Search

Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca2+ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca2+ cycling genes Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca2+ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.

Ca(2+) cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca(2+) handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca(2+) dynamics in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically-encoded Ca(2+) probe capable of simultaneously measuring cytosolic and mitochondrial Ca(2+) in real time. Using this novel probe, we determined and compared mitochondrial Ca(2+) activity and the coupling with cytosolic Ca(2+) dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca(2+) in sync with cytosolic Ca(2+),whereas hiPSC-CMs showed high cell-wide variability in mitochondrial Ca(2+) activity that is poorly coordinated with cytosolic Ca(2+). We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, are underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca(2+) dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca(2+) coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca(2+) coupling in hiPSC-CMs. PHYSIOLOGICAL RELEVANCE: The present study will advance our understanding of the role of mitochondria in Ca(2+) handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

Adeno-associated viruses (AAVs) have been well characterized and used to deliver therapeutic genes for diseases treatment in clinics and basic research. This study used the triple transient transfection of AAV-DJ/8 as a model expression system to develop and optimize the laboratory production of AAV for research and pre-clinical applications. Specifically, various production parameters, including host cell, transfection reagent, cell density, ratio of plasmid DNA and cells, gene size, and production mode, were tested to determine the optimal process. Our results showed that the adherent production using HEK 293AAV with calcium transfection generated the highest volumetric productivity of 7.86 × 109 gc/ml. The optimal suspensive production using HEK 293F had best AAV productivity of 5.78 × 109 gc/ml in serum-free medium under transfection conditions of transfection density of 0.4 × 106 cells/ml, plasmid DNA:cells ratio of 1.6 µg:106 cells and synthesized cationic liposomes as transfection reagent. The similar AAV productivity was confirmed at scales of 30–450 ml in shaker and/or spinner flasks. The in vitro transfection and in vivo infection efficiency of the harvested AAV-DJ/8 carrying luciferase reporter gene was confirmed using cell line and xenograft mouse model, respectively. The minimal or low purification recovery rate of AAV-DJ/8 in ion-exchange chromatography column and affinity column was observed in this study. In summary, we developed and optimized a scalable suspensive production of AAV to support the large-scale preclinical animal studies in research laboratories.

While mitochondrial dysfunction has been implicated in the pathogenesis of cardiac arrhythmias, how the abnormality occurring at the organelle level escalates to influence the rhythm of the heart remains incompletely understood. This is due, in part, to the complexity of the interactions formed by cardiac electrical, mechanical, and metabolic subsystems at various spatiotemporal scales that is difficult to fully comprehend solely with experiments. Computational models have emerged as a powerful tool to explore complicated and highly dynamic biological systems such as the heart, alone or in combination with experimental measurements. Here, we describe a strategy of integrating computer simulations with optical mapping of cardiomyocyte monolayers to examine how regional mitochondrial dysfunction elicits abnormal electrical activity, such as rebound and spiral waves, leading to reentry and fibrillation in cardiac tissue. We anticipate that this advanced modeling technology will enable new insights into the mechanisms by which changes in subcellular organelles can impact organ function.

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR+ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day−1·mouse−1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment. NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.

While optogenetic approaches have been widely used for remote control of cell membrane excitability and intracellular signaling pathways, their application in mitochondrial study has been limited, largely due to the challenge of effectively and specifically expressing heterologous light-gated rhodopsin channels in the mitochondria. Here, we describe the methods for expressing functional channelrhodopsin 2 (ChR2) proteins in the mitochondrial inner membrane with an unusually long mitochondrial leading sequence and characterizing optogenetic-mediated mitochondrial membrane potential (ΔΨ(m)) depolarization. We then illustrate how this next-generation optogenetic approach can be used to study the effect of ΔΨ(m) on mitochondrial functions such as mitophagy, programed cell death and preconditioning-mediated cytoprotection. We anticipate that this innovative technology will enable new insights into the mechanisms by which changes in ΔΨ(m) differentially impacts mitochondrial and cellular functions.

