Long-term Assays Research Articles

Advances in clinical chemistry patient-based real-time quality control (PBRTQC).

Patient-Based Real-Time Quality Control involves monitoring an assay using patient samples rather than external material. If the patient population does not change, then a shift in the long-term assay population results represents the introduction of a change in the assay. The advantages of this approach are that the sample(s) are commutable, it is inexpensive, the rules are simple to interpret and there is virtually continuous monitoring of the assay. The disadvantages are that the laboratory needs to understand their patient population and how they may change during the day, week or year and the initial change of mindset required to adopt the system. The concept is not new, having been used since the 1960s and widely adopted on hematology analyzers in the mid-1970s. It was not widely used in clinical chemistry as there were other stable quality control materials available. However, the limitations of conventional quality control approaches have become more evident. There is a greater understanding of how to collect and use patient data in real time and a range of powerful algorithms which can identify changes in assays. There are more assays on more samples being run. There is also a greater interest in providing a theoretical basis for the validation and integration of these techniques into routine practice.

Pilot-Scale Field Demonstration of Environmental Nanotechnology for Groundwater Defluoridation

Fluoride contamination in groundwater is a global issue. Nanoscale oxides of Zr(IV), Al(III), and Ti(IV) can form the inner-sphere complex with fluoride through ligand exchange, offering a new chance for efficient groundwater purification. However, pilot-scale field demonstration of nanotechnology in groundwater defluoridation is very rare. Herein, we conducted a 150-day field defluoridation study using the nanocomposite HZO@D201, which was prepared by encapsulating nano-hydrated zirconium oxide (HZO) inside the strongly basic anion exchanger D201. The HZO@D201 beads were packed in five-stage series columns (40 L for each column), and the groundwater was pumped through the columns sequentially. The effective treatment amount for the single column ([F–] < 1.0 mg/L) exceeded ∼3000 bed volume (BV). The exhausted HZO@D201 was fully refreshed through ex situ treatment with NaOH–NaCl solution for repeated use without significant loss in defluoridation reactivity. Owing to the specific interaction with HZO, the fluoride concentration could be steadily reduced below 1.0 mg/L throughout the 150-day field demonstration, while the common anions coexisting in groundwater (K+, Na+, Ca2+, Mg2+, SO42–, Cl–, etc.) remained almost unchanged. The operational cost for deep defluoridation was estimated as 0.574 RMB per ton of groundwater, much less than that of the widely studied reverse osmosis. TEM, XPS, and Fourier transform infrared (FTIR) analysis indicated that negligible change occurred in the structure and chemical composition of HZO@D201 after the long-term field assay. This study may inspire more attempts at the pilot scale and engineering demonstration of nano-enabled water treatment techniques.

How do Graphene Composite Surfaces Affect the Development and Structure of Marine Cyanobacterial Biofilms?

The progress of nanotechnology has prompted the development of novel marine antifouling coatings. In this study, the influence of a pristine graphene nanoplatelet (GNP)-modified surface in cyanobacterial biofilm formation was evaluated over a long-term assay using an in vitro platform which mimics the hydrodynamic conditions that prevail in real marine environments. Surface characterization by Optical Profilometry and Scanning Electron Microscopy has shown that the main difference between GNP incorporated into a commercially used epoxy resin (GNP composite) and both control surfaces (glass and epoxy resin) was related to roughness and topography, where the GNP composite had a roughness value about 1000 times higher than control surfaces. The results showed that, after 7 weeks, the GNP composite reduced the biofilm wet weight (by 44%), biofilm thickness (by 54%), biovolume (by 82%), and surface coverage (by 64%) of cyanobacterial biofilms compared to the epoxy resin. Likewise, the GNP-modified surface delayed cyanobacterial biofilm development, modulated biofilm structure to a less porous arrangement over time, and showed a higher antifouling effect at the biofilm maturation stage. Overall, this nanocomposite seems to have the potential to be used as a long-term antifouling material in marine applications. Moreover, this multifactorial study was crucial to understanding the interactions between surface properties and cyanobacterial biofilm development and architecture over time.

The RNA Binding Protein Musashi-2 (MSI2) Sustains the Growth and the Leukemogenic Potential of MLL-Rearranged Acute Lymphoblastic Leukemia, and It Is Involved in Glucocorticoid Resistance

