Vivo Imaging System Research Articles

Biomimetic modification of macrophage membrane-coated prussian blue nanoparticles loaded with SN-38 to treat colorectal cancer by photothermal-chemotherapy.

SN-38 is the active metabolite of irinotecan and acts as an effective topoisomerase I inhibitor with therapeutic effects on many malignant tumors, including some drug-resistant cancers. However, the poor solubility, low bioavailability, and severe dose-dependent toxicity limits the clinical application of SN-38. Currently, emerging macrophage membrane-coated nanoparticles provide an efficient biomimetic approach to develop novel SN-38 formulations for the reduction of its side effects. Photothermal therapy (PTT) is a promising methods in tumor treatment to thermally ablate tumors using various materials such Prussian blue nanoparticles (NPs) and can combined with chemotherapy to synergistically work. There is no report that combined SN38 and photothermal therapy for the treatment of colorectal cancer (CRC). SN38-PB@CM NPs were constructed by loading SN-38 into macrophage cell membrane-coated hollow mesoporous Prussian blue (PB) NPs. The morphology, size and zeta potential were evaluated by transmission microscopy and dynamic light scatter (DLS). Coomassie bright blue staining was performed to assess total protein profile. The photothermal properties of it were also investigated via near-infrared imaging. CCK8 and calcein-AM/PI staining were used to evaluate cell viability. Flow cytometry was performed to assess cell apoptosis. The fluorescent microscopy was used to observe cellular uptake of SN38-PB@CM NPs to assess its internalization in vitro. The biodistribution, tumor-targeting efficacy, antitumor efficacy and safety of SN38-PB@CM NPs in vivo were assessed in CT26 tumor-bearing mice via In Vivo Imaging System. SN38-PB@CM NPs were successfully constructed and exhibited a uniform size distribution (140.5 ± 4.3nm) and an excellent drug-loading capacity (5.61 ± 0.64%). SN38-PB@CM NPs showed stable release properties within 72h. It can also enhance the selective intracellular delivery of SN38 in vitro and showed good near-infrared (NIR) photothermal properties. And the NPs showed excellent tumor targeting, effective photothermal therapy, improved biosafety and antitumor efficacy on CT26-bearing mice. Multifunctional SN38-PB@CM NPs could achieve improved biosafety, great tumor-targeting, high-efficiency PTT and excellent antitumor efficacy, which provided a promising and attractive combination therapy for the treatment of CRC.

833-P: Duodenal Targeting by Oral Pharmacologic Duodenal Exclusion Therapy for Treatment of Type 2 Diabetes (T2D)

GLY-200 is an oral, non-absorbed polymeric drug designed to non-invasively and reversibly enhance the mucus lining of the proximal small intestine to mimic the effects of metabolic surgery and duodenal exclusion devices. In healthy volunteers and rat models of T2D (GK, ZDF, and DIO), GLY-200 produced reductions in postprandial glucose and bodyweight compared to control. This study aimed to visualize the retained duodenal barrier in rats using appropriate imaging modalities. Methods: Imaging of GLY-200 after gavage administration to fasted Sprague-Dawley rats was performed using Computed Tomography (CT) and an In Vivo Imaging System (IVIS). For CT imaging, GLY-200 was covalently conjugated with diatrizoate; images were obtained after transient isoflurane administration 1, 2, and 4 hours after gavage. For IVIS imaging, GLY-200 was reacted with FITC to produce a fluorescein-GLY-200 conjugate. Rats were euthanized 0.5, 1, 2, 4, and 8 hours after dosing and the GI tracts were immediately removed for imaging. Results: In CT imaging, GLY-200-diatrizoate was retained in the proximal small intestine 1 and 2 hours after administration, with an intense signal only in the duodenum. In comparison, a diatrizoate control was broadly distributed over the small intestine distal to the duodenum by 1 hour. In IVIS imaging, GLY-200-FITC showed intense fluorescence in the proximal small intestine that was predominantly associated with the gut wall and persisted for >4 hours after administration. In comparison, a FITC-dextran70 control was distributed broadly throughout the distal intestine and cecum by 30 minutes with no retention in the duodenum. Conclusion: Imaging studies confirm that GLY-200 conjugates are retained in the duodenum after oral administration. These data support the hypothesis that GLY-200 treatment results in a pharmacologic duodenal exclusion that could potentially recapitulate the glycemic and weight effects of metabolic surgery and duodenal exclusion devices. Disclosure A.Nimgaonkar: Employee; Glyscend Inc. C.Bryant: Employee; Glyscend Inc. T.Carlson: Employee; Glyscend Inc. F.V.Guerina: Employee; Glyscend Inc. K.Colbert: Consultant; LifeSprout, Employee; Glyscend Inc. S.Polomoscanik: None. J.S.Petersen: None. T.H.Jozefiak: Employee; Glyscend Inc., Stock/Shareholder; Glycologix, LLC. M.Fineman: Employee; Glyscend Inc., Stock/Shareholder; Glyscend Inc.

