In Vivo Imaging System Research Articles

In Vivo Imaging of Nipah Virus Infection in Small Animal Rodent Models.

In vivo imaging system (IVIS) is a powerful tool for the study of infectious diseases, providing the ability to non-invasively follow viral infection in an individual animal over time. Recombinant henipaviruses expressing bioluminescent or fluorescent reporter proteins can be used both to monitor the spatial and temporal progression of Nipah virus (NiV) infection in vivo as well as in ex vivo tissues. Virally produced luciferases react with systemically administered substrate to produce bioluminescence that can then be detected via IVIS imaging, while fluorescent reporters inherently generate detectable fluorescence without a substrate. Here we describe protocols applying bioluminescent or fluorescent reporter expressing recombinant viruses to in vivo or ex vivo imaging of NiV infection.

Establishing Brain Tumor Stem Cell Culture from Patient Brain Tumors and Imaging Analysis of Patient-Derived Xenografts.

Brain tumor stem cells (BTSCs) have been isolated from different types of brain tumors including glioblastoma. Although BTSCs share common characteristics with neural stem cells (NSCs), such as capacity to self-renew and undergo long-term proliferation, they have tumor-propagating capabilities. A small population of BTSC can give rise to secondary tumor when transplanted into severe immunodeficient (SCID) mice. The histological and cytological features, as well as genetic heterogeneity of the xenografted tumors in mice, closely resemble those of primary tumors in patients. Patient-derived xenografts (PDX), therefore, provide a clinically relevant model to study brain tumors. Here, we describe our protocol for establishing BTSC cultures following surgical excision of human brain tumors and the procedures to conduct PDX studies in SCID mice. We also provide our detailed step-by-step protocol on in vivo imaging system (IVIS) of the PDX tumors as a noninvasive method to trace the cells and tumor volume.

Efficient delivery of VEGF-A mRNA for promoting diabetic wound healing via ionizable lipid nanoparticles

Diabetes is often accompanied by chronic non-healing wounds, and vascularendothelial growth factor A (VEGF-A) is crucial in the treatment of chronic diabetic wounds. However, the application of VEGF-A protein in clinic is limited due to poor absorption and short half-life of protein macromolecule. Herein, we employed an emerging protein replacement therapy by delivering VEGF-A mRNA into the body to express the desired protein to accelerate diabetic wound healing. Primarily, VEGF-A mRNA was synthesized by an in vitro transcription (IVT) method and encapsulated with an ionizable lipid-mediated nanoparticles (LNP) delivery system via a microfluidic method. The resultant LNP/VEGF-A mRNA were characterized by using dynamic light scattering (DLS) and transmission electron microscope(TEM). The nanoparticles have regular spherical morphology with an average particle size of 101.17 nm, a narrow polydispersity (PDI) of 0.17 and negative Zeta potential of −3.05 mV. The bioactivities of the nanoparticles formulation were evaluated against HUVEC cells through cell proliferation, migration and tube formation assays. It was found that the LNP/VEGF-A mRNA nanoparticles could promote endothelial cell proliferation. In addition, they exhibited successful mRNA delivery and high VEGF-A protein expression in vitro and in vivo by means of Western Blot assay and in vivo imaging system (IVIS). Finally, C57BL/6 diabetic mice model was established and intradermally treated with the LNP/VEGF-A mRNA nanoparticles. It was found that the LNP/VEGF-A mRNA treated wounds were almost healed after 14 days with an average wound area of 2.4 %, compared with the PBS group of 21.4 %. Apparently, the nanoparticles formulation was able to significantly expedite diabetic wound healing. The histological analysis containing H&E, Masson’s trichrome staining and CD31 further confirmed the healing efficacy and low toxicity of the formulation. Taken together, the LNP/VEGF-A mRNA nanoparticles can be taken up by cells to express protein effectively and improve diabetic wound healing, which might have potential application in the treatment of chronic diabetic wounds as a protein replacement therapy.

Discovery of novel thyrointegrin αvβ3 antagonist fb-PMT (NP751) in the management of human glioblastoma multiforme.