Acute Lymphoblastic Leukemia occurring in infants and carrying the rearrangement of KMT2A, aka MLL (MLLr infant ALL) is a rare but very aggressive leukemia typically associated with poor outcome. Patients suffer from a dismal prognosis (overall survival: 20-40%) mainly due to the resistance to conventional therapy and a high incidence of relapse. The biological mechanisms involved in the pathogenesis and disease progression of infant MLLr leukemia are not completely understood and the identification of druggable genes is urgently needed for the development of novel therapeutic strategies. The RNA-binding protein Musashi-2 (MSI2) is a post-transcriptional regulator of mRNA translation to proteins. MSI2 has a pivotal role in normal hematopoiesis and in a variety of tumors, including leukemia, where it is involved in cell proliferation, differentiation and maintenance of the (cancer) stem cell pool. Although several studies strikingly demonstrated the involvement of MSI2 in chronic and acute myeloid leukemia, with regards to B-ALL, few papers reported over-expression of MSI2 associated with a poor outcome in both pediatric and adult B-ALL patients; however functional data are still missing. By retrospectively analyzing the gene expression profile of a large cohort of Italian patients with pediatric leukemia (n=314) we observed that MSI2 is highly expressed across the different cytogenetic subgroups, and the level of expression is even higher in ALL compared to AML cases; thus reinforcing the hypothesis that MSI2 might have a crucial role also in ALL. To unravel the functional role of MSI2 in MLL-r ALL, we successfully generated a model of MSI2 knock-out (MSI2 KO) using CRISPR/CAS9 genome editing in the MLL::AF4+ ALL cell line SEM. In a long-term competitive assay in vitro, in which the MSI2 KO and CNTRL cells were co-cultured at the initial 90:10 ratio (KO:CNTRL), we observed that after 20 passages in culture the CNTRL counterpart overgrew the MSI2 KO cells (which indeed extinguished); thus demonstrating that the lack of MSI2 leads to a proliferation disadvantage. In agreement with this, MSI2 KO cells show an impaired leukemia-initiating capacity and a lower disease burden in vivo, and mice transplanted with MSI2 KO cells display a prolonged survival compared to controls. Also, we performed a high-throughput drug screening using a manually curated library of 174 compounds (FDA-approved, currently used in the clinic) and we observed that MSI2 KO cells show a marked sensitivity to Glucocorticoids (GCs). Further validation studies in vitro demonstrated that MSI2 KO cells were significantly (100-fold) more sensitive to Prednisolone or Dexamethasone than their MSI2-expressing counterpart. In addition, the treatment of SEM cells (typically resistant to GCs) with the MSI2 inhibitor Ro 08-2750 phenocopied the sensitization to GCs observed in CRISPR-edited MSI2 KO cells, showing synergistic effects in combination with Prednisolone or Dexamethasone. As infant MLLr ALL patients are typically resistant to GCs, our findings suggest that targeting MSI2 might represent a potential novel approach to circumvent the therapy resistance. Finally, by further investigating the biological mechanisms of GC-sensitization, we unraveled a potential role for MSI2 in the regulation of the energetic metabolism of leukemic cells. Indeed, the confocal analysis of mitochondrial membrane potential and the profiling of cellular bioenergetics by Seahorse technology revealed that MSI2 KO cells display mitochondrial hyperactivity at steady-state compared to controls. Upon GCs exposure (which inhibits glycolysis in both MSI2 KO and CNTRL cells) mitochondrial respiration is increased in CNTRL cells to compensate ATP production, while it is severely impaired in MSI2 KO, leading to a mitochondrial crisis and energy failure. The importance of metabolic plasticity of tumor cells in adaptation to stress conditions is emerging as a crucial aspect of cancer biology. The putative role of MSI2 in regulating the energy metabolism of leukemia is a novel finding not previously investigated, which may pave the way for targeting the metabolic vulnerabilities of ALL as a novel therapeutic approach. In conclusion, our study demonstrates that MSI2 sustains the growth in vitro and the leukemogenic potential in vivo of MLLr ALL cells, and it is involved in GC-resistance by regulating the energy metabolism of leukemia.

Co-Stimulation of CD28/CD40 Signaling Molecule Potentiates CD19CAR-T Cell Functions and Stemness