Abstract 2387: Characterization of orthotopic bioluminescent patient-derived xenograft model of glioma for drug discovery

Abstract Introduction: Gliomas account for majority of primary brain tumors and are composed of different grades, which present distinct molecular alterations, histological characteristics, and response to treatments. Patient-derived xenografts (PDXs) are the most “close-to-patient” models for oncology studies which largely maintain the histo- and molecular- pathology of the original tumors and are widely applied in preclinical evaluations. Subcutaneously inoculated glioma PDX models provide convenient evaluation of treatments’ efficacy but are difficult to mimic the in-situ microenvironment and diffuse invasive growth to peripheral tissue in the brain. Orthotopic implantation of bioluminescent labelled glioma models provides a more relevant preclinical model to evaluate and quantify treatment response. Methods: A glioma PDX model was established by subcutaneously engrafting tumor directly from patient following surgery into immunodeficient mice. Once established and expanded through passaging, glioma cells were isolated from fresh PDX tumor and transduced with pLVX-IRES-ZsGreen1 lentiviral vector, which expressed both luciferase and GFP, rapidly. Cells were then subcutaneously inoculated into immunodeficient mice directly without in vitro culture and selection. Cell sorting of the following tumors based on GFP expression was performed to exclude the luciferase negative cells and enrich for stable luciferase signal in subsequent passages. The in vivo expansion and sorting of GFP+ tumor cells were repeated for two rounds. Cells from third passages of subcutaneous tumor were orthotopically inoculated in the brain of NOD.scid mice. The tumor progression in the brain was further confirmed by the IVIS® Spectrum In Vivo Imaging System. Response of orthotopically inoculated BN2289-Luc to temozolomide was also evaluated according to the bioluminescent signal in brain. Results: We successfully established a luciferase-GFP dual-labeled glioma PDX model BN2289-Luc which was stably passaged in vivo. The successful transduction of luciferase-GFP cascade was detected by in vitro luciferase assay and fluorescence imaging. Stable tumor progression and bioluminescent signal growth of BN2289-Luc were validated orthotopically and subcutaneously in immunodeficient mice, and a histological study revealed the consistency between orthotopic and subcutaneous tumors. Efficacy studies showed that orthotopically inoculated BN2289-Luc responded to temozolomide (25mg/kg, p.o., Day1-5/wkx4wks). Conclusion: An orthotropic glioma PDX model, BN2289-Luc displayed luciferase signal of in situ tumor progression and response to temozolomide treatment, providing an ideal approach for orthotopic investigation of gliomas and preclinical testing of new drugs. Citation Format: Jinxi Wang, Ling Chen, Jun Zhou, Likun Zhang, Jinyu Huang, Xueluan Zhu, Ludovic Bourre, Jingjing Wang. Characterization of orthotopic bioluminescent patient-derived xenograft model of glioma for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2387.

Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs

BackgroundWhile prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour.MethodsBMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ−/− (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology.ResultsFlow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP.ConclusionPC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology.

Targeted EV to Deliver Chemotherapy to Treat Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) are heterogeneous and metastatic, and targeted therapy is highly needed for TNBC treatment. Recent studies showed that extracellular vesicles (EV) have great potential to deliver therapies to treat cancers. This study aimed to develop and evaluate a natural compound, verrucarin A (Ver-A), delivered by targeted EV, to treat TNBC. First, the surface expression of epidermal growth factor receptor (EGFR) and CD47 were confirmed with immunohistochemistry (IHC) staining of patient tissue microarray, flow cytometry and Western blotting. EVs were isolated from HEK 293F culture and surface tagged with anti-EGFR/CD47 mAbs to construct mAb-EV. The flow cytometry, confocal imaging and live-animal In Vivo Imaging System (IVIS) demonstrated that mAb-EV could effectively target TNBC and deliver the drug. The drug Ver-A, with dosage-dependent high cytotoxicity to TNBC cells, was packed in mAb-EV. The anti-TNBC efficacy study showed that Ver-A blocked tumor growth in both 4T1 xenografted immunocompetent mouse models and TNBC patient-derived xenograft models with minimal side effects. This study demonstrated that the targeted mAb-EV-Ver-A had great potential to treat TNBCs.