Thyrointegrin αvβ3 receptors are unique molecular cancer therapeutic targets because of their overexpression on cancer and rapidly dividing blood vessel cells compared and quiescent on normal cells. A macromolecule, TriAzole Tetraiodothyroacetic acid (TAT) conjugated to polyethylene glycol with a lipophilic 4-fluorobenyl group (fb-PMT and NP751), interacts with high affinity (0.21 nM) and specificity with the thyrointegrin αvβ3 receptors on the cell surface without nuclear translocation in contrast to the non-polymer conjugated TAT. The following in vitro assays were carried out to evaluate NP751 including binding affinity to different integrins, transthyretin (TTR)-binding affinity, glioblastoma multiforme (GBM) cell adhesion, proliferation assays, nuclear translocations, chorioallantoic membrane model of angiogenesis, and microarray for molecular mechanisms. Additionally, in vivo studies were carried out to evaluate the anticancer efficacy of NP751, its biodistribution, and brain GBM tumor versus plasma levels kinetics. NP751 demonstrated a broad spectrum of antiangiogenesis and anticancer efficacy in experimental models of angiogenesis and xenografts of human GBM cells. Tumor growth and cancer cells' viability were markedly decreased (by > 90%; P < .001) in fb-PMT-treated U87-luc or 3 different primary human GBM xenograft-bearing mice based on tumor in vivo imaging system (IVIS) imaging and histopathological examination, without relapse upon treatment discontinuation. Additionally, it effectively transports across the blood-brain barrier via its high-affinity binding to plasma TTR with high retention in brain tumors. NP751-induced effects on gene expression support the model of molecular interference at multiple key pathways essential for GBM tumor progression and vascularization. fb-PMT is a potent thyrointegrin αvβ3 antagonist with potential impact on GBM tumor progression.

Single domain antibody‐based non‐invasive in vivo imaging of tau pathology

AbstractBackgroundNumerous studies have shown that the extent of pathological tau protein in the brain correlates strongly with the severity of Alzheimer’s dementia at the time of death, and similar association with functional phenotypes have been seen in other tauopathies. In recent years, several dye‐based imaging probes with selectivity for tau aggregates have been developed. However, all are β‐sheet binders with varying affinity for different amyloids, and thereby lack specificity for tau lesions. In contrast, single domain antibodies (sdAbs) are specific for their target and because of their small size have much greater access into the brain than standard whole antibodies.MethodTo examine sdAbs’ suitability as non‐invasive in vivo imaging probes for tauopathies, we immunized a llama with a full‐length recombinant tau protein followed by boosters with human brain‐derived pathological tau proteins. Phage display libraries were generated from its peripheral blood mononuclear cells and screened for binding to tau proteins using various assays, resulting in a prototype sdAb deemed promising for in vivo imaging. Subsequently, this anti‐tau sdAb was labeled with a near‐infrared tag (680 nm), and injected intravenously in tauopathy and control mice, followed by imaging using an In Vivo Imaging System (IVIS). After the imaging, brains were extracted for tissue analysis.ResultIntravenous injection of labeled sdAb resulted in a strong in vivo brain signal detected through the intact head in two different models of transgenic tauopathy mice, but not in wild‐type or transgenic α‐synucleinopathy mice. Importantly, the in vivo brain signal correlated strongly with insoluble (r = 0.9839, p &lt; 0.0001) and soluble (human tau (CP27) and phospho‐tau (PHF1): r = 0.9603 and 0.9459, p &lt; 0.0001) tau protein within the brain. Furthermore, postmortem analysis revealed extensive co‐localization of the sdAb imaging probe with tau aggregates within neurons in the endosomal‐lysosomal system, indicating their interaction in these degradation pathways.ConclusionOverall, this specific sdAb imaging ligand has great potential as an in vivo diagnostic marker for Alzheimer's disease and related tauopathies, and sdAb‐based imaging may be applicable to a variety of protein conformational disorders.

Clinical Risk Factors and Microbiological and Intestinal Characteristics of Carbapenemase-Producing Enterobacteriaceae Colonization and Subsequent Infection.