Introduction: Adoptive T-cell therapy of gene-modified to express CD19 chimeric antigen receptor (CD19CAR) has achieved promising clinical outcomes in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied including signaling domain modification to improve T-cell efficacy. The nuclear factor-κB (NF-κB) which interestingly exists in both T- and B-cells is a crucial signaling pathway for T-cell persistence. Furthermore, CAR-T memory signatures drive robust T-cell proliferation. Consequently, we developed the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to enhance CAR-T cell proliferation and tumoricidal activity. Methods: The anti-CD19CAR incorporated CD28/CD40 co-stimulatory domain; CD19scFv-CD28TM-CD28/CD40-CD3z-tEGFR, was generated. The CAR gene was subsequently cloned into retroviral packaging cells and transduced into primary CD3 cells. The CAR+-T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. These expanded cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells. Results: All CD19CAR genes were successfully transduced into primary CD3cells. We first determined the downstream T-cell signaling and found the higher baseline pNF-kB and pRelB of CD19.28.40z CAR-T cells compared to others. We then investigated the ability of CD19 CAR T-cell proliferation by coculturing with gamma-irradiated CD19-expressing K562 cells. Surprisingly, the novel CD19.28.40z exhibited robust T-cell proliferation and persistence throughout 2 weeks of the culture regardless of IL-2 supplementation. The long-term co-culture assay unveiled that CD19.28.40z CAR-T cells could effectively eradicate tumor cell growth until the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells. In term of cytokine secretion assays, the similar level of IL-2, TNF-a, and IFN-g concentrations were observed among CAR constructs after being stimulated with target cells. To mimic persistent antigen stimulation in vivo, we performed three consecutive weekly antigen stimulation that exhibited the greater CD19.28.40z CAR-T cell expansion without influencing T-cell fate and exhaustion. We interrogated the intrinsic genes underlying CD19.28.40z CAR-T cell responses compared to conventional CAR-T cells following CD19 antigen exposures using RNA-sequencing. The results confirmed the significant up-regulated genes associated with T-cell proliferation and differentiation, NF-κB pathway, WNT pathway, and CD4+ phenotypes as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway (Figure 1). Regarding T-cell memory phenotypes, we observed the enrichment of genes toward T-cell stemness particularly SELL, IL-7r, TCF7, and KLF2 compared to conventional CAR-T cells. To evaluate the potential anti-tumor efficiency in vivo, we studied NALM-6- and Raji-bearing NSG mice treated with a low CD19CAR-T cell dosage to assess the functional limits of different CAR structures. The CD19.28.40z CAR-T cells demonstrated potent tumoricidal activity as well as prolonged overall survival in both NSG mice models compared to control CAR-T cells (Figure 2). Conclusions: The CD28/CD40 costimulatory molecule potentiates NF-κB signaling and memory signatures enhancing CD19CAR-T cell proliferation and persistence which translates into greater anti-tumor efficacy in both in vitro and xenograft models. The simplified structural modification of dual T-/B-cell signaling molecule could be advantageous to maximize the CAR-T cell functions and stemness. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract PR019: Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence

Abstract Background: KRAS mutations drive tumorigenesis in &amp;gt;90% of pancreatic ductal adenocarcinomas (PDAC). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with metastatic KRAS-mutated solid tumors including PDAC. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. TCR therapies utilizing HLA class I-restricted TCRs often fail to harness CD4+ T cell activity vital for sustained anti-tumor immune responses. Here, we characterize HLA class I-restricted TCRs isolated from healthy donors and vaccinated patients targeting KRAS G12D or G12V, the most prevalent mKRAS variants observed in PDAC tumors. Methods: mKRAS-specific TCR sequences were identified from healthy donors or vaccinated PDAC patients enrolled on IRB-approved clinical studies at the University of Pennsylvania. Transgenic expression of synthetic TCRs was attained via lentiviral transduction of reporter cell lines or gene-edited (TCRabnull) primary CD8+ or CD4+ T cells. Limiting peptide dilution and combinatorial peptide library assays were used to assess antigen specificity, affinity and cross-reactivity. TCR reactivity to mKRAS cell lines was assessed by FACS analysis, in vitro cytotoxicity assays (4h 51Cr-release, xCELLigence Real Time Cell Analysis), and mouse xenograft tumor models. Results: TCRs exhibit specificity to the cognate KRAS G12D or G12V epitope without cross-reactivity to wild-type KRAS. TCRs are of high affinity and promote cytolysis of HLA-matched tumor cell lines harboring endogenous KRAS mutations irrespective of histologic origin. The adoptive transfer of gene-edited (TCRabnull) primary CD8+ T cells expressing synthetic TCRs exhibit potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. Select TCRs retain functional avidity independent of the CD8 co-receptor as determined by multimer binding and limiting peptide dilution assays. Disruption of the CD8:HLA binding interface via the introduction of D227A and T228A mutations within the HLA a3 domain has limited to no effect on TCR-mediated CD8+ T cell cytolytic activity. Transgenic expression of co-receptor independent TCRs by CD4+ T cells confers cytolytic activity against KRAS-mutated cell lines in both short- and long-term cytotoxicity assays, and flow cytometric analysis confirms antigen-specific expression of CD107a, IFN-g, TNF-a and IL-2 in a subset of CD4+ T cells. Conclusion: This study identifies TCRs with high therapeutic potency for the clinical development of TCR therapy trials targeting mKRAS in PDAC patients. Select TCRs exhibit CD8 co-receptor independence. CD4+ T cells redirected with co-receptor independent TCRs exhibit cytotoxic activity and express Th1 cytokines which may serve to enhance the persistence and anti-tumor activity of adoptive TCR therapy targeting mKRAS. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR019.