Immunomodulatory Effects of Perioperative Dexmedetomidine in Ovarian Cancer: An In Vitro and Xenograft Mouse Model Study

BackgroundThe surgical stress response (SSR) causes immunosuppression which may cause residual tumor growth and micrometastasis after cancer surgery. We investigated whether dexmedetomidine affects cancer cell behavior and immune function in an ovarian cancer xenograft mouse model.MethodsThe effect of dexmedetomidine on cell viability and cell cycle was assessed using SK-OV-3 cells at drug concentrations of 0.5, 0.1, 5, and 10 µg mL-1. BALB/c nude mice were used for the ovarian cancer model with the Dexmedetomidine group (n=6) undergoing surgery with dexmedetomidine infusion and the Control group (n=6) with saline infusion for 4 weeks. Natural killer (NK) cell activity, serum proinflammatory cytokines, and cortisol were measured at predetermined time points and tumor burden was assessed 4 weeks after surgery.ResultsDexmedetomidine had no effect on cell viability or cell cycle. Following a sharp decrease on postoperative day (POD) 1, NK cell activity recovered faster in the Dexmedetomidine group with significant difference vs. the Control group on POD 3 (P=0.028). In the Dexmedetomidine group, cortisol levels were lower on POD 3 (P=0.004) and TNF-α levels were lower at 4 weeks after surgery (P<0.001) compared to the Control group. The Dexmedetomidine group showed lower tumor burden at 4 weeks vs. the Control group as observed by both tumor weight (P<0.001) and the in vivo imaging system (P=0.03).ConclusionsDexmedetomidine infusion may improve ovarian cancer surgery outcome by suppressing the SSR and stress mediator release. Further studies are needed to elucidate the mechanisms by which dexmedetomidine acts on cancer and immune cells.

Targeted Liposomal Chemotherapies to Treat Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancers (TNBCs) are mainly treated with standard chemotherapies. Combined therapies have been demonstrated as a promising treatment strategy in clinics. The aim of this study was to develop a new formulation of combined chemotherapies facilitated with a targeted delivery vehicle. We found that the mertansine and gemcitabine with different anti-cancer mechanisms resulted in high cytotoxicity in TNBC cells. The in vivo evaluations using two TNBC xenograft models confirmed the anti-tumor efficacy, i.e., significantly reduced tumor growth rate. Furthermore, the antibody-tagged liposomes effectively delivered the therapeutic drugs to TNBC tumor, which could reduce the side effects. This study is highly translational and the targeted liposomal drug formulation can be further investigated in future clinical trials for TNBC treatment.Triple-negative breast cancers (TNBCs) are highly aggressive and recurrent. Standard cytotoxic chemotherapies are currently the main treatment options, but their clinical efficacies are limited and patients usually suffer from severe side effects. The goal of this study was to develop and evaluate targeted liposomes-delivered combined chemotherapies to treat TNBCs. Specifically, the IC50 values of the microtubule polymerization inhibitor mertansine (DM1), mitotic spindle assembly defecting taxane (paclitaxel, PTX), DNA synthesis inhibitor gemcitabine (GC), and DNA damage inducer doxorubicin (AC) were tested in both TNBC MDA-MB-231 and MDA-MB-468 cells. Then we constructed the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) tagged liposomes and confirmed its TNBC cell surface binding using flow cytometry, internalization with confocal laser scanning microscopy, and TNBC xenograft targeting in NSG female mice using In Vivo Imaging System. The safe dosage of anti-EGFR liposomal chemotherapies, i.e., <20% body weight change, was identified. Finally, the in vivo anti-tumor efficacy studies in TNBC cell line-derived xenograft and patient-derived xenograft models revealed that the targeted delivery of chemotherapies (mertansine and gemcitabine) can effectively inhibit tumor growth. This study demonstrated that the targeted liposomes enable the new formulations of combined therapies that improve anti-TNBC efficacy.

Abstract 3008: Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery

Abstract Background: Cancer is a complicated disease featuring geographic, genetic and immunological diversity. The tumor microenvironment (TME) is an essential part of tumor development, influencing tumor growth, metastasis and also therapeutic response. Patient-derived xenografts (PDX) and PDX-derived organoids (PDXO) are widely-used preclinically for drug evaluation with the prominent advantage of largely maintaining the histo- and molecular- pathology of the original tumor. Over decades of studies using PDX models, we found limitations in monitoring the orthotopic and metastatic pattern of cancer. With the development of gene engineering technologies (particularly for luciferase labeling), we now present the transduction of patient-derived models with firefly luciferase, to enable real-time monitoring of tumor burden in mice following orthotopic engraftment of either PDX tumor cells or PDXO in vivo. Methods: A panel of PDX cell slurry or PDXO cultures were transduced with lentiviral vectors carrying both the firefly luciferase gene as well as GFP. Mixed pools or GFP-sorted cells were used for in vivo validation. Bioluminescent PDX or PDXO were orthotopically and subcutaneously engrafted into immunodeficient mice. Real-time tumor growth and metastasis were monitored using the IVIS® Spectrum In Vivo Imaging System. Results: We have established a series of luciferase-labeled PDXO from different cancer types, including liver, ovarian, pancreatic and lung cancer and characterized their growth following orthotopic implantation. We have also established a series of luciferase-labeled PDX models from non-epithelial origin, including brain tumors, osteosarcoma and lymphomas. All of the models were confirmed with GFP signaling in vitro and showed significant luciferase signaling and growth in vivo. A mixed pool of bioluminescent PDXs or PDXOs presented heterogenous luciferase signals in tumors, while single clones or cell sorting by GFP or selected passage in vivo improved the consistency of the bioluminescent signal. Conclusions: PDX models are widely used as preclinical tools for clinical predictivity, however their application in orthotopic/metastatic modeling has been challenging due to the limitation in labelling PDXs for real-time monitoring. With the development of gene engineering technology in vitro and bioluminescent imaging (BLI) in vivo, we have developed and characterized a large panel of bioluminescent PDX and PDXO models from a range of different cancer types for real-time monitoring of orthotopic engrafted tumors in an organ-specific location to represent the TME, and the potential metastatic spread. Citation Format: Jessie Jingjing Wang, Jun Zhou, Ling Chen, Rongyun Zhou, Xiaoxi Xu, Henry Q. X. Li, Davy Xuesong Ouyang. Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3008.

