Tumor-implanted Mice Research Articles

Abstract 2808: Levels of isobutyric acid from gut butyrate-producing taxa are associated to Delta-24-RGDOX efficacy against malignant glioma

Abstract Glioblastoma (GBM) is the most aggressive malignant primary brain tumor worldwide. Current standard-of-care treatments allow for a median survival of 12 to 18 months. GBM has a poor survival prognosis, observing less than 6% of patients surviving 5 years post-diagnosis. Among recent treatment advancements, the oncolytic adenovirus Delta-24-RGDOX armed with the T-cell activator OX40L is promising. Studies have unveiled a relationship between the gut microbiome and high levels of short-chain fatty acids (SCFA), such as isobutyric and propionic acid, to immunotherapy response. Changes in the gut microbiome and metabolome associated to viroimmunotherapy efficacy remain unclear. Here, we assessed the gut microbial changes in addition to isobutyric and propionic acid levels regarding viroimmunotherapy efficacy. Immunocompetent C57BL/6 mice were intracranially implanted GL261-5 GBM cells and received intratumoral injections of PBS (control) or Delta-24-RGDOX. A naïve group with no tumor nor treatment was also included. Fecal pellets were collected at three different time points: (1) before tumor implantation, (2) before treatment, and (3) 14 days after the first dose of treatment. Genomic DNA was isolated with the QIAGEN DNeasy Powersoil Pro Kit from fecal pellets (n=75), followed by sequencing with the Illumina platform of the 16S ribosomal RNA V4 region. Quality assessment of the sequences was executed in QIITA. Bacterial community analysis with a sequencing depth of 1,470 reads, while using the taxonomic database SILVA, was followed downstream with QIIME2 and R. Metabolic profiling of SCFA of the fecal pellets was performed through Gas Chromatography. Results showed significant differences in bacterial community structure of viroimmunotherapy-treated mice with survival of >100 days compared to controls (ANOSIM p-value=0.006). Alpha diversity metrics showed significant differences in richness between the naive and tumor-implanted mice of the PBS (KW p-value=0.049) and Delta-24-RGDOX (KW p-value=0.019) groups, highlighting that tumor presence alters the gut microbiota. Additionally, viroimmunotherapy-treated mice with survival of >100 days had a significant increase of Lactobacillus and the butyrate-producing bacterias Erysipelatoclostridium and Ruminococcaceae. Also, we found significantly higher levels of isobutyric acid and lower levels of propionic acid in viroimmunotherapy-treated mice with survival of >100 days (p-value<0.05). These findings suggest that butyrate-producing bacteria coupled with higher levels of isobutyric acid may be important in the efficacy of Delta-24-RGDOX. This highlights the microbial and metabolic changes associated with oncolytic viral therapy efficacy. Sponsored by an award within The University of Puerto Rico/UT MD Anderson Cancer Center Partnership for Excellence in Cancer Research Grant (2U54CA096297-17). Citation Format: Natalie M. Meléndez-Vázquez, Xuejun Fan, Juan Fueyo, Candelaria Gomez-Manzano, Filipa Godoy-Vitorino. Levels of isobutyric acid from gut butyrate-producing taxa are associated to Delta-24-RGDOX efficacy against malignant glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2808.

A hepatitis B virus-derived peptide combined with HBsAg exerts an anti-HBV effect in an HBV transgenic mouse model as a therapeutic vaccine.

For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant. In this study, we explored the potential of Poly6 in combination with HBsAg as a therapeutic vaccine against hepatitis B virus infection. We investigated the immunotherapeutic potential of Poly6 combined with HBsAg vaccination against hepatitis B virus infection in C57BL/6 mice or an HBV transgenic mouse model. In C57BL/6 mice, Poly6 enhanced DC maturation and DC migration capacity in an IFN-I-dependent manner. Moreover, the addition of Poly6 to alum in combination with HBsAg also led to enhanced HBsAg-specific cell-mediated immune (CMI) responses, suggesting its potential as an adjuvant of HBsAg-based vaccines. In HBV transgenic mice, vaccination with Poly6 combined with HBsAg exerted a strong anti-HBV effect via induction of HBV-specific humoral and cell-mediated immune responses. In addition, it also induced HBV-specific effector memory T cells (TEM). Our data indicated that vaccination with Poly6 in combination with HBsAg exerts an anti-HBV effect in HBV transgenic mice, which is mainly mediated by HBV-specific CMI and humoral immune responses via IFN-I-dependent DC activation, suggesting the feasibility of Poly6 as an adjuvant for an HBV therapeutic vaccine.