Development of an Off-the-Shelf Chimeric Antigen Receptor (CAR)-T Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL) without Gene Editing

CAR-T cell therapy has revolutionized the treatment of B-lineage ALL and other B-cell malignancies, but the development of cell therapies for T-cell malignancies has lagged behind. For T-ALL, CD7 is an excellent CAR target because it is consistently and highly expressed in leukemic cells and is retained during chemotherapy and at relapse. However, CD7 is also expressed in normal T-cells and the expression of anti-CD7 CARs triggers T-cell fratricide, resulting in a marked decrease in viable and/or effective CAR-T cells. We developed an anti-CD7 CAR-T cell product (PCART7) by utilizing a CD7 protein expression blocker (PEBL) to downregulate CD7 surface expression and prevent fratricide. PEBLs consist of a target binding domain fused to an intracellular retention domain that enables relocalization of surface proteins intracellularly (Png et al. Blood Adv 2017; Kamiya et al. Blood Adv 2018; Kamiya et al., JCI 2019). Autologous PCART7 cells lacking surface CD7 and expressing anti-CD7 CARs can be effectively produced under cGMP conditions and the technology is being tested in clinical trials for patients with relapsed/refractory T-ALL or T-cell lymphoblastic lymphoma. Generation of autologous CAR-T cells, however, may be hampered by the presence of a high percentage of leukemic cells in peripheral blood and/or low T cell numbers. An allogeneic anti-CD7 CAR-T cell product available "off-the shelf" would represent a useful tool to reduce leukemic burden and provide a bridge to hematopoietic cell transplant or to autologous CAR-T cell therapy. Because allogeneic T-cells can be activated upon recognition of host tissue antigens by their T-cell receptor (TCR)/CD3 complex, their infusion carries a risk of causing graft-versus-host disease (GvHD). We optimized a CD3 PEBL that can effectively downregulate surface TCR/CD3. We then developed a lentiviral-based process that allows simultaneous expression of CD3 and CD7 PEBLs together with anti-CD7 CAR. The PEBLs retained surface CD7 and TCR/CD3 intracellularly, thereby minimizing fratricide and abrogating TCR-mediated antigen recognition. PCART7 cells lacking TCR/CD3 were manufactured from healthy donor T-cells by using a double transduction with a bicistronic lentiviral vector delivering CD7 PEBL and anti-CD7 CAR (PCART7 LVV), and a second lentiviral vector delivering CD3 PEBL. Robust expansion of PCART7-CD3 PEBL cells was observed and more than 90% of the cells were of the desired CAR+CD7-CD3- phenotype. Following depletion of residual CD3/TCRαβ+ cells, CD3- cell purity was >99% (Fig. 1A). These results were reproduced across multiple donors. The efficient downregulation of surface TCR/CD3 by CD3 PEBL was similar to that achieved by CRISPR knockout of the TCR alpha chain locus (TRAC) and completely abrogated activation in response to TCR stimulation. In vitro short- and long-term assays confirmed strong cytotoxic potency against CD7+ leukemic cells but not CD7- target cells. Importantly, PCART7 cells were equally cytotoxic regardless of whether they were co-transduced with CD3 PEBL. In a xenograft model using the CCRF-CEM T-ALL cell line, PCART7-CD3 PEBL cells, like PCART7 cells, effectively suppressed tumor growth and improved survival (Fig. 1B). This study demonstrates the feasibility of using two PEBLs simultaneously for intracellular protein retention without perturbing critical CAR-T cell functions. The use of PEBL technology to generate allogeneic anti-CD7 CAR-T cells fits seamlessly in cGMP manufacturing protocols and avoids the risk of genotoxicity arising from the use of gene-editing techniques. Because the resulting allogeneic anti-CD7 CAR-T cells had high potency and were devoid of alloreactivity potential, they represent an attractive new tool for off-the-shelf CAR-T cell therapy of T-ALL and other CD7+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Short- and long-term single and combined effects of microplastics and chromium on the freshwater water flea Daphnia magna

In this study, we investigated the individual and combined effects of microplastics (MPs) and chromium (Cr) on the freshwater water flea Daphnia magna by measuring mortality, bioaccumulation, antioxidative response, multixenobiotic resistance activity, and sestrin-related mitochondrial biogenesis in short-term assays and in vivo endpoints including reproduction and adult survival rate in long-term assays. Exposure to MPs, Cr, and their combination caused significant deleterious effects and acute toxicity in D. magna. Alterations in oxidative stress occurred in the groups treated with MPs and Cr alone and together. However, upon co-exposure to MPs, the Cr concentration, measured by inductively coupled plasma optical emission spectroscopy, decreased, suggesting that MPs and Cr interact with each other. Based on enzymatic activities, we noted a decrease in MP egestion via inhibition of P-glycoprotein activity in the MP-exposed groups, and multidrug resistance–associated protein activity increased in some of the MP-exposed animals depending on Cr concentration. On the other hand, MP exposure seemed to lead to mitochondrial transcription dysfunction induced by Cr via sestrin-related mitochondrial biogenesis. Overall, these results indicate that co-exposure to MPs and Cr causes acute toxicity in D. magna but lacks the chronic toxicity (21 days) and mitochondrial dysfunction caused by Cr exposure alone.

The Use of 3D Optical Coherence Tomography to Analyze the Architecture of Cyanobacterial Biofilms Formed on a Carbon Nanotube Composite.

The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.