While mitochondrial dysfunction has been implicated in the pathogenesis of cardiac arrhythmias, how the abnormality occurring at the organelle level escalates to influence the rhythm of the heart remains incompletely understood. This is due, in part, to the complexity of the interactions formed by cardiac electrical, mechanical, and metabolic subsystems at various spatiotemporal scales that is difficult to fully comprehend solely with experiments. Computational models have emerged as a powerful tool to explore complicated and highly dynamic biological systems such as the heart, alone or in combination with experimental measurements. Here, we describe a strategy of integrating computer simulations with optical mapping of cardiomyocyte monolayers to examine how regional mitochondrial dysfunction elicits abnormal electrical activity, such as rebound and spiral waves, leading to reentry and fibrillation in cardiac tissue. We anticipate that this advanced modeling technology will enable new insights into the mechanisms by which changes in subcellular organelles can impact organ function.

Glioblastomas, accounting for approximately 50% of gliomas, comprise the most aggressive, highly heterogeneous, and malignant brain tumors. The objective of this study was to develop and evaluate a new targeted therapy, i.e., highly potent natural compound verrucarin A (Ver-A), delivered with monoclonal antibody-directed extracellular vesicle (mAb-EV). First, the high surface expression of epidermal growth factor receptor (EGFR) in glioblastoma patient tissue and cell lines was confirmed using immunohistochemistry staining, flow cytometry, and Western blotting. mAb-EV-Ver-A was constructed by packing Ver-A and tagging anti-EGFR mAb to EV generated from HEK293F culture. Confocal microscopy and the In Vivo Imaging System demonstrated that mAb-EV could penetrate the blood–brain barrier, target intracranial glioblastoma xenografts, and deliver drug intracellularly. The in vitro cytotoxicity study showed IC50 values of 2–12 nM of Ver-A. The hematoxylin and eosin staining of major organs in the tolerated dose study indicated minimal systemic toxicity of mAb-EV-Ver-A. Finally, the in vivo anti-tumor efficacy study in intracranial xenograft models demonstrated that EGFR mAb-EV-Ver-A effectively inhibited glioblastoma growth, but the combination with VEGF mAb did not improve the therapeutic efficacy. This study suggested that mAb-EV is an effective drug delivery vehicle and natural Ver-A has great potential to treat glioblastoma.

Triple-negative breast cancers (TNBCs) are highly aggressive and recurrent. Standard cytotoxic chemotherapies are currently the main treatment options, but their clinical efficacies are limited and patients usually suffer from severe side effects. The goal of this study was to develop and evaluate targeted liposomes-delivered combined chemotherapies to treat TNBCs. Specifically, the IC50 values of the microtubule polymerization inhibitor mertansine (DM1), mitotic spindle assembly defecting taxane (paclitaxel, PTX), DNA synthesis inhibitor gemcitabine (GC), and DNA damage inducer doxorubicin (AC) were tested in both TNBC MDA-MB-231 and MDA-MB-468 cells. Then we constructed the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) tagged liposomes and confirmed its TNBC cell surface binding using flow cytometry, internalization with confocal laser scanning microscopy, and TNBC xenograft targeting in NSG female mice using In Vivo Imaging System. The safe dosage of anti-EGFR liposomal chemotherapies, i.e., &lt, 20% body weight change, was identified. Finally, the in vivo anti-tumor efficacy studies in TNBC cell line-derived xenograft and patient-derived xenograft models revealed that the targeted delivery of chemotherapies (mertansine and gemcitabine) can effectively inhibit tumor growth. This study demonstrated that the targeted liposomes enable the new formulations of combined therapies that improve anti-TNBC efficacy.

The optimization of cell culture modes and basal media is very important to improve the production of therapeutic monoclonal antibodies (mAbs) by Chinese hamster ovary (CHO) cells, but the underlying cellular mechanism has not been well investigated. The objective of this study was to elucidate the interaction between the intracellular proteome and key process parameters by employing comparative proteomics. Three cell culture operations and four basal media were characterized in the production of anti-HER2 mAb using CHO DG44. The representative host cell proteins that demonstrated significant responses to various culture modes and media, including the enzymes involved in carbohydrate metabolism, transcription, translation, and post translational modification, were described. The fed-batch culture in a 2-L bioreactor produced mAb with a titer of 2411 mg/L using Dynamis medium. The purified mAb showed strong and specific targeting to HER2+ MDA-MB-361 cell and similar anti-cancer toxicity to the FDA approved Trastuzumab. Moreover, this proteomics study also indicated several key considerations for future rationally engineering a mAb production process, which could benefit biopharmaceutical manufacturing.