Co-Administration of nalbuphine to improve morphine tolerance in mice with bone cancer pain.

Kappa-opioid receptor (KOR) agonists are known for having opposite and/or different effects compared with Mu-opioid receptor (MOR) agonists. This study is aimed at clarifying the analgesic effect and tolerance of nalbuphine combined with morphine, and quantifying the mRNA and protein expression of spinal MOR and KOR in a mouse bone cancer pain (BCP) model treated with nalbuphine and morphine. BCP model was prepared in C3H/HeNCrlVr Mice by implanting the sarcoma cells into the intramedullary space of the femur. The paw withdrawal thermal latency (PWL) measured by thermal radiometer was used to assess thermal hyperalgesia. PWL testing was performed after implantation and drug administration according to the protocol. Hematoxylin-eosin staining in the spinal cord and x-ray in the femoral intramedullary canal was detected. Real-time PCR and western blot analysis played a role in detecting spinal MOR and KOR expression changes. In tumor-implanted mice, the spinal MOR and KOR protein and mRNA expression was down-regulated when compared to that in sham-implanted mice (p < 0.05). Morphine therapy can lead to a decrease in spinal μ receptor expression. Similarly, the nalbuphine therapy can lead to a decrease in the expression of κ receptor protein and mRNA at the spinal cord level (p < 0.05). Morphine, nalbuphine, or nalbuphine co-administration with morphine all can extend the paw withdrawal thermal latency (PWL) to radiant thermal stimulation in tumor-implanted mice (p < 0.05). Compared with the morphine treatment group, nalbuphine co-administration with morphine delayed the reduction of PWL value again (p < 0.05). BCP itself may induce down-regulation of the spinal MOR and KOR expression. A low dose of nalbuphine co-administration with morphine led to the delayed emergence of morphine tolerance. The part of the mechanism may be due to the regulation of spinal opioid receptors expression.

TMIC-59. GLIOBLASTOMA INDUCES THE DIFFERENTIATION AND RECRUITMENT OF NON-CANONICAL ANTI-TUMORAL NEUTROPHILS FROM SKULL BONE MARROW

Abstract The effects of tumor-associated neutrophils (TANs) on glioblastoma biology remain poorly understood. Flow cytometric analysis of 5 newly diagnosed glioblastoma fresh tissue specimens surprisingly revealed a high fraction (21.0-34.1%) of TANs expressed MHCII, a marker of antigen-presenting cells not classically associated with neutrophils and not expressed in matched peripheral blood (PBNs). Transcriptomic profiling confirmed that patient TANs upregulated expression of MHCII subunits (HLA-DR), chaperones (HLA-DM), and costimulatory ligands (CD86/83). Ex vivo cocultures further demonstrated that TANs activated patient-matched naïve T cells in an MHCII-dependent manner, while PBNs did not (CD25 MFI fold-change: 1.1 vs 3.2, p&amp;lt; 0.001). The antitumoral relevance of this property was confirmed in a syngeneic mouse glioma model, wherein αLy6G-mediated neutrophil depletion in T-cell-competent mice (Balb/c, n= 13) yielded endpoint tumors that had reduced CD8+ T cell infiltration (p= 0.0024) and were 2.4-fold larger by BLI (p= 0.0383) than controls, but had no bearing on tumor burden in T-cell-deficient (athymic) mice. Given the absence of MHCII+ neutrophils in circulation, we interrogated the inducibility of this phenotype by tumor-conditioned media in murine marrow/blood, finding that only immature Ly6Glow bone marrow neutrophils had sufficient plasticity to express MHCII and process foreign antigen as measured by DQ-ovalbumin uptake/proteolysis. Because of the non-inducibility of PBNs, we investigated the hypothesis that MHCII+ TANs were recruited to the glioblastoma microenvironment from adjacent skull bone marrow by labeling this space with CFMDA in tumor-implanted mice; at 72h, skull marrow-derived neutrophils contributed disproportionately to MHCII+ TANs compared to all TANs (41.7% vs 9.3%). As confirmation, in the first-ever scRNA-seq of human TANs, we demonstrated via pseudotime analysis that MHCII+ neutrophils represent a developmental lineage seen in TANs but not matched PBNs. Given the immunostimulatory effects of this population, mechanisms to promote neutrophil egress and tumor infiltration from the skull marrow may have therapeutic value.