Efficiency of ant-control agents in colony-level oral toxicity tests using Tetramorium tsushimae (Hymenoptera: Formicidae) for post-establishment control of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae)

The red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) causes serious damage worldwide as an invasive alien species. The species has expanded its range to the Pacific Rim since 2000s and Japan has faced its multiple introductions since 2017. While colony-level control methods are urgently needed, testing living colonies of the unestablished species is challenging especially due to various restrictions under the COVID-19 pandemic. Here, we proposed alternative long-term toxicity assays using artificial colonies of Tetramorium tsushimae Emery (Hymenoptera: Formicidae), a Japanese native species belonging to the same subfamily (Myrmicinae) as S. invicta. We conducted an acute toxicity test to determine if T. tsushimae is a suitable substitute for S. invicta using fipronil and found the LD50 value in T. tsushimae was close to that in S. invicta. Then, we conducted the long-term toxicity test with fipronil and two insect growth regulators (pyriproxyfen and etoxazole) using artificial colonies of T. tsushimae. All workers and larvae in the fipronil-treated colonies died within 3 days of treatment initiation. Emergence of new workers was observed after 18 days in the etoxazole-treated and control colonies, but not in the pyriproxyfen-treated colonies. We concluded that fipronil was the most promising insecticide for post-establishment control, and pyriproxyfen was effective as a toxic-bait agent for colony-level control.

Short and long-term effects of nanobiomaterials in fish cell lines. Applicability of RTgill-W1

Nanobiomaterials (NBMs) are nanostructured materials for biomedical applications that can reach aquatic organisms. The short and long-term effects of these emerging contaminants are unknown in fish. The RTgill-W1 cell line has been proposed as a model to predict the acute toxicity of chemicals to fish (OECD Test Guideline nº 249). We assessed the applicability of this cell line to study the short and long-term toxicity of 15 NBMs based on hydroxyapatites (HA), lipid (LSNP/LNP), gold, iron oxide, carbon, poly l-Lactide acid (PLLA) fibers with Ag and poly (lactide-co-glycolide) acid. Two more rainbow trout cell lines (RTL-W1, from liver, and RTS-11, from spleen) were exposed, to identify possible sensitivity differences among cells. Exposures to a range of concentrations (0.78–100 μg/mL) lasted for 24 h. Additionally, the RTgill-W1 was used to perform long-term (28 d exposure) and recovery (14 d exposure/14 d recovery) assays. Cells were exposed to the 24 h-IC20 and/or to 100 μg/mL. A triple cytotoxicity assay was conducted. After 24 h, only PLLA Fibers-Ag showed cytotoxicity (IC50 < 100 μg/mL). However, the NBMs in general provoked concentration-dependent effects after long-term exposures, except the LSNPs. A recovery of viability was only observed for AuNPs, AuNRods, Fe3O4PEG-PLGA, MgHA-Collag_Scaffolds, Ti-HA and TiHA-Alg NPs.These results evidenced the need to test the long-term toxicity of NBMs and showed differences in cytotoxicity probably associated to different mechanisms of toxic action. The RTgill-W1 was useful to screen short and long-term toxicities of NBMs and appears as a promiseful model to assess possible toxicity of NBMs in fish.

Evaluation of 3,5-Diphenyl-2-Pyrazolines for Antimitotic Activity by Inhibition of Tubulin Polymerization

Chalcones have a skeleton of diphenyls connected via an α,β-unsaturated carbonyl group. Many chalcone derivatives have been shown to interact with tubulin. Based on previous reports, chalcones derived by substitution of a carbonyl group with 2-pyrazoline can be expected to inhibit tubulin polymerization. Therefore, 3,5-diphenyl-2-pyrazolines were prepared to investigate their ability to inhibit tubulin polymerization. The clonogenic long-term survival assay showed that derivative 4, 5-(3,5-dimethoxyphenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, was the most effective at inhibiting the clonogenicity of HCT116 human colon cancer cells. Derivative 4 induced G2/M cell cycle arrest. In addition, derivative 4 caused dispersed microtubules, a disorganized spherical arrangement of chromosomes, and inhibition of mitotic spindle formation. The binding mode between tubulin and derivative 4 was elucidated by in silico molecular docking. Derivative 4 was superimposed with colchicine and entered the colchicine-binding site well. These results suggest that derivative 4 inhibits tubulin polymerization by binding to the colchicine binding site of tubulin, thus preventing mitotic spindle formation during mitosis in HCT116 colon cancer cells. We propose that derivative 4 could be used as a promising antimitotic chemotherapeutic agent.

