alpha-helix content and three distinct domains (I, II, and III). HSA offers a great promise in drug delivery with enhanced permeability and retention effect. But it is hindered by protein denaturation during drug entrapment or conjugation that result in distinct cellular transport pathways and reduction of biological activities. Here we report using a protein design approach named reverse-QTY (rQTY) code to convert specific hydrophilic alpha-helices to hydrophobic to alpha-helices. The designed HSA undergo self-assembly of well-ordered nanoparticles with highly biological actives. The hydrophilic amino acids, asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y) in the helical B-subdomains of HSA were systematically replaced by hydrophobic leucine (L), valine (V), and phenylalanine (F). HSArQTY nanoparticles exhibited efficient cellular internalization through the cell membrane albumin binding protein GP60, or SPARC (secreted protein, acidic and rich in cysteine)-mediated pathways. The designed HSArQTY variants displayed superior biological activities including: i) encapsulation of drug doxorubicin, ii) receptor-mediated cellular transport, iii) tumor cell targeting, and iv) antitumor efficiency compare to denatured HSA nanoparticles. HSArQTY nanoparticles provided superior tumor targeting and antitumor therapeutic effects compared to the albumin nanoparticles fabricated by antisolvent precipitation method. We believe that the rQTY code is a robust platform for specific hydrophobic modification of functional hydrophilic proteins with clear-defined binding interfaces.

Senomorphic properties of miR-181b in aged human aortic smooth muscle cells

Introduction: Age associated large artery stiffness (LAS) is an independent predictor of adverse cardiovascular health outcomes in humans and has also been shown to be associated with the development of heart failure, kidney disease, and even dementia. Furthermore, the development of cellular senescence in the aorta, the permanent arrest of the cell cycle and development of pro-inflammatory, pro-fibrotic senescence associated secretory phenotype (SASP), has also been shown to contribute to age associated LAS. MicroRNAs (miR) are small, non-coding RNAs that down-regulate protein expression in a semi-specific manner. miR-181b has been shown to be downregulated with aging, targets many of the secreted factors associated with the aortic SASP and has previously been shown to have a beneficial effect on vascular health. With smooth muscle cells being the predominant cell type in the large arteries, we wanted to address the hypothesis that miR-181b has the potential to act as a senomorphic in human aortic smooth muscle cells (HASMCs) and promote beneficial changes in individual cells that influence LAS. Methods: To test our hypothesis, we obtained young and old HASMCs and transfected them with miR-181b or a scrambled miR control utilizing a lipofectamine transfection protocol. Total RNA was isolated from the HASMCs and quantitative PCR was performed to assess changes of the SASP profile. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology. Lastly, individual HASMC stiffness measurements were made using twisting magnetic microscopy. Results: Transfection of HASMCs with miR-181b resulted in reduced expression of markers of cell cycle arrest (p16 and p21) and the SASP profile (IL-6, TNF-alpha, MCP1, TGF-b1, IL-1alpha, and MMP2) when compared to scrambled control (p<0.05). HASMC proliferation was decreased in old HASMCs compared to young (p<0.05) and was increased with miR-181b transfection when compared to scramble control (p<0.05). Lastly, individual HASMC stiffness was lower in young cells compared to old (0.4382 ± 0.29 vs. 0.9959 ± 0.72 Pa/nm, n=120, p<0.001). Cell stiffness was decreased in old HASMCs when treated with miR-181b when compared with control (0.7856 ± 0.144 vs. 1.18 ± 0.288 Pa/nm, n=76, p<0.001), but there was no difference in young HASMCs. Conclusions: miR-181b appears to act as a senomorphic agent when transfected in old HASMCs. This is evidenced by noticeable changes in the expression of markers of cellular senescence which corresponded with positive changes in cellular characteristics that are associated with LAS, namely the improvement in cellular proliferation and reduction of individual cell stiffness in the old HASMC groups. Together, these results suggest the modulation of miR-181b may be a potential therapeutic target to improve age associated LAS through an effect on cellular senescence. K08 AG070281 (ET), Western Institute for Veterans Research (ET), U-CARES (ET), R01 AG048366 (LAL), R01 AG050238 (AJD), R01 AG060395 (AJD) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3664: Mechanism for bone marrow failure differs between acute myeloid and lymphoid leukemia