A Biopolymer‐Fused Form of Vascular Endothelial Growth Factor‐B (VEGFB) For the Treatment of Preeclampsia

Introduction Preeclampsia is clinically characterized by the development of elevated blood pressure combined with proteinuria or other symptoms during the second and third trimester of pregnancy. sFlt-1, a soluble form of the extracellular domain of the Flt-1 receptor, is up-regulated in the blood of preeclamptic women and functions as a trap for vascular endothelial growth factor (VEGF), preventing healthy VEGF signaling to endothelial cells and contributing to endothelial dysfunction. Previously, our lab has shown that administration of a VEGF isoform, VEGF-A, bound to a carrier protein called elastin-like polypeptide (ELP), significantly reduced free s-Flt1 levels and normalized blood pressure in a rodent model of preeclampsia. However, VEGF-A administration has a host of side effects mediated thorough its activation of the Flk-1 receptor. In response to this, we have developed a separate therapeutic chimera which utilizes ELP bound to a different isoform of VEGF, VEGF-B, that has no known interaction with the Flk-1 receptor but still possess the potential to bind to sFlt-1. In this study we have conducted pharmacokinetic, biodistribution, and efficacy experiments to test our hypothesis that a biopolymer chimera of VEGF-B (ELP-VEGF-B) would function as a sequestrant of sFlt-1 in order to lower blood pressure in the reduced uterine perfusion pressure (RUPP) model of preeclampsia. Methods Pharmacokinetics were determined by injecting pregnant rats with labeled protein and measuring clearance by repeated tail vein blood draws. Biodistribution and placental transfer of ELP-VEGF-B was determined by ex vivo imaging of organs, placentae, and pups of animals injected with labeled protein using an IVIS Spectrum In Vivo Imaging System (IVIS). Efficacy studies were conducted in pregnant SD rats from day 13 to day 18 of gestation. Pregnant SD rats received either the RUPP or sham procedure and were subsequently administered ELP-VEGF-B (50mg/kg) or sterile saline, subcutaneously, on days 13, 15, and 17 of gestation. Body weights were monitored daily, and blood pressure was measured, through carotid catheters, on day 18 of gestation. Animals were then sacrificed and harvested on day 18 of gestation. Results Plasma clearance of ELP-VEGF-B fit well into a two-compartment pharmacokinetic model, resulting in half lives of 48.4hr(terminal) and 0.22hr (distribution). ELP-VEGF-B accumulated primarily in the maternal kidney, liver, and placenta, with undetectable amounts in the pups. ELP-VEGF-B administration had no significant effect on maternal body weight, pup weight, or placental weight amongst any of the treatment or control groups. However, administration of ELP-VEGF-B in a rodent model of placental ischemia did achieve a significant reduction in blood pressure when compared to saline treated animals following induction of placental ischemia (123.38 ± 11.4 versus 139.98 ± 10.56 mmHg, p=0.0129). Conclusions The biopolymer fusion peptide, ELP-VEGF-B, has a pharmacokinetic profile similar to our previously tested therapeutic, ELP-VEGF-A. Additionally, ELP-VEGF-B was sequestered from crossing the placenta and was able to significantly reduce blood pressure in a rodent model of placental ischemia.

Dual-Targeted Extracellular Vesicles to Facilitate Combined Therapies for Neuroendocrine Cancer Treatment.

Neuroendocrine (NE) cancers arise from cells within the neuroendocrine system. Chemotherapies and endoradiotherapy have been developed, but their clinical efficacy is limited. The objective of this study was to develop a dual-targeted extracellular vesicles (EV)-delivered combined therapies to treat NE cancer. Specifically, we produced EV in stirred-tank bioreactors and surface tagged both anti-somatostatin receptor 2 (SSTR 2) monoclonal antibody (mAb) and anti-C-X-C motif chemokine receptor 4 (CXCR4) mAb to generate mAbs-EV. Both live-cell confocal microscopy imaging and In Vivo Imaging System (IVIS) imaging confirmed that mAbs-EV specifically targeted and accumulated in NE cancer cells and NE tumor xenografts. Then the highly potent natural cytotoxic marine compound verrucarin A (Ver-A) with IC50 of 2.2–2.8 nM and microtubule polymerization inhibitor mertansine (DM1) with IC50 of 3.1–4.2 nM were packed into mAbs-EV. The in vivo maximum tolerated dose study performed in non-tumor-bearing mice indicated minimal systemic toxicity of mAbs-EV-Ver-A/DM1. Finally, the in vivo anticancer efficacy study demonstrated that the SSTR2/CXCR4 dual-targeted EV-Ver-A/DM1 is more effective to inhibit NE tumor growth than the single targeting and single drug. The results from this study could expand the application of EV to targeting deliver the combined potent chemotherapies for cancer treatment.