An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation

Therapeutic cancer vaccines (TCVs) should induce robust tumor-specific T cell responses. To achieve this, TCVs incorporate T cell epitopes and strong adjuvants. Here, we report an all-in-one adjuvanted cancer vaccine platform that targets the intracellular compartment of dentritic cells and subsequently induces effective cytotoxic T cell responses. We screened a novel peptide (DCpep6) that specifically binds and transmits into CD11c+ cells through a novel in vivo phage biopanning. We then engineered a protein-based TCV (DEF) consisting of DCpep6 (D), an optimized HPV E7 tumor antigen (E), and a built-in flagellin adjuvant (F) as a single molecule. DEF was stably expressed, and each component was functional. In vivo-administered DEF rapidly biodistributed in draining LNs and internalized into CD11c+ cells. DEF immunization elicited strong antitumor T cell responses and provided long-term survival of TC-1 tumor-implanted mice. The DEF-mediated antitumor effect was abolished in NLRC4−/− mice. Taken together, we propose a protein-based all-in-one TCV platform that intracellularly codelivers tumor antigen and inflammasome activator to DCs to induce long-lasting antitumor T cell responses.

Enhanced Antitumor Immune Responses via a Self-Assembled Carrier-Free Nanovaccine.

Nanovaccines have emerged as promising agents for cancer immunotherapy. However, insufficient antitumor immunity caused by inefficient antigen/adjuvant loading and complicated preparation processes are the major obstacles that limit their clinical application. Herein, two adjuvants, monophosphatidyl A (MPLA) and CpG ODN, with antigens were designed into a nanovaccine to overcome the above obstacles. This nanovaccine was constructed with adjuvants (without additional materials) through facile self-assembly, which not only ensured a high loading efficacy and desirable safety but also facilitated clinical translation for convenient fabrication. More importantly, the selected adjuvants could achieve a notable immune response through synergistic activation of Toll-like receptor 4 (TLR4) and TLR9 signaling pathways, and the resulting nanovaccine remarkably inhibited the tumor growth and prolonged the survival of tumor-implanted mice. This nanovaccine system provides an effective strategy to construct vaccines for cancer immunotherapy.

Over 1000 nm Near-Infrared Multispectral Imaging System for Laparoscopic In Vivo Imaging.

In this study, a laparoscopic imaging device and a light source able to select wavelengths by bandpass filters were developed to perform multispectral imaging (MSI) using over 1000 nm near-infrared (OTN-NIR) on regions under a laparoscope. Subsequently, MSI (wavelengths: 1000–1400 nm) was performed using the built device on nine live mice before and after tumor implantation. The normal and tumor pixels captured within the mice were used as teaching data sets, and the tumor-implanted mice data were classified using a neural network applied following a leave-one-out cross-validation procedure. The system provided a specificity of 89.5%, a sensitivity of 53.5%, and an accuracy of 87.8% for subcutaneous tumor discrimination. Aggregated true-positive (TP) pixels were confirmed in all tumor-implanted mice, which indicated that the laparoscopic OTN-NIR MSI could potentially be applied in vivo for classifying target lesions such as cancer in deep tissues.

Applications of Mesoporous Silica-Encapsulated Gold Nanorods Loaded Doxorubicin in Chemo-photothermal Therapy.

We investigate the chemo-photothermal effects of gold nanorods (GNRs) coated using mesoporous silica (mSiO2) loading doxorubicin (DOX). When the mesoporous silica layer is embedded by doxorubicin drugs, a significant change in absorption spectra enables to quantify the drug loading. We carried out photothermal experiments on saline and livers of mice having GNRs@mSiO2 and GNRs@mSiO2-DOX. We also injected the gold nanostructures into many tumor-implanted mice and used laser illumination on some of them. By measuring the weight and size of tumors, the distinct efficiency of photothermal therapy and chemotherapy on treatment is determined. We experimentally confirm the accumulation of gold nanostructures in the liver.