OS08.8.A Integrin-specific CAR T cells for the treatment of glioblastoma

Abstract Background Current standard of care for glioblastoma patients has limited therapeutic efficacy and novel innovative treatment strategies are urgently needed. One such strategy is chimeric antigen receptor (CAR) T cell therapy that has shown great success in hematological malignancies. αv integrins are overexpressed in several neoplasms and have already been used as therapeutic targets for small molecule inhibitors and antibodies, which did not cause major toxicities. Herein, we propose αv integrins as an ideal target for CAR T cell therapy in glioblastoma. Material and Methods CAR T cells targeting specific heterodimers (αvβ3, αvβ5 or αvβ8) were generated by transducing primary human T cells from healthy donors with a lentiviral vector expressing a second-generation CAR. Activity and specificity of CAR T cells was determined by co-culture assays with different glioma cells. Efficacy of CAR T cells to control tumor growth in vivo was investigated in clinically relevant orthotopic xenograft glioma mouse models. Additionally, we generated CAR T cells from T cells from a glioblastoma patient and measured their activity against the patient’s autologous tumor cells. Results All newly generated integrin-targeting CAR T cells exerted strong anti-glioma activity in vitro. Long-term and repetitive killing assays as well as cytokine-release measurements demonstrated highest activity of αvβ5 and αvβ8 integrin-specific CAR T cells. Antigen specificity of these cells was confirmed, as glioma cells with a CRISPR/Cas9-mediated knockout of the target antigen were resistant to CAR T cell-mediated cytotoxicity. Intratumoral injection of αvβ5 or αvβ8 CAR T cells significantly prolonged the survival and cured a substantial fraction of glioma-bearing mice in two different xenograft models. When used in a patient-derived setting, matched CAR T cells exerted strong anti-glioma activity. Conclusion We show strong and integrin-specific anti-glioma activity of CAR T cells developed from healthy donor T cells and glioblastoma-patient-derived T cells in vitro. αvβ5- and αvβ8-specific CAR T cells exerted the best therapeutic activity in two different xenograft glioma models in vivo. These data support the evaluation of integrin-specific CAR T cells as a therapeutic strategy in clinical neuro-oncology.

Comparing different surface modifications of zinc oxide nanoparticles in the developmental toxicity of zebrafish embryos and larvae

Nanotechnology allows for a greater quality of life, but may also cause environmental and organismic harm. Zinc oxide nanoparticles (ZnONPs) are one of the most commonly used metal oxide nanoparticles for commercial and industrial products. Due to its extensive use in various fields, there has already been much concern raised about the environmental health risks of ZnONPs. Many studies have investigated the toxicological profile of ZnONPs in zebrafish embryonic development; however, the specific characteristics of ZnONPs in zebrafish embryonic/larval developmental damage and their molecular toxic mechanisms of liver development are yet to be fully elucidated. This study aimed to reveal the hazard ranking of different surface modifications of ZnONPs on developing zebrafish and the toxicological mechanisms of these modified ZnONPs in liver tissue. The ~30 nm ZnONPs with amino- (NH2- ZnONPs) or carboxyl- (COOH-ZnONPs) modification were incorporated during the embryonic/larval stage of zebrafish. Severe toxicity was observed in both ZnONP groups, especially NH2-ZnONPs, which presented a higher toxicity in the low concentration groups. After prolonging the exposure time, the long-term toxicity assay showed a greater retardation in body length of zebrafish in the NH2-ZnONP group. Response data from multiple toxicity studies was integrated for the calculation of the EC50 values of bulk ZnO and ZnONPs, and the hazard levels were found to be decreasing in the order of NH2-, COOH-ZnONPs and bulk ZnO. Notably, NH2-ZnONPs induced ROS burden in the developing liver tissue, which activated autophagy-related gene and protein expression and finally induced liver cell apoptosis to reduce liver size. In conclusion, our findings are conducive to understanding the hazard risks of different surface modifications of ZnONPs in aquatic environments and will also be helpful for choosing the type of ZnONPs in future industrial applications.

Table olive elicits antihypertensive activity in spontaneously hypertensive rats.

Table olives are a food with a high content of bioactive compounds with cardioprotective properties, such as oleic acid, polyphenols, and pentacyclic triterpenes. Here, we investigate the effect of the intake of table olives on blood pressure (BP) and body weight in spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar Kyoto (WKY) rats. 'Arbequina' table olives (3.85 g kg-1 ) were administered by gavage to SHR and WKY rats in short-term (1day) and long-term (7 weeks) experiments. BP was measured by the tail-cuff method, and polyphenols and triterpenes were determined in olives and plasma by liquid chromatography-mass spectrometry. Administration of 'Arbequina' olives to WKY rats did not exert any change in BP in any of the experiments. However, in SHR, the single dose induced a transient reduction in BP of approximately 15 mmHg, from the second to the tenth hour after the administration. In the long-term assay, a similar decrease was established in the second week and was maintained throughout the experiment. Moreover, the daily administration of olives to rats did not affect their body weight when compared with controls in either the WKY rats or SHR. The determination of polyphenols and triterpenes in plasma indicated that, at the end of the experiment, only maslinic acid, oleanolic acid, hydroxytyrosol, and luteolin were found, all of them being compounds with already described capacity to decrease BP. The results suggest that the daily intake of table olives could decrease BP in hypertension without affecting body weight, indicating that table olives could contribute to improving cardiovascular health. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Toxicity mitigation and biodistribution of albumin corona coated graphene oxide and carbon nanotubes in Caenorhabditis elegans