Anti-EGFR antibody-drug conjugate for triple-negative breast cancer therapy.

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

Abstract 2788: Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics

Abstract Background: The rapid development of immuno-oncology (I/O) therapies for various cancer types has transformed the paradigm of cancer treatment from targeting the tumor to targeting the immune system, and now to combination strategies. Numerous Phase I-III trials with immunotherapeutics and combinations are being conducted; however, a lot of these are failing. More preclinical models are required to help investigate efficacy, uncover mechanisms of action, and to design more rational combination strategies. The most widely used models for immunotherapy evaluation are models with functional murine immunity such as syngeneic allograft models and genetically engineered mouse models (GEMMs). Syngeneic models are used in nearly all I/O therapy programs as these are well-characterized and straightforward to set-up as subcutaneous models. However, one of the major disadvantages of subcutaneous models is the lack of a clinically relevant tumor microenvironment (TME) including stromal cells, immune cells, extracellular matrix etc. The dynamic interplay of these cells drive alterations in cellular functions and impact study outcomes. Using bioluminescent imaging (BLI), we have developed and characterized a panel of orthotopic models where tumor cells are inoculated in a relevant organ-specific location to recapitulate the immune and stromal component interactions with the tumor, which can also facilitate metastatic spread. These models can be used to evaluate various therapies in both the orthotopic and metastatic settings. Method: A panel of syngeneic cell lines were labeled with bioluminescence by transducing the cell lines with a lentiviral vector carrying the firefly luciferase gene. This panel represents a diverse range of cancer types including liver (Hepa 1-6 and HT22), breast (4T1), colon (CT26.WT), brain (GL261), prostate (RM-1), and pancreas (Pan02). These bioluminescent cells were orthotopically implanted into immune competent mice. In-life tumor growth and metastasis were assessed using the IVIS® Spectrum In Vivo Imaging System. Tumor-infiltrating lymphocytes were assessed by flow cytometry (BD LSRFortessa™) and multiplex IHC. Results: Orthotopic implantation of syngeneic models was successfully demonstrated for all models as confirmed by in-life imaging and ex vivo imaging at termination. Baseline response towards various checkpoint inhibitors and chemotherapies were established. For the Hepa 1-6 liver model, response to an anti-PD-1 antibody was significant in both the subcutaneous and orthotopic setting. In contrast, the H22 liver model responded to both sorafenib and anti-mCTLA-4 antibody in the subcutaneous model but not in the orthotopic setting. In addition, anti-mCTLA-4 treatment led to more significant inhibition of pancreatic Pan02 tumor growth in the orthotopic model in comparison with its corresponding subcutaneous setting. Conclusion: Bioluminescent syngeneic models in the orthotopic setting provide a valuable tool for testing immunotherapies, with a more clinically relevant TME in comparison to the corresponding subcutaneous model. The establishment of a range of models covering diverse cancer types allows for a thorough interrogation of immunotherapeutic potential. Citation Format: Diandong Jiang, Bryan Miller, Yanrui Song, Rajendra Kumari, Annie An, Jie Cai, Davy Xuesong Ouyang, Henry Qixiang Li, Yinfei Yin. Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2788.

Temozolomide and Pazopanib Combined with FOLFOX Regressed a Primary Colorectal Cancer in a Patient-derived Orthotopic Xenograft Mouse Model

Purpose: The goal of the present study was to determine the efficacy of temozolomide (TEM) and pazopanib (PAZ) combined with FOLFOX (oxaliplatin, leucovorin and 5-fluorouracil) on a colorectal cancer patient-derived orthotopic xenograft (PDOX) mouse model. Materials and Methods: A colorectal cancer tumor from a patient previously established in non-transgenic nude mice was implanted subcutaneously in transgenic green fluorescence protein (GFP)-expressing nude mice in order to label the tumor stromal cells with GFP. Then labeled tumors were orthotopically implanted into the cecum of nude mice. Mice were randomized into four groups: Group 1, untreated control; group 2, TEM + PAZ; group 3, FOLFOX; group 4, TEM + PAZ plus FOLFOX. Tumor width, length, and mouse body weight were measured weekly. The Fluor Vivo imaging System was used to image the GFP-lableled tumor stromal cells in vivo. H&E staining and immunohistochemical staining were used for histological analysis. Results: All three treatments inhibited tumor growth as compared to the untreated control group. The combination of TEM + PAZ + FOLFOX regressed tumor growth significantly more effectively than TEM + PAZ or FOLFOX. Only the combination of TEM + PAZ + FOLFOX group caused a decrease in body weight. PAZ suppressed lymph vessels density in the colorectal cancer PDOX mouse model suggesting inhibition of lymphangiogenesis. Conclusion: Our results suggest that the combination of TEM + PAZ + FOLFOX has clinical potential for colorectal cancer patient.