Abstract B81: Efficacy of nivolumab, pembrolizumab, and atezolizumab against MC38 colon cancer expressing human PD-1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes

Abstract The human checkpoint genes PD-1 and PD-L1 have been the target of extensive research for the treatment of human malignancies. Checkpoint inhibitor therapy has shown great promise in the clinic. Animal models that can use the human clinical antibodies will be beneficial for evaluating new therapies. A novel transgenic mouse model was developed that expresses the human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We evaluated the efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. The tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 μg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. We have shown that both PD-1 and PD-L1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. Citation Format: Murray Stackhouse, Ted Green, Jay Liu, Charlotte Hammond, LaJuana Durbin, Anna Chen, Mike Koratich. Efficacy of nivolumab, pembrolizumab, and atezolizumab against MC38 colon cancer expressing human PD-1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B81.

Laser Therapy Enhances Blood-Brain Barrier and Blood-Tumor Barrier Permeability in a Mouse Model of Glioblastoma

Abstract INTRODUCTION A main challenge in the treatment of glioblastoma is the blood-brain barrier (BBB). In this study, we established a laser interstitial thermotherapy (LITT) platform in mice to test the hypothesis that LITT can increase BBB and blood-tumor barrier (BTB) permeability, which could potentially be exploited to enhance delivery of antitumor agents. METHODS We established a preclinical mouse model for LITT using an Nd-YAG laser coupled to a 600 μm fiber optic and thermocouple probe in C57BL/6 mice. Both the fiber optic and temperature probes were inserted through a small burrhole to target somatosensory cortex. In other experiments, mice were stereotactically implanted with syngeneic GL261 tumor cells prior to LITT. Post-LITT, brain tissue was assessed for BBB and BTB permeability through quantitative measurement of brain fluorescein (FL) after intraperitoneal injection and immunohistochemistry (IHC) after intravenously injected dextran (MWs 10 and 70 kDa) and human IgG (MW 150 kDa) to define spatial characteristics. Electron microscopy and IHC was used to delineate the mechanisms of BBB break down. RESULTS Using BBB permeability tracers, noninjected mice and mice orthotopically implanted with GL261 tumor cells were used to characterize the effect of laser therapy on the BBB and BTB, respectively. We found LITT substantially disrupts the BBB in the first day post-treatment and is sustained for 4 wk. Additionally, tumor-implanted mice demonstrated an increase in Dextran 10 and 70 kDa and human IgG extravasation after IV injection of these tracers. Mechanistically, we provide evidence, by IHC and electron microscopy, that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis to increase overall BBB permeability. CONCLUSION Our data suggest that LITT increases BBB and BTB permeability over a defined time window in a mouse model and that large molecular size agents, including human IgG, can infiltrate brain parenchyma and brain tumors after systemic administration. Future experiments will focus on testing delivery of antitumor agents following LITT to enhance survival in brain tumor-bearing mice.

Abstract 1504: Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1

Abstract The human checkpoint targets PD-1 and PDL-1 continue to demonstrate great promise in the clinic. With much of the current focus turning toward combination regiments, an animal model using the human clinical antibodies will be beneficial for evaluating new combination strategies. Here we continue characterization of a C57BL/6 transgenic mouse model expressing both the human PD-1 and PD-L1 combined with a modified murine MC38 colon tumor cell line expressing human PD-L1. Both PD-1 and PD-L1 expression of the transgenic mice were verified in ex vivo stimulated splenocytes by flow cytometry analysis. Expression of PD-L1 on the genetically modified MC38 cells was also demonstrated by flow cytometry. For in vivo efficacy evaluation, tumor-implanted mice were treated with the clinical agents nivolumab and pembrolizumab at 100 µg and atezolizumab at 1 mg on Days 3, 7, 10, and 14 post implant. Treatment with nivolumab and pembrolizumab initiated tumor regression by Day 17. Complete tumor regressions were seen at Day 28 in nivolumab, 62.5% complete regression, and pembrolizumab, 71.4% complete regression. Growth inhibition was 83.9%, 69.7%, and 95.0% on Day 28 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To compare the human anti-PD-1 and PD-L1 clinical agent specificity, the non-transgenic parent C57BL/6 mouse strain was implanted with the unmodified MC38 colon tumor cells. Treatment with pembrolizumab and atezolizumab was conducted as with the transgenic animals. Through 28 days no growth inhibition, tumor size 105.9% of control, was seen in the pembrolizumab treated group, demonstrating lack of cross reactivity of the human therapeutic in the standard mouse model. Atezolizumab did demonstrate a 56.9% growth inhibition compared to controls and is consistent with the known cross reactivity of atezolizumab between human and mouse. We have shown a genetically modified MC38 colon tumor expressing human PD-L1 in transgenic mice expressing both human PD-1 and PD-L1 to be a suitable model for checkpoint inhibitor evaluation of the human form of the particular checkpoint therapeutic. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. Citation Format: Michael Koratich, Ted Green, Jie Liu, Charlotte Hammond, LaJuana Durbin, Jerry Zhou, Anna Chen, Murray Stackhouse. Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1 [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 1504.

