Long noncoding RNA CRART16 confers 5-FU resistance in colorectal cancer cells by sponging miR-193b-5p

Background The emergence of chemoresistance to 5-fluorouracil (5-FU)-based chemotherapy is the main cause of treatment failure in advanced and metastatic colorectal cancer (CRC) patients. Long noncoding RNAs (lncRNAs) have been reported to be involved in 5-FU resistance. Previously, we first detected that lncRNA cetuximab resistance-associated RNA transcript 16 (CRART16) could contribute to cetuximab resistance by upregulating V-Erb-B2 erythroblastic leukemia viral oncogene homologue 3 (ERBB3) expression by sponging miR-371a-5p in CRC cells. The current study aimed to explore the role of CRART16 in acquired 5-FU resistance in CRC cells and its possible mechanism. Methods Quantitative real-time PCR (RT-qPCR) was used to measure the expression levels of CRART16 in a 5-FU-resistant CRC cell subline (SW620/5-FU) and the parent cell line. Lentivirus transduction was performed to establish SW620 and Caco-2 cells stably overexpressing CRART16. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were applied to measure cell chemosensitivity to 5-FU. Flow cytometric and immunofluorescence staining were adopted to assess cell apoptosis induced by 5-FU. The dual-luciferase reporter assay was used to validate the direct interactions between CRART16 and miR-193b-5p and between miR-193b-5p and high-mobility group AT-hook-2 (HMGA2). The expression levels of HMGA2, apoptosis-associated proteins and p-ERK were examined by western blotting. The statistical differences within any two groups were used Student’s t test. Results CRART16 was upregulated in SW620/5-FU cells. Overexpression of CRART16 reduced the sensitivity of CRC cells to 5-FU by attenuating apoptosis. In addition, CRART16 promoted 5-FU resistance by suppressing the expression of miR-193b-5p. Furthermore, CRART16 modulated the expression of HMGA2 by inhibiting miR-193b-5p and activated the MAPK signaling pathway. Conclusions CRART16 confers 5-FU resistance in CRC cells through the CRART16/miR-193b-5p/HMGA2/MAPK pathway.

Regulation of the integrin αVβ3- actin filaments axis in early osteogenesis of human fibroblasts under cyclic tensile stress

Background Integrins play a prominent role in osteogenic differentiation by transmitting both mechanical and chemical signals. Integrin expression is closely associated with tensile stress, which has a positive effect on osteogenic differentiation. We investigated the relationship between integrin αVβ3 and tensile stress. Methods Human fibroblasts were treated with c (RGDyk) and lentivirus transduction to inhibit function of integrin αVβ3. Y-15, cytochalasin D and verteporfin were used to inhibit phosphorylation of FAK, polymerization of microfilament and function of nuclear YAP, respectively. Fibroblasts were exposed to a cyclic tensile stress of 10% at 0.5 Hz, once a day for 2 h each application. Fibroblasts were harvested on day 4 and 7 post-treatment. The expression of ALP, RUNX2, integrin αVβ3, β-actin, talin-1, FAK, vinculin, and nuclear YAP was detected by Western blot or qRT-PCR. The expression and distribution of integrin αVβ3, vinculin, microfilament and nuclear YAP. Results Cyclic tensile stress was found to promote expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation downregulated the rearrangement of microfilament and the expression of ALP, RUNX2 and nuclear YAP. When the polymerization of microfilament was inhibited the expression of ALP, RUNX2 and nuclear YAP were decreased. The phosphorylation of FAK induced by cyclic tensile stress reduced by the inhibition of integrin αVβ3. The expression of ALP and RUNX2 was decreased by inhibition of phosphorylation of FAK and inhibition of nuclear YAP. Conclusions Cyclic tensile stress promotes osteogenesis of human fibroblasts via integrin αVβ3-microfilament axis. Phosphorylation of FAK and nuclear YAP participates in this process.

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

Currently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond simple binding. This situation has impeded the next generation of cancer immunotherapeutics, such as bispecific T cell engager (BiTE) antibodies or agonist antibodies against costimulatory receptors, from reaching their full potential. Here, we developed a highly efficient droplet-based microfluidic platform combining a lentivirus transduction system that enables functional screening of millions of antibodies to identify potential hits with desired functionalities. To showcase the capacity of this system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with two rounds of screening. Furthermore, the versatility of the system was demonstrated by combining an anti-Her2 × anti-CD3 BiTE antibody library with functional screening, which enabled efficient identification of active anti-Her2 × anti-CD3 BiTE antibodies. The platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research.