Pazopanib Inhibits Tumor Growth, Lymph-node Metastasis and Lymphangiogenesis of an Orthotopic Mouse of Colorectal Cancer.

Pazopanib (PAZ) can inhibit tumor progression, but whether PAZ inhibits lymph node metastasis and lymphangiogenesis in colorectal cancer is still unknown. The aim of the present study was to determine the efficacy of PAZ on tumor growth, lymph node metastasis and lymphangiogenesis in an orthotopic nude mouse model in colorectal cancer. CT-26-green fluorescence protein (GFP)-expressing mouse colon cancer cells were injected into nude mice to establish a subcutaneous colorectal cancer model and were treated with saline and PAZ. Additionals subcutaneous tumors were harvested and cut into 5 mm3 fragments, then tumor fragments were implanted orthotopically in the cecum to establish an orthotopic colorectal-cancer nude mouse model. Orthotopic mice were randomized into two groups for the treatment with saline and PAZ, respectively. Tumor width, length and mouse body weight was measured twice a week. The Fluor Vivo imaging system was used to image the GFP. Hematoxylin & eosin staining and immunohistochemical staining was used for histological analysis. PAZ inhibited the growth of subcutaneous colorectal cancer, as wells as orthotopic transplanted colorectal cancer tumors. PAZ suppressed lymph node metastasis and lymphangiogenesis in the orthotopic colon cancer model. No significant changes were observed in the body weight between the control and the mice treated with PAZ. PAZ can inhibit the growth of colorectal cancer and inhibit lymph node metastasis and lymphangiogenesis in orthotopic colon cancer nude mouse models.

Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp.

This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.

A network pharmacology approach to discover action mechanisms of Yangxinshi Tablet for improving energy metabolism in chronic ischemic heart failure.

Most cardiovascular diseases ultimately result in heart failure, an intractable problem in modern medicine. Yangxinshi tablet (YXS) is a Chinese medicine formula that is used clinically to treat coronary heart disease. However, the active compounds, potential targets, and pharmacological and molecular mechanism of its anti-heart failure activity remain unclear. Therefore, further investigation is required. Active ingredients and potential targets of YXS for treating heart failure have been reported previously. However, the molecular functions or biological processes of YXS in energy metabolism have not been discovered. To date, no experimental study to validate the potential anti-heart failure mechanism of YXS. The aim of this study was to study the therapeutic effect of YXS on rats with chronic ischemic heart failure by evaluating rat cardiac function and exercise tolerance, and to explore its potential mechanism by network pharmacology, western blotting, quantitative RT-PCR and histological analysis. In this investigation, chronic ischemic heart failure rats were randomly assigned to five groups: control group (sham operation), model group (0.5% CMC-Na), trimetazidine group (positive control) and two YXS groups (low- and high-dose groups). Experimental rats were treated by gavage with 10 mg/kg/d (clinical equivalent dose) trimetazidine (TMZ), 500 mg/kg/d (clinical equivalent dose) YXS and 1000 mg/kg/d YXS, respectively, for 5 weeks. The cardiac functions of rats were detected by High-Resolution In Vivo Imaging System. We elucidated novel understanding of the active compounds of YXS in rat plasma and predicted the energy metabolism related targets and processes for heart failure. Then, we validated experimentally the targets and mechanism of YXS on these pathological processes in vivo. It was found that YXS was able to effectively improve cardiac LVIDs, LVEDV, LVESV and EF, decrease myocardial oxygen consumption and reduce myocardial infarct size in rats with chronic ischemic heart failure was similar to that of TMZ. We identified 63 major candidate targets for YXS that are closely to heart failure progression. Enrichment analysis revealed key targets for YXS associated to oxygen delivery, glucose utilization, and mitochondrial biogenesis. Meanwhile, we validated that YXS could promote the expression of downstream HIF-1α, PGC1α and GLUT4 by increasing phosphorylation of PI3K, Akt, mTOR, rpS6 and AMPK. The results show that YXS could activate related PI3K/Akt/mTOR/rpS6/HIF-1α and AMPK/PGC1α/GLUT4 signaling pathways in chronic ischemic heart failure rats. Further experiments demonstrated that YXS increased mitochondrial biogenesis in chronic ischemic heart failure rats and improved exercise tolerance CONCLUSION: YXS treated chronic ischemic heart failure through activating its targets which play pivotal roles in oxygen delivery, glucose utilization and mitochondrial biogenesis to improve energy metabolism through a multi-component, multi-level, multi-target, multi-pathway and multi-mechanism approaches.