Gastrointestinal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is always a prerequisite for the development of translocated infections. Here, we sought to screen for fecal carriage of CRE and identify the risk factors for CRE colonization as well as subsequent translocated pneumonia in critically ill patients admitted to the intensive care unit (ICU) of a university hospital in China. We further focused on the intestinal flora composition and fecal metabolic profiles in CRE rectal colonization and translocated infection patients. Animal models of gastrointestinal colonization with a carbapenemase-producing Klebsiella pneumoniae (carbapenem-resistant K. pneumoniae [CRKP]) clinical isolate expressing green fluorescent protein (GFP) were established, and systemic infection was subsequently traced using an in vivo imaging system (IVIS). The intestinal barrier, inflammatory factors, and infiltrating immune cells were further investigated. In this study, we screened 54 patients hospitalized in the ICU with CRE rectal colonization, and 50% of the colonized patients developed CRE-associated pneumonia, in line with the significantly high mortality rate. Upon multivariate analysis, risk factors associated with subsequent pneumonia caused by CRE in patients with fecal colonization included enteral feeding and carbapenem exposure. Furthermore, CRKP colonization and translocated infection influenced the diversity and community composition of the intestinal microbiome. Downregulated propionate and butyrate probably play important and multiangle roles in regulating immune cell infiltration, inflammatory factor expression, and mucus and intestinal epithelial barrier integrity. Although the risk factors and intestinal biomarkers for subsequent infections among CRE-colonized patients were explored, further work is needed to elucidate the complicated mechanisms. IMPORTANCE Carbapenem-resistant Enterobacteriaceae have emerged as a major threat to modern medicine, and the spread of carbapenem-resistant Enterobacteriaceae is a clinical and public health problem. Gastrointestinal colonization by potential pathogens is always a prerequisite for the development of translocated infections, and there is a growing need to assess clinical risk factors and microbiological and intestinal characteristics to prevent the development of clinical infection by carbapenem-resistant Enterobacteriaceae.

Establishment of a Novel Rat Model of Gram-Negative Periprosthetic Joint Infection Using Cementless Hip Hemiarthroplasty.

Gram-negative periprosthetic joint infections (GN-PJIs) present unique challenges. Our aim was to establish a clinically representative GN-PJI model that recapitulates biofilm formation in vivo. We also hypothesized that biofilm formation on the implant surface would affect its ability to osseointegrate. Three-dimensionally-printed medical-grade titanium hip implants were used to replace the femoral heads of male Sprague-Dawley rats. GN-PJI was induced using 2 bioluminescent Pseudomonas aeruginosa strains: a reference strain (PA14-lux) and a mutant biofilm-defective strain (ΔflgK-lux). Infection was monitored in real time using an in vivo imaging system (IVIS) and magnetic resonance imaging (MRI). Bacterial loads were quantified utilizing the viable colony count. Biofilm formation at the bone-implant interface was visualized using field-emission scanning electron microscopy (FE-SEM). Implant stability, as an outcome, was directly assessed by quantifying osseointegration using microcomputed tomography, and indirectly assessed by identifying gait-pattern changes. Bioluminescence detected by the IVIS was focused on the hip region and demonstrated localized infection, with greater ability of PA14-lux to persist in the model compared with the ΔflgK-lux strain, which is defective in biofilm formation. This was corroborated by MRI, as PA14-lux induced relatively larger implant-related abscesses. Biofilm formation at the bone-implant interface induced by PA14-lux was visualized using FE-SEM versus defective-biofilm formation by ΔflgK-lux. Quantitatively, the average viable colony count of the sonicated implants, in colony-forming units/mL, was 3.77 × 108 for PA14-lux versus 3.65 × 103 for ΔflgK-lux, with a 95% confidence interval around the difference of 1.45 × 108 to 6.08 × 108 (p = 0.0025). This difference in the ability to persist in the model was reflected significantly on implant osseointegration, with a mean intersection surface of 4.1 × 106 ± 1.99 × 106 μm2 for PA14-lux versus 6.44 × 106 ± 2.53 × 106 μm2 for ΔflgK-lux and 7.08 × 106 ± 1.55 × 106 μm2 for the noninfected control (p = 0.048). To our knowledge, this proposed, novel in vivo biofilm-based model is the most clinically representative for GN-PJI to date, since animals can bear weight on the implant, poor osseointegration was associated with biofilm formation, and localized PJI was assessed by various modalities. This model will allow for more reliable testing of novel biofilm-targeting therapeutics.