Abstract Acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) are aggressive forms of blood cancer that impair normal hematopoiesis and are typically associated with bone marrow (BM) failure, reflected by peripheral blood pancytopenia. While BM failure is thought to be mediated by the loss of hematopoietic stem and progenitor cells (HSPCs) due to leukemic expansion, with leukemic cells “crowding out” normal HSPCs, the mechanisms underlying this process remain unclear. To elucidate these mechanisms, we examined the effects of AML or ALL on residual normal or wild type (WT) hematopoiesis. The 961C cell line is an AML cell line derived from a NUP98-PH23 (NP23) transgenic mouse, and the T259 cell line is a B cell precursor ALL (BCP ALL) cell line also derived from an NP23 mouse. AML and BCP ALL were generated by transplantation of 961C or T259 cell lines into congenic recipients without cytotoxic conditioning. The use of congenic mice allowed engraftment of immunocompetent mice, and the lack of cytotoxic conditioning prevented damage to the BM microenvironment. Mice with AML demonstrated severe anemia, thrombocytopenia, and depletion of HSPCs, along with a marked reduction of total BM cellularity (WT + AML cells), suggesting that BM suppression was not due to a physical “crowding” mechanism. Instead, BM suppression in the absence of BM replacement with myeloblasts suggested that hematopoiesis may be inhibited by a factor secreted by the AML blasts. The inflammatory cytokines TNF-A and IL-6 were markedly increased in the peripheral blood of mice with AML, suggesting these were candidates for a factor responsible for decreased cellularity and pancytopenia. In contrast, mice with BCP ALL demonstrated normal hemoglobin and leukocyte counts, even with significant ALL engraftment. The loss of WT hematopoietic cells in the BM was equivalent to the gain of leukemic cells in the BM, and the total number of leukemic cells in mice with BCP ALL was 14-fold higher than the number of leukemic cells in mice with AML. Of note, the spleen of mice with BCP ALL displayed a 100x increase in WT HSPCs compared to non-transplanted mice with healthy BM. These findings suggest that BCP ALL depletes WT HSPCs in the BM through a physical expansion mechanism. WT splenic HSPCs from mice with AML were shown to have normal numbers of functional hematopoietic stem cells via a limiting dilution assay. In addition, WT HSPCs harvested from mice with BCP ALL generated similar numbers and types of colonies in vitro compared to WT HSPC harvested from non-leukemic mice. Both experiments suggest that neither BCP ALL nor AML induced a permanent change in the functionality of WT HSPCs. In conclusion, our results suggest that AML induces BM failure independent of leukemic burden, possibly via a secreted cytokine, whereas BCP ALL induces BM failure through physical expansion that replaces WT HSPCS with leukemic cells. Citation Format: Ryan Matthew Bertoli, Peter D. Aplan, Yang Jo Chung. Mechanism for bone marrow failure differs between acute myeloid and lymphoid leukemia. [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 3664.

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [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 1370.

The p52-ZER6/G6PD axis alters aerobic glycolysis and promotes tumor progression by activating the pentose phosphate pathway

Abnormal glucose metabolism is a highlight of tumor metabolic reprogramming and is closely related to the development of malignancies. p52-ZER6, a C2H2-type zinc finger protein, promotes cell proliferation and tumorigenesis. However, its role in the regulation of biological and pathological functions remains poorly understood. Here, we examined the role of p52-ZER6 in tumor cell metabolic reprogramming. Specifically, we demonstrated that p52-ZER6 promotes tumor glucose metabolic reprogramming by positively regulating the transcription of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). By activating the PPP, p52-ZER6 was found to enhance the production of nucleotides and nicotinamide adenine dinucleotide phosphate, thereby providing tumor cells with the building blocks of ribonucleic acids and cellular reductants for reactive oxygen species scavenging, which subsequently promotes tumor cell proliferation and viability. Importantly, p52-ZER6 promoted PPP-mediated tumorigenesis in a p53-independent manner. Taken together, these findings reveal a novel role for p52-ZER6 in regulating G6PD transcription via a p53-independent process, ultimately resulting in tumor cell metabolic reprogramming and tumorigenesis. Our results suggest that p52-ZER6 is a potential target for the diagnosis and treatment of tumors and metabolic disorders.

The influence of light on the availability of photoreactive and photostable iron(III)–ligand complexes to diatoms

AbstractUptake of iron (Fe) by eukaryotic phytoplankton is suggested to proceed by a reductive pathway in which Fe(III) bound to inorganic and organic ligands is reduced prior to Fe internalization. Photochemical reduction of the Fe–ligand complexes can increase Fe bioavailability, but the role of light in regulating physiological processes related to Fe acquisition is not well known. Here, we report how model Fe–ligand complexes differing in photolability affected growth, cellular Fe reduction, and Fe uptake rates of two marine diatoms as a function of irradiance. Growth rates of Thalassiosira oceanica and Phaeodactylum tricornutum in media amended with Fe complexed with ethylenediaminetetraacetic acid (Fe‐EDTA) and desferrioxamine B (FeDFB) increased linearly with light between 50 and 400 μmol photons m−2 s−1 under Fe‐limiting conditions. Steady‐state Fe uptake rates (ρSS) were also light dependent, increasing proportionally with growth irradiance for both Fe–ligand complexes. Photolysis of the Fe‐EDTA complex could explain the increase in ρSS in Fe‐EDTA‐amended medium because it increased [Fe(III)’], but it could not account for the faster rate of Fe uptake from FeDFB at high light because FeDFB is photostable. Short‐term Fe uptake rates measured in the absence of photochemically mediated Fe(III) reduction showed that cells preconditioned to high growth irradiance took up Fe faster than cells grown at low light. Thus, growth at high light induced a physiological response that increased Fe uptake. High growth irradiance also increased the rate of cellular Fe(III)’ reduction in the dark, similar to its stimulatory effect on short‐term Fe uptake. These results identify a previously unrecognized Fe–light interaction that could be important in enhancing Fe availability to diatoms. We suggest that the light effect could be the result of a light‐dependent regulation of cellular Fe(III) reduction.