Abstract 2994: In vivo targeting of mesothelioma’s molecular signature with nanoparticle functionalized with nucleolin-binding peptide

Abstract Mesothelioma is a malignant tumor of the mesothelium, often associated with asbestos exposure, with an overall survival of only 8.8 months. A combination of cisplatin and pemetrexed has been used as an adjuvant treatment to surgical resection or in patients who have inoperable disease. This combination remains as the current standard of care, regardless the sub-type of mesothelioma, with only a 9% 5-year survival rate. Herein, it is hypothesized that a novel targeted treatment, based on a doxorubicin (DXR)-containing nanoparticle functionalized with the nucleolin-binding F3 peptide (named PEGASEMP), will have significant a therapeutic impact against the most aggressive subtype of mesothelioma (biphasic). The hypothesis relies on nucleolin deregulated overexpression in cancer and endothelial cells from tumor blood vessels. Human biphasic mesothelioma cells were stably transduced with luciferase-reporter gene, and orthotopically injected intrapleurally into female immunocompromised mice. Three weeks (Early Stage) and 4 weeks (Advanced Stage) after cell injection, animals were randomly allocated to different treatment groups: vehicle, PEGASEMP at 5.6 or 7 mg of DXR/kg alone (q7dx5w); cisplatin at 4.0 mg/kg alone or combined with PEGASEMP at 5.6 mg of DXR/kg (q7dx5w); and the standard of care, a combination of cisplatin at 4.0 mg/kg (q7dx5w) plus pemetrexed at 100.0 mg/kg (q2dx3x5w). Bioluminescence was monitored weekly with IVIS Spectrum In Vivo Imaging system. Differential gene expression upon PEGASEMP treatment of advanced stage tumors was also performed in comparison with non-treated animals using the GeneChip Human Transcriptome Array 2.0. In the early stage of development, PEGASEMP, at 7.0 mg/kg, inhibited tumor growth by 2,713-fold relative to standard of care, which translated into a 1223-fold decrease in tumor burden. At an advanced stage of tumor development, the combination of PEGASEMP and cisplatin enabled 107-fold tumor growth inhibition, enabling a 55-fold reduction in tumor burden, relative to standard of care. In both cases, no decrease of the body weight was observed. Furthermore, 75% (146) gene transcripts were found to be deregulated (while the remaining 25% were upregulated), involving the downregulation of cell division, including ontologies such as cell cycle or chromatin organization and assembly. Specifically, downregulation of topoisomerase 2, cyclin B1 or Ki67 transcripts, part of the described molecular signature of mesothelioma, was observed and was consistent with the mechanism of action of doxorubicin. Overall, the novel mechanism of action associated with PEGASEMP, enables a significant benefit in terms of efficacy and safety in the treatment of biphasic mesothelioma, as compared with the current standard of care, affecting the molecular signature of the disease and thus supporting future clinical evaluation. Citation Format: Nuno A. Fonseca, Vera Moura, Fabiana Colelli, Daniela Pesce, Giacomo Signorino, Laura Focareta, Alessandra Fucci, Francesco Cardile, Claudio Pisano, Sérgio Simões, João Nuno Moreira. In vivo targeting of mesothelioma’s molecular signature with nanoparticle functionalized with nucleolin-binding peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2994.

Abstract 1421: Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells. METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo. CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC. Citation Format: Teruki Shimizu, Mako Tomogane, Yusuke Sano, Daiki Shimizu, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara. Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1421.

Abstract 537: Antibody drug conjugate to treat triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is characterized by rapid growth, metastasis, and high recurrence. The severe adverse effects and drug resistance associated with standard cytotoxic chemotherapies minimize their clinical benefits. The anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) had been developed and tested to treat TNBC, but no significant efficiency was demonstrated in clinical trials. The goal of this study was to develop a novel antibody-drug conjugate (ADC) to treat TNBC. Specifically, we established a platform to conjugate anti EGFR mAb with mertansine, which inhibits cell division by blocking tubulin polymerization, and also improved the conjugation efficiency by optimizing the linker and drug-to-antibody ratio. Flow cytometry and live cell confocal laser scanning microscopy analysis confirmed that the anti-EGFR ADC can specifically bind to multiple TNBC subtypes, is effectively internalized, and release potent drug intracellularly within 1 hour after mixing. The IC50 study showed that the anti-EGFR ADC killed &amp;gt;90% TNBC cells with dose of 5 nM. The TNBC xenograft mouse model also showed that the constructed ADC had high anticancer efficacy and successfully inhibited tumor growth. Moreover, the live-animal imaging using In Vivo Imaging System and the tissue microarray samples with immunohistochemical analysis of TNBC and organs indicated that our ADC specifically target TNBC in vivo. Further evaluations, such as maximal tolerated dose (MTD), pharmacokinetics (PK), and biodistribution, are needed. To summarize, we developed an EGFR-targeted ADC, which could provide an effective targeted therapy to treat TNBC and improve the life quality and survival rate of patients with TNBC. Note: This abstract was not presented at the meeting. Citation Format: Yingnan Si, Nghi Dang, Seulhee Kim, Lufang Zhou, Xiaosi Han, Runhua Liu, Xiaoguang (Margaret) Liu. Antibody drug conjugate to treat triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 537.