SURG-21. DEVELOPMENT OF MICROSURGICAL FORCEPS EQUIPPED WITH HAPTICS TECHNOLOGY FOR IN SITU DIFFERENTIATION OF TUMOR TISSUE FROM NORMAL BRAIN DURING MICROSURGERY BY TISSUE STIFFNESS

Abstract Neurosurgeons perform tumor resection based on their tactile perception. However, many years of experience and training are required to appropriately differentiate tumor tissue from surrounding parenchymal tissue. It was reported that the stiffness of human cancer tissue strongly correlates with pathological tissue and it can be used as a biomarker. Comprehensive analysis also revealed that the molecular expression differs greatly between hard and soft tumors.Haptics technology measures and uses the stimulus that causes the sensation as a physical quantity. Force and tactile sensation is a sensation caused by mechanical contact with an object. Real Haptics is our main research field, which is a general term for technologies that quantify and utilize the force-tactile stimuli generated by contact with objects that exist in the real world and / or the virtual world.We have developed a microsurgical forceps equipped with haptics technology to differentiate tumor tissue from surrounding brain tissue during tumor dissection. We generated and used three glioblastoma cell lines (SF126, U87, U251) expressing proteins of firefly luciferase and mCherry. Each cell line was transplanted into forebrain of 5 nude mice. Tumor progression was regularly monitored with In Vivo Imaging System (IVIS) and mice were euthanized at the appropriate stage. Tumor and normal brain stiffness were measured by using the Master-slave integrated haptic forceps device. Importantly, all of the three glioblastomas were shown to be harder than the normal brain (p=0.047). The median stiffness of each tissue was as follows. Normal brain (n=15): 149.98 N/m, SF126 (n=5): 207.70 N/m, U87 (n=5): 214.40 N/m, U251 (n=5): 188.86 N/mThis pilot study showed that brain tumors could be differentiated by their stiffness from normal brain, and the forceps equipped with haptics technology might help the neurosurgeons to dissect tumor margin along the appropriate plane.

CSIG-41. SENSITIZING CANCER CELLS TO TUMOR TREATING FIELDS (TTFIELDS) BY INHIBITION OF PI3K

Abstract Tumor Treating Fields (TTFields) are alternating electric fields, which disrupt cellular process critical for cancer cell survival and tumor progression. TTFields therapy is approved for the treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma, and is being tested in clinical studies for the treatment of other solid tumors, including ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The current study aimed to detect potential mechanisms that may reduce cellular sensitivity to TTFields, and target these pathways in order to re-sensitize the cells to TTFields. Cancer cells (Ovarian A2780, GBM U-87 MG, and NSCLC H1299) that display reduced sensitivity to TTFields were generated by continuous long-term TTFields application (7 or 13 days, depending on the cell line). A Luminex multiplex assay revealed activation of the PI3K/AKT/mTOR signaling pathway in these cells, with significant increases in phosphorylation levels of AKT and RPS6. This elevation was also observed by immunohistochemistry in tumor sections from N1S1 HCC tumor-bearing rats treated with TTFields relative to sham. Treatment of cells with PI3K inhibitors re-sensitized them to TTFields and downregulated the phosphorylation of AKT. Concomitant application of TTFields with the PI3K inhibitor alpelisib in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells resulted in enhanced efficacy, as determined by In Vivo Imaging System (IVIS) measurements of tumor volume. Overall, this study demonstrated that the PI3K/AKT/mTOR signaling pathway is involved in reduced cancer cell sensitivity to long-term application of TTFields, and that re-sensitization may be achieved with relevant inhibitors. The results provide a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors.

Combined pembrolizumab and bevacizumab therapy effectively inhibits non-small-cell lung cancer growth and prevents postoperative recurrence and metastasis in humanized mouse model

Antibodies targeting the programmed cell death protein 1/programmed cell death ligand-1 (PD-1/PD-L1) pathway have dramatically changed the treatment landscape of advanced non-small cell lung cancer (NSCLC). However, combination approaches are required to extend this benefit beyond a subset of patients. In addition, it is of equal interest whether these combination therapy can be applied to neoadjuvant therapy of early-stage NSCLC. In this study, we hypothesized that combining immunotherapy with anti-angiogenic therapy may have a synergistic effect in local tumor control and neoadjuvant therapy. To this end, the effect of combination of bevacizumab and pembrolizumab in humanized mouse models was evaluated. Furthermore, we innovatively constructed a neoadjuvant mouse model that can simulate postoperative recurrence and metastasis of NSCLC to perform neoadjuvant study. Tumor growth and changes in the tumor vasculature, along with the frequency and phenotype of tumor-infiltrating lymphocytes, were examined. Additionally, in vivo imaging system (IVIS) was used to observe the effect of neoadjuvant therapy. Results showed that combination therapy could inhibited tumor growth by transforming tumor with low immunoreactivity into inflamed (‘hot’) tumor, as demonstrated by increased CD8+granzyme B+ cytotoxic T cell infiltration. Subsequent studies revealed that this process is mediated by vascular normalization and endothelial cell activation. IVIS results showed that neoadjuvant therapy can effectively prevent postoperative recurrence and metastasis. Taken together, these preclinical studies demonstrated that the combination of bevacizumab and pembrolizumab had a synergistic effect in both advanced tumor therapy and neoadjuvant setting and therefore provide a theoretical basis for translating this basic research into clinical applications.