Abstract 4087: Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes

Abstract Human checkpoint genes PD-1 and PD-L1 are the target of extensive research for treating human cancers. Checkpoint inhibitor therapy shows great promise in the clinic. Combining checkpoint inhibitors with standard chemotherapeutic agents is an important for these inhibitors. Animal models that use human clinical antibodies and chemotherapeutic agents will be beneficial for evaluating new therapies. A novel transgenic mouse model is described that expresses human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We initially evaluated early treatment efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. Tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 µg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To extend the utility of the model we implanted mice and allowed the tumors to grow to 63 to 126 mg before treatment initiation (Day 10). Similar results were observed in established tumors as for early treatment with the PD-1 inhibitors nivolumab and pembrolizuman. In contrast there was little activity from the PD-L1 inhibitor atezolizumab in established tumors. To prepare for combination studies, we also tested cisplatin, irinotecan, gemcitabine, decitabine, rucaparib, and thiarabine near the MTD for each agent with various treatment schedules against the established tumors. The doses for cisplatin, irinotecan and decitabine resulted in excess animal deaths and require reduced doses for combination therapies. Gemcitabine, decitabine, and thiarabine (in house development drug) were too active with complete regressions observed; the doses for combination treatment will be reduced. Due to the effectiveness of the PD-1 checkpoint inhibitors the number of doses will be reduced from four to two treatments in combination studies; no change for atezolizumab. We have shown that PD-1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice in early treatment and in established tumors while the PD-L1 inhibitor was only effective in early treatment. We also have identified doses and schedules of five chemotherapeutic agents to use in future combination studies in this model. Citation Format: Murray A. Stackhouse, Ted Green, Jay Liu, Charlotte Hammond, LaJuana Durbin, Ana Chen, Jerry Zhou, Mike Koratich. Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes [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 4087.

Abstract 545: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD20 for the treatment of B cell lineage cancer

Abstract Tumor cells over expressing CD47 can evade immune surveillance through delivering a “Don’t eat me” signal to macrophages. Developing CD47-targeted modalities has been a hot pursue in the pharmaceutical industry. However due to the ubiquitous expression of CD47 in normal tissues, especially in red blood cells (RBC), severe antigenic sink as well as hemotoxicity including anemia set up a higher bar for this particular targeted therapy development. To overcome these hurdles, we have developed a bispecific recombinant antibody-trap, IMM0306, designed to simultaneously target CD47 and CD20 on B cells but avoid binding to human RBCs. Extensive in vitro characterization demonstrated that IMM0306 binds to both CD47 and CD20 with affinity 3-8 folds lower than either single-targeted molecule, but has strong pro-phagocytosis activity over CD47-positive target cells, and even stronger ADCC activity than Rituximab. Intriguingly, IMM0306 has no binding activity at all toward human RBCs, albeit much lower binding activity toward monkey RBCs. Treatment of tumor-implanted SCID mice with IMM0306 significantly inhibited tumor growth and led to eradication of the tumor cells from 5 out of 8 mice, which is much more effective than Rituximab and than co-administration of Rituximab and SIRPα-Fc (IMM01), although profound synergistic effect has been observed for the co-administration. Preclinical study in non-human primates demonstrated a favorable pharmacokinetic profile with no obvious hemotoxicity following single as well as multiple administrations at different dosage. Our study suggests that antibody-trap like IMM0306 might be an ideal approach for CD47-targeted immunotherapy development since selective avoidance of RBC mediated antigen-sink as well as anemia could be achieved along with the robust anti-tumor activity. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Guangjun Liang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Jie Weng, Gui Zhao, Dandan Liu, Huiqin Guo, Chunmei Yang, Ruliang Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD20 for the treatment of B cell lineage 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 545.