Pegilodecakin, a Pegylated Human IL-10 (AM0010), Enhances the Cytotoxicity of CAR-T Cells in Vitro and In Vivo

▪IntroductionClinical outcomes with immune cell therapies, including chimeric antigen receptor (CAR) T cells, rely on expansion of the anti-tumor T cells for successful therapeutic outcomes. Recent studies on Pegilodecakin, a pegylated interleukin-10 (IL-10), have demonstrated that doses greatly exceeding typical endogenous levels can drive a productive tumor specific T cell expansion and response. In a large Phase I/Ib study, Pegilodecakin achieved objective responses across multiple tumor types, alone and in combination with chemotherapies and immune checkpoint inhibitors, including Programmed Cell Death-1 (PD-1). Agents that improve the functional expansion of CAR-T cells, post adoptive transfer, hold promise to improve the therapeutic efficacy of the CAR-T therapies in patients. Here we report on early studies which demonstrate that Pegilodecakin significantly enhances the anti-tumor cytotoxic T lymphocyte (CTL) activity of CAR-T cells.MethodsIn Vitro RTCA Assays: The engineered Primary CD19 CAR-T cells were generated by isolating human T cells from whole blood and transducing these T cells with a CD19-targeted CAR. The CD19 CAR-T cells were then activated, expanded, and tested in a Real-time Cytotoxicity Assay (RTCA) against HeLa cells stably expressing human CD19 (CD19-HeLa), at multiple effector-to-target (E:T) ratios. The CTL activity or cytotoxicity was measured through cell-sensor impedance in an electronic microtiter plate. Cytotoxicity was measured for CD19 CAR-T alone or in combination with varied concentrations of Pegilodecakin, and was directly compared with controls, including Pegilodecakin alone or Pegilodecakin with non-transduced T cells. To functionally validate the CTL activity, we measured the levels of Granzyme-B and Interferon-gamma (IFNg) in the culture supernatants at the end of the RTCA by ELISA.In Vivo Raji Leukemia Model: 6-8 week old female NSG Mice NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NOD scid gamma), 5 mice per group, were injected with 0.5E6 Raji cells engineered to express luciferase (Raji-luc) on Day 0, IV. Tumor progression was imaged on Day 0 and weekly thereafter for 6 weeks using the Spectrum In Vivo Imaging System (IVIS). Mice were treated with varying numbers of non-transduced/CD19 CAR-T cells alone or in combination with 0.5mg/kg Pegilodecakin. Specifically, non-transduced T/CD19 CAR-T cells were administered on Days 2 and 9, with daily Pegilodecakin where indicated. Blood was collected weekly for immunophenotypic analyses. Serum was analyzed for IFNg and Granzyme B via ELISA.ResultsThe RTCA assay revealed that Pegilodecakin in combination with the CD19 CAR-T showed a significant increase (p<0.001) in CTL activity against CD19-HeLa cells as compared to the CD19-CAR-T alone. Functionally, when combined with Pegilodecakin, CD19-CAR-T cells produced significantly higher levels of Granzyme-B (p<0.0005) and IFNg (p<0.02) in the RTCA Cytotoxicity Assay. Controls, including Pegilodecakin alone (without CAR-T or non-transduced T cells) and non-transduced T cells in combination with Pegilodecakin, had significantly lower CTL activity by RTCA or ELISA (Granzyme-B or IFNg).In the Raji-leukemia model, the Pegilodecakin combination with 5E6 CD19 CAR-T had a significant tumor control benefit by IVIS imaging, as directly compared with CD19 CAR-T monotherapy. A modest dose dependent benefit with 2.5E6 CD19 CAR-T was also observed.ConclusionsWe demonstrate that when combined with Pegilodecakin, the cytotoxic activity of the CD19-CAR-T is significantly improved in in vitro and in vivo models. Also, this combination demonstrates significantly improved functional activity of these CD19-CAR T cells via enhanced Granzyme-B and IFNg activity. The in vivo synergistic activity of Pegilodecakin with CD19 CAR-T demonstrates superior tumor control with potentially better depth of response. These results suggest Pegilodecakin/CAR-T therapy combination may provide longer in vivo persistence of the CAR T products offering better clinical outcomes. DisclosuresMcCauley:ARMO Biosciences: Employment. Verma:ARMO Biosciences: Employment. Oft:ARMO Biosciences: Employment.