PI3K Inhibition Sensitized Cancerous Cells to Tumor Treating Fields (TTFields)

<h3>Purpose/Objective(s)</h3> Tumor Treating Fields (TTFields) are alternating electric fields, approved for treatment of patients with newly diagnosed glioblastoma (GBM), recurrent GBM, or unresectable malignant pleural mesothelioma. Additional clinical trials are underway in other tumor types, including ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). While TTFields therapy has been demonstrated to extend life, tumor progression eventually occurs in most patients. The current research aimed to identify molecular mechanisms involved in reduced cancer cellular sensitivity to TTFields, and targeting these potential pathways in order to re-sensitize the cells to TTFields. <h3>Materials/Methods</h3> Ovarian A2780, GBM U-87 MG, and NSCLC H1299 cells with reduced sensitivity to TTFields were generated by continuous long-term application of TTFields (7 or 13 days, depending on the cell line). Changes in signaling pathways in these cells were examined by Luminex multiplex assay, and specific pathway markers were validated by Western blot of the cell lysates as well as by immunohistochemistry in tumor sections from N1S1 HCC tumor-bearing rats treated with sham or TTFields. Next, TTFields were applied in conjunction with relevant inhibitors, followed by cell count measurements and western blot examinations. Finally, the concomitant application of TTFields with a relevant inhibitor was evaluated in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end using the In Vivo Imaging System (IVIS) to detect the luciferin signal. <h3>Results</h3> Sensitivity of cancer cells to TTFields was decreased following continuous long-term application of TTFields. PI3K/AKT/mTOR signaling pathway was activated in these cells, with significant increases in phosphorylation levels of AKT and RPS6. This elevation was also observed in tumor sections from rats treated with TTFields. Treatment with PI3K inhibitors re-sensitized the cells to TTFields and downregulated the phosphorylation of AKT. In vivo, concomitant application of TTFields with the PI3K inhibitor alpelisib resulted in enhanced efficacy. <h3>Conclusion</h3> The current study demonstrated that the PI3K/AKT/mTOR signaling pathway is involved in reduced cancer cell sensitivity to long-term application of TTFields. Furthermore, re-sensitization to TTFields could be achieved with PI3K inhibitors, providing a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors.

A new isolate of Streptomyces lateritius (Z1-26) with antibacterial activity against fish pathogens and immune enhancement effects on crucian carp (Carassius auratus).

The Streptomyces lateritius Z1-26 was isolated from soil samples which showed broad-spectrum antibacterial activity against a broad range of fish pathogens. The In Vivo Imaging System (IVIS) monitored that strain Z1-26 could survive and colonize in the gills and abdomen of crucian carp. The effects of dietary supplementation with strain Z1-26 were evaluated with respect to the growth performance, antioxidant capacity, and immune response of crucian carp. The results showed that the Z1-26-fed fish had a significantly higher growth rate than the fish fed the control diet. The immune and antioxidant parameters revealed that the non-specific immune indicators (AKP, SOD, and LZM) of the serum, the expression of immune-related genes (IgM, C3, and LZM), and antioxidant-related genes (Nrf2 and Keap1) of the immune organs were significantly increased, whereas the expression of pro-inflammatory factors (IL-1β, IL-8, and TNF-α) of the immune organs was significantly down-regulated in crucian carp fed strain Z1-26 compared with fish fed a control diet. Moreover, fish fed with Z1-26 supplemented diets showed a significantly improved survival rate after Aeromonas hydrophila infection. In addition, the whole genome analysis showed that strain Z1-26 possesses 28 gene clusters, including 6 polyketide synthetase (PKS), 4 non-ribosomal peptide-synthetase (NRPS), 1 bacteriocin, and 1 lantipeptide. In summary, these results indicated that strain Z1-26 could improve the growth performance and disease resistance in crucian carp, and has the potential to be developed as a candidate probiotics for the control of bacterial diseases in aquaculture.