Abstract P6-01-15: Breast cancer patients with different hormone receptor subtypes receiving neoadjuvant chemotherapy (NAC) experience differential overall survival according to their resistance to NAC

Abstract Background: Response to neoadjuvant chemotherapy (NAC) is an indicator of outcomes and can be quantified as achievement of pathologic complete response (pCR) (absence of residual invasive disease in breast and lymph nodes) and residual percent cellularity. However, the significance of residual tumor cellularity post-NAC is not well understood. We assessed the impact of NAC-induced reduction in tumor cellularity among hormone receptor subtypes and the effect of these reductions on overall survival (OS). Methods: An IRB-approved retrospective review identified demographics, disease presentation, response to treatment, and outcomes. ER and PR status were categorized as low positive (1-9%), positive (≥ 10%) and negative. Treatment response was noted as percent residual cellularity (complete 0%, almost complete < 10%, good 10-30%, moderate >30-80% and poor >80%) in the surgical specimen and pathologic stage. We examined measures of response to NAC within breast tumor subtypes and the effect of these responses on associations with OS among hormone receptor subtypes. Results: The clinical series comprised 384 patients who received curative intent NAC. This series was diverse in presentation, displayed considerable variability in response to NAC (Table 1). 88 (23.6%) patients did not experience tumor down staging, and of those presenting with clinical nodal Stage 1 or higher (n=197), 94 (47.7%) did not experience nodal down staging. Although Triple Negative Breast Cancer (TNBC) status was not significantly associated with post-NAC residual tumor cellularity (p=0.74), ER, PR, and HER2 status were individually associated with this measure of response to NAC (p=0.04, 0.01, and 0.01, respectively). However, none of these associations explained more than 2.5% of the variability in this marker of treatment response. Median non-censored follow-up time was 4.26 years. Accounting for censoring, median survival was 13.65 years (lower 95% confidence limit was 11.79 years). Differential associations with OS were observed for three hormone receptor subtypes (ER: p< 0.001, HER2: p=0.04, and TNBC: p< 0.001) according to residual tumor cellularity, classified by percent residual cellularity categories of complete or almost complete response versus all others. Importantly, although ER negative patients with poor residual cellularity response had worse OS than ER positive patients with good response (Hazard Ratio [HR], 95% Confidence Interval [CI] = 4.74, 2.2-10.2), ER negative patients with good response did not have significantly worse OS than ER positive patients with good response (HR, 95% CI = 1.37, 0.57-3.26). Similar patterns were seen for patients with HER2 negative breast cancer or TNBC. Chemo resistant TNBC patients had higher risks for mortality than if they were not chemo resistant (HR, 95% CI: 2.41, 1.02-5.71). Conclusions: Our data suggest that OS associated with different hormone subtypes differs according to response to NAC. Differential OS according to response to NAC is greatest for patients classified according to ER status influences, with good response eliminating much of the differential mortality risk between ER negative and positive patients. There is a similar difference according to TNBC, although the difference appears to be less complete. Further studies should focus on understanding the chemo resistance of ER negative tumors, and perhaps TNBC, to identify mechanisms that may ultimately drive treatment response and survival. Table 1. Demographic and other characteristics Citation Format: Sangeetha Prabhakaran, V. Shane Pankratz, Christopher McNicoll, Nadja Falk, Jacklyn Nemunaitis, Jain Zhou, Payton Sandoval-Belt. Breast cancer patients with different hormone receptor subtypes receiving neoadjuvant chemotherapy (NAC) experience differential overall survival according to their resistance to NAC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-15.

Aldh1a2 + fibroblastic reticular cells regulate lymphocyte recruitment in omental milky spots.

Lymphoid clusters in visceral adipose tissue omentum, known as milky spots, play a central role in the immunological defense in the abdomen. Milky spots exhibit hybrid nature between secondary lymph organs and ectopic lymphoid tissues, yet their development and maturation mechanisms are poorly understood. Here, we identified a subset of fibroblastic reticular cells (FRCs) that are uniquely present in omental milky spots. These FRCs were characterized by the expression of retinoic acid-converting enzyme, Aldh1a2, and endothelial cell marker, Tie2, in addition to canonical FRC-associated genes. Diphtheria toxin-mediated ablation of Aldh1a2+ FRCs resulted in the alteration in milky spot structure with a significant reduction in size and cellularity. Mechanistically, Aldh1a2+ FRCs regulated the display of chemokine CXCL12 on high endothelial venules (HEVs), which recruit blood-borne lymphocytes from circulation. We further found that Aldh1a2+ FRCs are required for the maintenance of peritoneal lymphocyte composition. These results illustrate the homeostatic roles of FRCs in the formation of non-classical lymphoid tissues.