Exosomal circRNA derived from gastric tumor promotes white adipose browning by targeting the miR-133/PRDM16 pathway.

Cancer-related cachexia is a metabolic syndrome characterized by a wasting disorder of adipose and skeletal muscle and is accompanied by body weight loss and systemic inflammation. The treatment options for cancer cachexia are limited, and the molecular mechanism remains poorly understood. Circular RNAs (circRNAs) are a novel family of endogenous noncoding RNAs that have been proposed to regulate gene expression in mammals. Exosomes are small vesicles derived from cells, and recent studies have shown that circRNAs are stable in exosomes. However, little is known about the biological role of circRNAs in exosomes. In our study, we showed that circRNAs in plasma exosomes have specific expression features in gastric cancer (GC), and ciRS-133 is linked with the browning of white adipose tissue (WAT) in GC patients. Exosomes derived from GC cells deliver ciRS-133 into preadipocytes, promoting the differentiation of preadipocytes into brown-like cells by activating PRDM16 and suppressing miR-133. Moreover, knockdown of ciRS-133 reduced cancer cachexia in tumor-implanted mice, decreasing oxygen consumption and heat production. Thus, exosome-delivered circRNAs are involved in WAT browning and play a key role in cancer-associated cachexia.

Preclinical Development of a Bispecific Antibody that Safely and Effectively Targets CD19 and CD47 for the Treatment of B-Cell Lymphoma and Leukemia.

CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20-targeted therapy. Mol Cancer Ther; 17(8); 1739-51. ©2018 AACR.

MiR-103 Functions as a Tumor Suppressor by Directly Targeting Programmed Cell Death 10 in NSCLC.

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. Absence of miR-103 has recently been identified to be associated with metastatic capacity of primary lung tumors. However, the exact role of miR-103 in NSCLC and the molecular mechanism are unclear. In the present study, we showed that miR-103 expression was reduced in NSCLC tissues and cells. miR-103 expression was negatively correlated with tumor size and stage. The overall survival was longer in patients with higher miR-103 level than in those with lower miR-103 expression. miR-103 inhibited cell proliferation in A549 cells, decreased tumor weight and volume, and prolonged survival of tumor-implanted nude mice. miR-103 increased apoptotic cell death in A549 cells. Furthermore, miR-103 decreased the invasion and migration abilities in A549 cells, as evidenced by Transwell and wound healing results. Downregulation of miR-103 significantly reduced the level of programmed cell death 10 (PDCD10). We found a significant decrease in the relative luciferase activity of the reporter gene in A549 cells cotransfected with the miR-103 mimic and pGL3-PDCD10 WT 3′-UTR, but not pGL3-PDCD10 mut 3′-UTR. We showed that overexpression of PDCD10 significantly inhibited miR-103-induced inhibition of cell proliferation, increased apoptosis, and decreased invasion and migration in A549 cells. Moreover, we found that PDCD10 expression was increased in NSCLC tissues and cells. PDCD10 expression was positively correlated with tumor size and stage. Overexpression of PDCD10 increased cell proliferation and inhibited apoptosis in A549 cells. The data demonstrated that dysregulation of the miR-103/PDCD10 signal may be a novel therapeutic target for the treatment of NSCLC.

Pharmaceutical Production of Anti-tumor and Immune-potentiating Enterococcus faecalis-2001 β-glucans: Enhanced Activity of Macrophage and Lymphocytes in Tumor-implanted Mice.