Electroacupuncture of the trigeminal nerve causes N-methyl-D-aspartate receptors to mediate blood-brain barrier opening and induces neuronal excitatory changes.

The blood-brain barrier (BBB) is an important structure for maintaining environmental stability in the central nervous system (CNS). Our previous study showed that specific parameters of electroacupuncture (EA) at the head points Shuigou (GV26) and Baihui (GV20) can open the BBB; however, the mechanism by which stimulation of body surface acupuncture points on the head results in peripheral stimulation and affects the status of the central BBB and the neuronal excitatory changes has not been elucidated. We used laser spectroscopy, the In Vivo Imaging System (IVIS), immunofluorescence and immunoblotting to verified the role of the trigeminal nerve in BBB opening during EA, and we applied the central N-methyl-D-aspartate (NMDA) receptors blocker MK-801 to verify the mediating role of NMDA receptors in EA-induced BBB opening. Next, electroencephalogram (EEG) and in vivo calcium imaging techniques were applied to verify the possible electrical patterns of BBB opening promoted by different intensities of EA stimulation. The results showed that the trigeminal nerve plays an important role in the alteration of BBB permeability promoted by EA stimulation of the head acupoints. Brain NMDA receptors play a mediating role in promoting BBB permeability during EA of the trigeminal nerve, which may affect the expression of the TJ protein occludin, and thus alter BBB permeability. The analysis of the electrical mechanism showed that there was no significant change in the rhythm of local field potentials (LFP) in different brain regions across frequency bands immediately after EA of the trigeminal nerve at different intensities. However, the local primary somatosensory (S1BF) area corresponding to the trigeminal nerve showed a transient reduction in the delta rhythm of LFP with no change in the high-frequency band, and the action potential (spike) with short inter spike interval (ISI) varied with EA intensity. Meanwhile, EA of the trigeminal nerve resulted in rhythmic changes in calcium waves in the S1BF region, which were influenced by different EA intensities. This study provides a research perspective and a technical approach to further explore the mechanism of EA-induced BBB opening and its potential clinical applications.

P10.11.B Re-sensitizing cancer cells to Tumor Treating Fields (TTFields) through PI3K/AKT/mTOR pathway inhibition

Abstract Background Tumor Treating Fields (TTFields) are alternating electric fields disrupting cellular processes critical for cancer cell replication and tumor progression. TTFields therapy is approved for treatment of newly diagnosed glioblastoma (GBM), recurrent GBM, and unresectable malignant pleural mesothelioma, and is currently under clinical investigation for treating other solid tumors, such as ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The research herein aimed to identify potential treatment escape mechanisms and explore the possibility of targeted inhibition of these pathways for re-sensitizing the cells to TTFields. Material and Methods GBM U-87 MG, ovarian A2780, and NSCLC H1299 cells were treated with TTFields (1.7 V/cm RMS, 200 or 150 kHz) for continuous long-term application (7 or 13 days, specific conditions depending on the cell line). Changes in signaling pathways in these cells relative to cells exposed to short term TTFields application (3 or 7 days, depending on cell line) were examined by Luminex multiplex assay. Specific pathway markers were examined by immunohistochemistry of tumor sections from sham or TTFields-treated rats bearing N1S1 HCC tumors. TTFields were then co-applied with relevant pathway inhibitors, followed by cell count measurements and western blot examinations for specific pathway markers. The concomitant application of TTFields with a selected inhibitor was tested in mice inoculated orthotopically with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end by luciferin signal detection using the In Vivo Imaging System (IVIS). Results Cancer cells exposed to long-term application of TTFields displayed decreased sensitivity to TTFields. The PI3K/AKT/mTOR pathway was activated in these cells, with significant increases in AKT and RPS6 phosphorylation levels also observed in HCC tumors from rats treated with TTFields. PI3K inhibitors re-sensitized the cells to TTFields cytotoxicity, with associated down regulation of AKT phosphorylation. Application of TTFields concomitant with the PI3K inhibitor alpelisib resulted in enhanced efficacy in the ovarian cancer mouse model. Conclusions The current research demonstrated that PI3K/AKT/mTOR signaling pathway activation was involved in the response to long-term application of TTFields, with increased phosphorylation of key proteins observed both in vitro and in vivo. Concomitant treatment with PI3K inhibitors and TTFields re-sensitized the cells to TTFields, as it alleviated TTFields-induced AKT activation.

Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 biology and the impact of antiviral therapeutics is a robust method to detect the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2-expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals is limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively) or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bioreporter-expressing rSARS-CoV-2 for the study of SARS-CoV-2 in vitro and in vivo.IMPORTANCE Despite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use in specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualized SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).

The synthesis and evaluation of thiolated alginate as the barrier to block nutrient absorption on small intestine for body-weight control.

Obesity is the most common health concern all over the world. However, till now, there is no promising way to manage obesity or body-weight control. The aim of the study is to develop an edible gel as a health supplement that temporarily attaches to the mucus of the intestines, forming an absorption barrier to block the nutrients. We modify the alginate with the thiol group as thiolated alginate (TA) that may stay on the mucosa layer for a much longer time to reduce nutrient absorption. In this study, the TA is synthesized successfully and proved a good mucosal adhesion to serve as a barrier for nutrient absorption both in vitro and in vivo. The results of in vivo imaging system (IVIS) show that the synthesized TA can be exiled from the gastrointestinal tract within 24 h. The animal study shows that the TA by daily oral administration can effectively reduce body weight and fat deposition. The biosafety is evaluated in vitro at the cellular level, based on ISO-10993, and further checked by animal study. We do believe that the TA could have a greater potential to be developed into a safe health supplement to manage obesity and for body-weight control.

EP-368 Intraoperative identification of colorectal cancer lesions using indocyanine green labelled antibodies in preclinical murine models

Abstract Aims The ability to identify cancerous tissue intraoperatively has the potential to allow for real time identification of involved margins, lymph node involvement and extra-colonic deposits. Here we investigate whether antibodies targeting a novel receptor, anonymously referred to as LN1, a frequently over expressed protein in colorectal cancer (CRC), labelled with indocyanine green (ICG) can be used for the intraoperative detection of malignant tissue in preclinical models. Methods Representative metastatic CRC models were generated in 20 NSG mice using HCT116 human CRC cells. Mice were treated with either intravenous ICG labelled LN1 antibodies (LN1-ICG) or control treatments (untreated, free ICG or cotreatment unlabelled LN1 blocking antibody + LN1-ICG). Signal intensity of normal and malignant tissue was quantified from near infrared (NIR) endoscopy and IVIS® in-vivo imaging system. Results LN1-ICG labelled antibodies was highly effective in identifying cancerous lesions down to 500nm diameter. Quantification of NIR and IVIS images demonstrate significantly greater tumour signal in the LN1-ICG group versus controls (p=0.004 and p=0.001 respectively). Examination of normal organ tissues demonstrated signal in the liver and digestive tract in keeping with hepatobiliary clearance of ICG but nil further off-site accumulation of LN1-ICG. No difference in signal from the liver, small bowel or colon was noted between groups (p&amp;gt;0.05). Conclusion LN1-ICG labelled antibodies were highly effective at identifying even small cancerous deposits in murine models. It is highly likely that these effects could be replicated in human procedures which may allow for perioperative identification of extra-colonic deposits, involved tumour margins or lymph node involvement.