Aims The FDA-approved histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been shown to induce cardiomyocyte autophagy and blunt ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, the precise mechanisms underlying the cardioprotective activity of SAHA are unknown. Mitochondrial dysfunction and oxidative damage are major contributors to myocardial apoptosis during I/R injury. We hypothesize that SAHA protects the myocardium by maintaining mitochondrial homeostasis and reducing reactive oxygen species (ROS) production during I/R injury. Methods Mouse and cultured cardiomyocytes (neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes) I/R models were used to investigate the effects of SAHA on mitochondria. ATG7 knockout mice, ATG7 knockdown by siRNA and PGC-1α knockdown by adenovirus in cardiomyocytes were used to test the dependency of autophagy and PGC-1α-mediated mitochondrial biogenesis respectively. Results Intact and total mitochondrial DNA (mtDNA) content and mitochondrial mass were significantly increased in cardiomyocytes by SAHA pretreatment before simulated I/R. In vivo, I/R induced >50% loss of mtDNA content in the border zones of mouse hearts, but SAHA pretreatment and reperfusion treatment alone reverted mtDNA content and mitochondrial mass to control levels. Moreover, pretreatment of cardiomyocytes with SAHA resulted in a 4-fold decrease in I/R-induced loss of mitochondrial membrane potential and a 25%–40% reduction in cytosolic ROS levels. However, loss-of-function of ATG7 in cardiomyocytes or mouse myocardium abolished the protective effects of SAHA on ROS levels, mitochondrial membrane potential, mtDNA levels, and mitochondrial mass. Lastly, PGC-1α gene expression was induced by SAHA in NRVMs and mouse heart subjected to I/R, and loss of PGC-1α abrogated SAHA's mitochondrial protective effects in cardiomyocytes. Conclusions SAHA prevents I/R induced-mitochondrial dysfunction and loss, and reduces myocardial ROS production when given before or after the ischemia. The protective effects of SAHA on mitochondria are dependent on autophagy and PGC-1α-mediated mitochondrial biogenesis.

The adoptive transfer of human T cells or genetically-engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The objective of this study was to develop a novel T cell biomanufacturing platform using stirred-tank bioreactor for large-scale and high-quality cellular production. First, various factors, such as bioreactor parameters, media, supplements, stimulation, seed age, and donors, were investigated. A serum-free fed-batch bioproduction process was developed to achieve 1000-fold expansion within 8 days after first stimulation and another 500-fold expansion with second stimulation. Second, this biomanufacturing process was successfully scaled up in bioreactor with dilution factor of 10, and the robustness and reproducibility of the process was confirmed by the inclusion of different donors’ T cells of various qualities. Finally, T cell quality was monitored using 12 surface markers and 3 intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. In this study, a new biomanufacturing platform was created to produce reliable, reproducible, high-quality, and large-quantity (i.e. > 5 billion) human T cells in stirred-tank bioreactor. This platform is compatible with the production systems of monoclonal antibodies, vaccines, and other therapeutic cells, which provides not only the proof-of-concept but also the ready-to-use new approach of T cell expansion for clinical immune therapy.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of &gt, 98% to meningioma CH157-MN cells but a low binding rate of &lt, 5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of &lt, 10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Triple-negative breast cancers (TNBCs) are highly aggressive, heterologous and frequently recurrent. The standard radiotherapy and chemotherapy have limited clinical benefits while the combined therapies or antibody-drug conjugates (ADCs) are promising to treat TNBC. The objective of this study was to develop a combined therapy, CD276/CD47-targeted ADCs, for TNBC treatment. Specifically, the CD276 and CD47 surface receptors were identified with immunohistochemistry staining of drug-sensitive and resistant patient tissues and confirmed in other assays. Two innovative monoclonal antibodies (mAbs), i.e. anti-CD276 mAb and anti-CD47 mAb that target the N-glycosylated extracellular domains (Leu29-Pro245 and Gln19-Glu141) of surface receptors, were developed using hybridoma technology. The top mAb clones with high surface binding, drug delivery and affinity were screened, produced in fed-batch culture, purified using liquid chromatography, and conjugated with payload mertansine (DM1). Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the CD276/CD47 ADCs effectively target human and mouse TNBC cells and xenograft models. The in vitro anti-cancer cytotoxicity was confirmed in multiple cell lines (MDA-MB-468, MDA-MB-231, 4T1), and in vivo evaluations using TNBC cell line-derived xenograft models and patient-derived xenograft (PDX) models demonstrated high anti-tumor efficacy and tumoral immunity of the dually targeted ADCs. In addition, whole blood analysis indicated that neither CD276 mAb nor CD47 mAb showed general immune toxicity in immunocompetent model. The maximal tolerated dose (MTD), pharmacokinetics (PK) and biodistribution will be further investigated. This study indicated that the ADCs dually targeting CD276 and CD47 have great potential to treat TNBCs. Citation Format: Yingnan Si, Kai Chen, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. CD276/CD47-targeted antibody-drug conjugates to treat triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5958.

Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

The cellulolytic Clostridium cellulovorans has been engineered to produce n -butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans . First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n -butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.

Chinese hamster ovary (CHO)1 cells have been widely used to express heterologous genes and produce therapeutic proteins in biopharmaceutical industry. Different CHO host cells have distinct cell growth rates and protein expression characteristics. In this study, the expression of about 1,307 host proteins in three sublines, i.e. CHO K1, CHO S and CHO/dihydrofolate reductase (dhfr)−, were investigated and compared using proteomic analysis. The proteins involved in cell growth, glycolysis, tricarboxylic acid cycle, transcription, translation and glycosylation were quantitated using Liquid chromatography tandem-mass spectrometry (LC-MS/MS). The key host cell proteins that regulate the kinetics of cell growth and the magnitude of protein expression levels were identified. Furthermore, several rational cell engineering strategies on how to combine the desired features of fast cell growth and efficient production of therapeutic proteins into one new super CHO host cell have been proposed.

Background: Previous studies show that mitochondrial network excitability, or the propagation of ROS signals, is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial network abnormality, the effect of mitochondrial-targeted antioxidant, such as mitoquinone (MitoQ), on mitochondrial network in pressure overload hearts has not been demonstrated. We hypothesize that MitoQ improves mitochondrial networks in HF via regulation of redox-related cardiac remodeling-associated non-coding RNAs. Methods and results: To test the hypothesis, C57BL/6J mice were subjected to ascending aortic constriction (AAC) to induce left ventricular (LV) pressure overload, followed by 7 days of MitoQ treatment (2 μmol). Doppler echocardiography revealed severe LV dilation and decreased ejection fraction following AAC, which were attenuated by MitoQ. Electron microscopy and immunostaining showed that inter-mitochondrial and mitochondria-sarcoplasmic reticulum (SR) network structure were altered in HF myocardium, in parallel with reduced expression of mitofusin proteins (e.g., MFN1 and MFN2) compared to sham-operated animals. MitoQ blunted mitofusin protein downregulation and improved mitochondrial networks. Our data also identified a MitoQ-mediated mechanism of mitofusin expression in HF by ameliorating the dysregulation of redox-related cardiac remodeling-associated long non-coding RNAs and microRNAs (i.e. Plscr4-miR-214 axis). Conclusion: The present study indicates that MitoQ improves inter-mitochondrial and mitochondrial-SR structural organization in pressure overload hearts by attenuating the dysregulation of cardiac remodeling-associated non-coding RNAs.

Introduction: Calcium cycling plays a critical role in regulating function of cardiomyocytes (CMs) under both physiological and pathological conditions. Among their many important functions, mitochondria are known to involve in calcium handling. However, little is known about the calcium dynamics of human stem cell-derived cardiomyocytes (hiPSC-CM) and what little is known shows much greater variability than what is seen in adult cardiomyocytes. Methods: We produced adenovirus encoding the red Ca 2+ indicator RCaMP prepended with a mitochondrial targeting sequence fused to the green Ca 2+ indicator GCaMP with a cleavable linker. We then used this virus to induce expression of this calcium probe in CMs & recorded calcium activity with a confocal microscope. Results & Discussion: Mitochondrial targeting was confirmed by confocal imaging showing GCaMP (green), mRCaMP (red), and costaining with Mitotracker Deep Red (Magenta) (Figure 1A, 1B). Probe functionality was validated by observing beat-by-beat calcium dynamics in paced neonatal rat CMs with and without blocking mitochondrial calcium uptake with the mitochondrial calcium uniport inhibitor Ru360. Recording GCaMP and mRCaMP dynamics in adult CMs (Figure 1C) showed synchronous beat-by-beat calcium activity in the cytosol and mitochondria. However, iPSC-CMs (Figure 1D) showed variable mitochondrial activity, with some synchronous mitochondria and some showing inverse activity. Conclusions: hiPSC-CMs and adult CMs show differences in beat-by-beat mitochondrial calcium dynamics, offering a potential investigatory target for improving the therapeutic effect of stem cell-derived heart therapies.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs(+RI)) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.