Different Biological Activities of Histidine-Rich Peptides Are Favored by Variations in Their Design

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences.

PINK1 Activation Attenuates Impaired Neuronal-Like Differentiation and Synaptogenesis and Mitochondrial Dysfunction in Alzheimer’s Disease Trans-Mitochondrial Cybrid Cells

Background: Mitochondrial dysfunction, bioenergetic deficit, and extensive oxidative stress underlie neuronal perturbation during the early stage of Alzheimer’s disease (AD). Previously, we demonstrated that decreased PTEN-induced putative kinase 1 (PINK1) expression is associated with AD pathology in AD-affected human brains and AD mice. Objective: In the present study, we highlight the essential role of PINK1 in AD-relevant mitochondrial perturbation and neuronal malfunction. Methods: Using trans-mitochondrial “cybrid” (cytoplasmic hybrid) neuronal cells, whose mitochondria are transferred from platelets of patients with sporadic AD, we observed the effect of PINK1 in neuronal-like differentiation and synaptogenesis and mitochondrial functions. Results: In AD cybrid cells, the downregulation of PINK1 is correlated to the alterations in mitochondrial morphology and function and deficit in neuronal-like differentiation. Restoring/increasing PINK1 by lentivirus transduction of PINK1 robustly attenuate mitochondrial defects and rescue neurite-like outgrowth. Importantly, defective PINK1 kinase activity fails to reverse these detrimental effects. Mechanistically, AD cybrid cells reveal a significant decrease in PINK1-dependent phosphorylated mitofusin (Mfn) 2, a key mitochondrial membrane protein that participates in mitochondrial fusion, and an insufficient autophagic activity for clearance of dysfunctional mitochondria. Overexpression of PINK1, but not mutant PINK1 elevates phosphorylation of Mfn2 and autophagy signaling LC3-II. Accordingly, PINK1-overexpressed AD cybrids exhibit increases in mitochondrial length and density and suppressed reactive oxygen species. These results imply that activation of PINK1 protects against AD-affected mitochondrial dysfunction and impairment in neuronal maturation and differentiation. Conclusion: PINK1-mediated mitophagy is important for maintaining mitochondrial health by clearance of dysfunctional mitochondria and therefore, improves energy homeostasis in AD.

Utility of SARS-CoV-2 infection models using in vitro and in vivo ACE2-lentivirus transduction

SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. hACE2-lentivirus transduction of C57BL/6J, IFNAR-/- and IL-28RA-/- mice lungs illustrated that removing type I or III interferon responses had no significant effect on virus replication. However, RNA-Seq analyses illustrated they were required for the inflammatory responses, which were similar to those seen in COVID-19 patients. We also illustrate the utility of hACE2 transduction for vaccine evaluation in C57BL/6J mice. In summary, we establish a hACE2-lentivirus mouse model which has broad application in SARS-CoV-2 research, while also offering the inherent advantages of lentiviral transduction such as stable genomic integration.

High throughput functional screening for next generation cancer immunotherapy using droplet-based microfluidics

Currently high throughput approaches are lagged for isolation of antibodies whose function goes beyond simple binding, which have prevented the next generation cancer immunotherapeutics, such as bispecific T cell engager antibodies or agonist antibody of costimulatory receptor, from reaching their full potential. Here we developed a highly efficient droplet-based microfluidics platform combining with lentivirus transduction system that enables functional screening of millions of antibodies. To showcase the capacity of the system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with 2 rounds of screening. To demonstrate its versatility, an anti-Her2/anti-CD3 bispecific antibody library was established using bispecific T cell Engager (BiTE) platform and functional screening enabled efficient identification of potent anti-Her2/anti-CD3 BiTE antibodies. The platform could revolutionize the next generation cancer immunotherapy drug development and research world.

Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19, CD22, or B cell maturation antigen (BCMA). Antigen density is an important factor modulating CAR T cell responses, and therefore antigen expression below a certain threshold may fail to induce optimal CAR-T cell response. This presents a significant problem as antigen expression varies highly in many cancers including lymphoma, leukemia, multiple myeloma, and solid tumors, both between and within patients. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two antigens will result in enhanced CAR T cell signaling, allowing them to attack low antigen density (LAD) tumors that would otherwise escape CAR T cells. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 (FMC63), CD22 (CD22-12), or both, herein referred to as CAR19, CAR22, or CAR19/22, respectively. Tumor cells expressing variegated antigen densities were generated using lentivirus transduction or mRNA electroporation. For in vitro functional characterization, co-culture assay of T cells and tumor cells was performed, and CAR T cell anti-tumor potency was determined by assessing T cell functional parameters including T cell cytotoxicity in real-time using the Incucyte® systems for live-cell imaging and analysis. In addition, cytokine production and CFSE-based proliferation were also assessed using flow cytometry. RESULTS Using CAR19/22, we found LAD tumors are indeed more susceptible to bi-specific CAR T cells compared to either CAR19 or CAR22 T cells, as demonstrated by significantly higher amounts of cytokines produced by bi-specific CAR T cells such as IL-2 (p&lt;0.0001) and IFNg (p&lt;0.0001) compared to CAR19 or CAR22 T cells. Results from cytotoxicity assay revealed increased killing of LAD tumors by CAR19/22 T cells compared to CAR19 (p&lt;0.0001) or CAR22 (p=0.0002) T cells. Further, we found bi-specific CAR19/22 T cells have increased proliferation capacity (p&lt;0.0001) compared to either of the mono-specific CAR T cells. Using NanoString-based RNA analysis, we confirmed that graded antigen density on tumor cells modulates CAR T cell gene expression, with cytotoxicity-associated genes being more susceptible to modulation by antigen expression than cytokine-associated genes. Upcoming experiments aim to test and compare therapeutic efficacy of bi-specific and mono-specific CAR T cells in vivo against LAD tumors. Further characterization of the immune synapse using confocal microscopy as well as analyses of early and late events in downstream CAR signaling will further illuminate the mechanism by which bi-specific CARs can overcome the challenge of low tumor-associated antigen density. CONCLUSIONS Here we showed that antigen density on tumors modulates T cell transcriptional profiles. Our results demonstrated that bi-specific CAR19/22 T cells are superior than mono-specific CAR19 or CAR22 T cells against LAD tumors, as demonstrated by their enhanced cytokine-producing function, cytotoxic capacity and proliferation. Results from this study will provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing the risk of antigen-negative escape. Disclosures Ruella: Abclon, BMS, NanoString: Consultancy; Abclon: Consultancy, Research Funding; UPenn/Novartis: Patents & Royalties. Gill:Tmunity Therapeutics: Research Funding; Sensei: Consultancy; Aileron: Consultancy; Fate: Consultancy; Carisma Therapeutics: Patents & Royalties, Research Funding; Novartis: Research Funding.

Establishment and Application of Engineered NIH 3T3 Cell Line with Stable Human RAGE Expression

Aim NIH3T3 cell line with expression of human receptor for advanced glycation end-products (hRAGE) transduced with lentivirus vectors was used to analyze affinity, biological activity, and/or molecular mechanisms of molecules targeting the hRAGE pathway. Method The DNA fragment coding for hRAGE gene was integrated into the genome of NIH3T3 cells using lentivirus transduction. Cells expressing hRAGE were selected with puromycin, and the level of hRAGE expression was analyzed by Western blot. To establish a stable cell line, colonies of hRAGE-expressing cells were generated, and the level of RAGE expression in each engineered cell line was analyzed within 20 generations. Flow cytometry assay was used to verify affinity of anti-hRAGE antibody binding to hRAGE on the surface of engineered cells. The engineered NIH3T3 cell line was applied to assess effects of anti-hRAGE blocking antibody on amyloid β-induced cells apoptosis by CCK-8 assay. Results The engineered NIH3T3 cell line (hRAGE-NIH3T3) could stably express human RAGE. Commercial anti-RAGE polyclonal antibody could recognize and bind to human RAGE on the surface of hRAGE-NIH3T3 but not original NIH3T3 cells. In addition, hRAGE-NIH3T3 was more sensitive to RAGE pathway-dependent stimulation. Our data show that the hRAGE-NIH3T3 cell line established is an excellent tool in the study of RAGE-targeting molecules based on the cellular level, biological function, and RAGE-mediated molecular mechanisms.