In this work, the toxicity and biodistribution of graphene oxide (GO) and oxidized multi-walled carbon nanotubes (MWCNT) were investigated in Caenorhabditis elegans. Bovine serum albumin (BSA) was selected as a model protein to evaluate the influence of protein corona formation on materials physicochemical properties, colloidal stability, and toxicity. Biological assays were performed to assess the effects of bare and albumin corona coated materials on survival, oxidative stress, intestinal barrier permeability, growth, reproduction, and fertility. Critical alterations in topography, surface roughness and chemistry of GO and MWCNT were observed due to albumin corona formation. These modifications were associated with changes in colloidal stability of materials and prevention of their aggregation and sedimentation in nematode testing medium. Both GO and MWCNT caused damage to nematode survival, growth, reproduction, and fertility, as well as enhanced oxidative stress and permeability of the intestinal barrier. But GO was more toxic than MWCNT to C. elegans, especially at long-term assays. Albumin corona mitigated 100% of acute and chronic effects of MWCNT. In contrast, the negative effects of GO were not completely mitigated; GO inhibited 16.2% of nematode growth, 86.5% of reproduction, and 32.0% of fertility at the highest concentration evaluated (10 mg L−1), while corona coated GO mitigated 50% and 100% of fertility and growth, respectively. Confocal Raman spectroscopy imaging was crucial to point out that bare and albumin corona coated GO and MWCNT crossed the C. elegans intestinal barrier reaching its reproductive organs. However, BSA corona protected the nematodes targeted organs from negative effects from MWCNT and blocked its translocation to other tissues, while coated GO was translocated inside the nematode affecting the functionality of crucial organs. In addition, coated MWCNT was excreted after 2 h of food resumption, whereas coated GO still accumulated in the nematode intestine. Our results demonstrate that the materials different translocation and excretion patterns in C. elegans had a relation to the impaired physiological functions of primary and secondary organs. This work is a contribution towards a better understanding of the impacts of protein corona on the toxicity of graphene oxide and carbon nanotubes; essential information for biological applications and nanosafety.

Joint action of binary mixtures based on parameter k·ECx from concentration-response curves in long-term toxicity assay

A previous acute toxicity study of binary mixtures showed that the combined toxicity can be predicted with the parameter k∙ECx. To systematically investigate the ability of k∙ECx to predict the chronic combined toxicity of binary mixtures, the toxicity of six contaminants and five binary mixtures was determined by long-term microplate toxicity analysis (L-MTA) using Aliivibrio fischeri as the test organism. The independent action model (IA) and the relative model deviation ratio (rMDR) were employed to determine the relationship between the Δ(k∙ECx)% and rMDRx. The results showed that these two factors conformed to the exponential function in long-term toxicity. Owing to the time-dependence of toxicity, the mixture type of chronic toxicity changes to the relative type of acute toxicity. If the acute toxicity of binary mixtures changes their mode of joint action throughout the concentration range, the chronic toxicity will also change their mode of joint action, and vice versa. This study clarified the change rules of the joint action of binary mixtures in acute and chronic toxicity which can promote research on chronic toxicity of binary mixtures.

P1400: BOOSTING MITOCHONDRIAL RECYCLING REJUVENATES HEMATOPOIETIC AND IMMUNE SYSTEMS

Background: Hematopoietic stem cells (HSCs) generate all blood lineages and ensure the correct homeostasis between myeloid and lymphoid lineages during the entire life of an organism. However, HSCs decrease their regenerative potential and undergo toward a myeloid-biased fate skewing with age. This phenomenon leads to a reduction of B and T cell compartments and immune dysfunction. Consequently, elderly patients are more susceptible to severe viral and fungal infections. It was reported that HSC aging is associated with defective autophagy and accumulation of damaged mitochondria. And emerging studies showed that dysregulation of mitochondria metabolism correlates closely with hematopoietic aging process. However, the clear causal relationship between mitochondrial metabolism and HSC aging remains unknown. Aims: We were wondering if metabolic modulator can rejuvenate aged HSCs, rescue loss-of-function of aged HSCs and improve overall immune response. Methods: We used mito-QC reporter mice and mitochondrial stain to analyze mitochondrial network upon in vitro treatment of metabolic modulator. In vivo long-term blood reconstitution assays and LCMV infection model were used to estimate the effect of metabolic modulator on HSC functionality. Results: Here we reported that interventions aimed to modulate mitochondria recycling and metabolism can rejuvenate aged HSCs and thus preserving the lymphoid compartments and immune function. In vitro administration of the mitophagy inducer Urolithin A on aged HSCs restore their blood and immune reconstitution capability. Moreover, Urolithin A supplemented in the mouse diet, improves bone marrow functionality, expands the lymphoid compartment and boost immune response against viral infection in old mice. Finally, we discovered that HSCs rejuvenation is associated with mitophagy induction and re-establishment of mitochondria homeostasis and fitness in aged HSCs. Summary/Conclusion: In summary, we identified mitophagy as potent regulator of aging process and as valuable target for rejuvenating approaches in HSCs.