Abstract 2659: Inhibition of PI3K sensitized cancer cells to Tumor Treating Fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are alternating electric fields, delivered noninvasively to the tumor site. TTFields therapy is currently approved for treatment of patients with newly diagnosed glioblastoma (GBM), recurrent GBM, or unresectable malignant pleural mesothelioma. Investigations are ongoing in additional tumor types, including non-small cell lung carcinoma (NSCLC), ovarian cancer, and hepatocellular carcinoma (HCC). Although TTFields have been demonstrated to extend life, most patients will eventually progress. The current research aimed to identify molecular mechanisms involved in reduced cancer cellular sensitivity to TTFields, and the potential of targeting these pathways to re-sensitize the cells to TTFields. Methods: Continuous long-term application of TTFields (7 or 13 days, depending on the cell line) generated cancer cells with reduced sensitivity to TTFields. Luminex multiplex assay was used to detect changes in signaling pathways in ovarian A2780 and GBM U-87 MG cells, and relevant pathway markers were validated by Western blot. Further validation was performed by immunohistochemistry of tumor sections from N1S1 HCC tumor-bearing rats treated with sham or TTFields. The significance of the identified pathways in reducing cancer cell sensitivity to TTFields was evaluated through in vitro combination treatment with PI3K inhibitors, followed by cell count measurements. Finally, the concomitant application of TTFields and the PI3K inhibitor Alpelisib was evaluated in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured using the In Vivo Imaging System (IVIS) to detect the luciferin signal, before and after treatment. Results: Cancer cell sensitivity to TTFields was reduced following continuous long-term application of TTFields. This was accompanied by activation of the PI3K/AKT/mTOR signaling pathway, with significant increases in the levels of phosphorylated AKT and RPS6 observed in cell cultures and in rat tumor sections following application of TTFields. Application of PI3K inhibitors re-sensitized the cells to TTFields in vitro. In vivo, concomitant application of TTFields with Alpelisib resulted in enhanced efficacy. Conclusions: The current study demonstrated that reduced cancer cell sensitivity to long-term application of TTFields is mediated by activation of the PI3K/AKT/mTOR signaling pathway. Furthermore, PI3K inhibitors were shown to re-sensitize the cells to TTFields, providing a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Kerem Wainer-Katsir, Alexandra Volodin, Bella Koltun, Boris Brant, Yiftah Barsheshet, Tal Kan, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Inhibition of PI3K sensitized cancer cells to Tumor Treating Fields (TTFields) [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 2659.

Abstract 339: Anti-doppel monoclonal antibody as a tumor endothelial cell-specific angiogenesis inhibitor

Abstract Introduction: Approved angiogenesis inhibitors generally target certain growth factors or their receptors, which exist in both normal and cancerous cells; this results in suppression of cell-signaling pathways throughout the body and causes adverse effects. For this reason, there is a need for a new angiogenesis inhibitor that can specifically target the tumor vasculature. In our previous study, we revealed that doppel, a prion-like protein, was overexpressed specifically in the tumor vasculatures, but not in normal endothelium, and its expression enhanced blood vessel formation. To develop our new anti-angiogenic agent, we produced monoclonal antibodies which target doppel as the antigen. Methods: We evaluated whether the doppel antibody inhibits angiogenesis by a spheroid assay. We cultured human colonic tumor-associated endothelial cells (HCTEC) and collected the cells by a hanging drop method. After producing compact aggregates, the spheroids were collected and embedded into collagen-based 3D cultures. The culture media/growth factor/doppel antibody mixture was added to each well that contained 3D spheroid cultures. After 24 hours, the number of sprouts was counted and analyzed. Further, HCTECs were incubated with culture media, growth factor, and doppel antibodies for investigating the underlying mechanisms. Each well was lysed, and we conducted western blot and microarray experiments using the lysates. In addition, we injected fluorescent dye-conjugated doppel antibody into tumor xenograft mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS). Results: In spheroid assays, we found that doppel antibody SNU-H01 and SNU-H02 inhibit angiogenesis (by 32% and 29%, respectively) compared to control. In anti-phosphorylation assays using western blot, the amount of phosphorylated VEGFR2 and phosphorylated FGFR1 was decreased in doppel antibody SNU-H01 and SNU-H02-treated groups. Also, when SNU-H02-treated lysate was added to the microarray kit, STAT5A and beta-catenin were inhibited. Inhibition of STAT5A may promote apoptosis and increase sensitivity to anticancer drugs, and inhibition of beta-catenin may further increase suppression of angiogenesis. In addition, doppel antibody SNU-H04 accumulates in the tumor significantly higher compared to IgG injected mouse group. Conclusion: Doppel antibodies in development, SNU-H01, -H02, -H03, -H04, will be screened and selected for their anti-angiogenic efficacy and favorable pharmacodynamic features. We hope that doppel antibody is expected to be a new and superior tumor vasculature-specific angiogenesis inhibitor that can overcome the limitations of existing anti-angiogenic agents. Citation Format: Ha Kyeong Lee, Ruby Maharjan, Yeojin Jeon, Jeong Uk Choi, Youngro Byun. Anti-doppel monoclonal antibody as a tumor endothelial cell-specific angiogenesis inhibitor [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 339.

Abstract 5958: CD276/CD47-targeted antibody-drug conjugates to treat triple-negative breast cancers

Abstract 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.