Abstract 4190: Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma

Abstract Resistance to CAR-T cell therapy through antigen loss and clonal heterogeneity is a major limiting factor in achieving durable responses, even in highly persistent cell therapies. Simultaneous targeting of multiple tumor antigens with a single therapeutic modality offers the potential to treat heterogeneous tumor populations, prevent antigen escape, and induce durable clinical remission. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of multiple myeloma (MM) using an off-the-shelf induced pluripotent stem cell (iPSC) derived NK cell platform. The iPSC background has been functionally enhanced and can be combined with therapeutic antibodies. A CAR targeting B cell maturation antigen (BCMA), a well-defined TAA in MM, was designed utilizing a previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). This CAR was combined with a companion CAR targeting the pan-TAAs, MICA and MICB. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding by blocking the putative cleavage site (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against a broad range of tumor types. To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression of BCMA and MICA/B on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden the targeting approach of malignant plasma cells. In a reductionist approach, dual CAR-iNK cells exhibited antigen-specific activation, degranulation and cytotoxicity against a Nalm6 target line constitutively expressing the BCMA and MICA/B. Similar trends were observed in a series of long-term cytotoxicity assays against several MM lines, consistent with antibody staining on target cells, illustrating that co-targeting of MICA/B and BCMA expands the breadth of coverage against MM. Known modulators of antigen expression were tested for their ability to provide further depth of response, and therapeutic antibodies such as anti-CD38 were tested in combination to exploit the non-cleavable CD16 and CD38KO edits in the iPSC backbone. These combinations were further tested in vivo against a disseminated model of multiple myeloma where BCMA/MICA dual CAR-iNK cells demonstrated superior tumor control relative to single-CAR controls, and TGI was augmented with the addition of Daratumamab. These data highlight the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Citation Format: Ketan Mathavan, John Reiser, Sajid Mahmood, Yijia Pan, Bryan Hancock, Robert Blum, Wen-I Yeh, Andrew Houk, Chia-Wei Chang, Tom Lee, Bobby Goulding, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Uta Hoepken, Armin Rehm, Kai Wucherpfennig. Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma [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 4190.

Abstract 1300: Targeted therapies prime lung cancer cells for macrophage-mediated destruction

Abstract The CD47/SIRPa axis is an immune checkpoint that regulates macrophage anti-tumor function. Therapies that block CD47 on cancer cells show promise in clinical trials for solid and hematologic malignancies, particularly when combined with other anticancer agents. However, the best combination strategies for using CD47-blocking therapies remain unknown. In this study, we developed a novel in vitro screening platform to identify drugs that render cancer cells more vulnerable to macrophage attack. We performed an unbiased screen of 800 FDA-approved drugs using primary human macrophages and PC9 cells, a human EGFR mutant lung cancer cell line. We identified EGFR tyrosine kinase inhibitors (TKIs) as drugs that act on the cancer cells and specifically enhance macrophage-mediated cytotoxicity (&amp;gt;4-fold enhancement, p&amp;lt;0.01). In contrast, conventional chemotherapy drugs either showed no significant enhancement or abrogated macrophage activation. The combination of EGFR TKIs with anti-CD47 antibodies elicited maximal phagocytosis across a range of cell lines and conditions. In long-term co-culture assays with macrophages, the combination of EGFR TKIs and anti-CD47 antibodies eliminated persister cells to prevent TKI resistance. Importantly, these findings extended to lung cancers with other RTK-MAPK pathway mutations, such as ALK-rearranged cancers (treated with lorlatinib, alectinib, or crizotinib) or KRAS G12C mutant cancers (treated with sotorasib or adagrasib). In xenograft and syngeneic mouse models, the combination of targeted therapies with CD47 ablation was able to dramatically reduce tumor burden. To understand the mechanism of synergy, we generated 8 different lung cancer cell lines resistant to EGFR, ALK, or KRAS inhibitors. The resistant lines significantly upregulated CD47 and concomitantly became more sensitive to macrophage attack in vitro and in vivo. By RNA sequencing, we identified multiple mechanisms contributing to vulnerability, including secretion of the cytokine MIP-3 by the cancer cells and alteration of other immunoregulatory molecules. Overall, we have identified a novel therapeutic strategy to enhance the efficacy of RTK-MAPK pathway inhibitors by combining them with anti-CD47 therapies. Our findings suggest cross-sensitivity occurs such that lung cancer cells that become resistant to targeted therapies also become more sensitive to macrophage attack. Our findings provide rationale for testing this combination approach in patients with lung cancers bearing driver mutations. Citation Format: Kyle Vacarro, Juliet Allen, Asaf Maoz, Sarah Reeves, Aaron Hata, Kipp Weiskopf. Targeted therapies prime lung cancer cells for macrophage-mediated destruction [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 1300.