Enterococcus faecalis 2001 is a probiotic lactic acid bacterium and has been used as a biological response modifier (BRM). From physiological limitation of bacterial preservation in storage and safety, the live E. faecalis 2001 has been heat-treated and the BRM components containing high level of β-glucan, named EF-2001, were prepared. The heat-treated EF-2001 has been examined for the antioxidative potential for radical scavenging and anti-tumor activities as well as immune-enhancing response in mice. Lymphocyte versus polymorphonuclear leukocyte ratio was increased in mice upon treatment with EF-2001. The number of lymphocytes was increased in the EF-2001-treated group. In the mice bearing two different Ehrlich solid and Sarcoma-180 carcinomas, the treatment with EF-2001 resulted in anti-tumor action. Tumor-suppressive capacity upon treatment with EF-2001 was significantly increased compared to normal controls. During the time interval administration of 5 weeks between the priming and secondary administration of EF-2001, the expression and production levels of TNF-α were also observed in the EF- 2001-administered mice. Additionally, anti-tumor activity examined with the intravenous administration of EF 2001 with a 34 times interval was also observed, as the growth of Sarcoma180 cells was clearly inhibited by the EF-2001. From the results, it was suggested that the immune response is enhanced due to antioxidative activity caused by the EF-2001 and anti-tumor activity by NK cells and TNF-α.

Abstract 2682: IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer-related deaths in the United States with metastasis to the liver as the major cause of mortality. While the propensity of PDAC to spread to the liver may reflect mechanical trapping of tumor cells that enter the portal circulation, primary tumor cells have also been suggested to secrete factors that may promote recruitment of myeloid cells to establish a pro-metastatic niche. In this study, we used the LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mouse model of PDAC to investigate the impact of PDAC development on the formation of a pro-metastatic niche in the liver. We found that KPC mice (compared to age- and gender-matched control mice) demonstrated an increased susceptibility to tumor seeding in the liver even prior to development of invasive PDAC. Examination of the liver of KPC mice revealed diffuse activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling, particularly in hepatocytes. Although hepatocytes are recognized as important regulators of inflammation, their role in establishing a pro-metastatic niche is unknown. To define changes in the liver associated with development of a pro-metastatic niche, we performed QuantSeq analysis on RNA isolated from the liver of KPC versus control PC mice. Our results showed increased transcriptional levels of myeloid chemoattractants, particularly serum amyloid A proteins that are predominantly produced by hepatocytes. Consistent with this finding, we observed an accumulation of F4/80+ and Ly6G+ myeloid cells in the liver of KPC mice by immunofluorescence microscopy. We next determined the role of tumor cells in driving cellular activation seen in the liver by establishing intraperitoneal and orthotopic models of PDAC. Using these models, we found that implantation of pancreatic tumor cells induced STAT3 activation in hepatocytes and stimulated F4/80+ and Ly6G+ myeloid cell recruitment to the liver. To determine whether cellular activation in the liver was associated with systemic release of soluble factors, we performed parabiotic joining of tumor-implanted mice and control wild type mice, and we found evidence of STAT3 activation and myeloid recruitment to the liver in parabiotic pairs. As interleukin-6 (IL-6) is a key inflammatory cytokine that can activate STAT3 signaling, we hypothesized a role for IL-6 directed STAT3 activation in hepatocytes for development of a pro-metastatic niche in the liver. Consistent with this hypothesis, we found that IL-6 receptor blocking antibodies administered after tumor implantation reduced STAT3 activation in hepatocytes and decreased transcriptional levels of hepatocyte-derived chemoattractants. Together, our findings support a role for IL-6/STAT3 signaling in hepatocytes in driving a pro-metastatic niche in the liver during PDAC development. Citation Format: Jae W. Lee, Paige M. Porrett, Chad A. Komar, Whitney L. Gladney, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2017-2682

Necroptosis DAMPens anti-tumor immunity.

Necroptosis DAMPens anti-tumor immunity.

Oligoesculin fraction induces anti-tumor effects and promotes immune responses on B16-F10 mice melanoma.

Laccase was used to enzymatically polymerize esculin. Oligoesculin fraction was obtained after ultrafiltration through a 5-kDa membrane. Several studies have been carried out to prove the effectiveness of natural substances such as immunomodulators to promote the anti-cancer activity in situ. The purpose of our report was to explore whether the anti-tumor potential of the oligoesculin fraction in vitro and in vivo is linked to its immunological mechanisms in melanoma-bearing mice. We revealed that oligoesculin fraction reduced B16-F10 proliferation and migration in vitro in a dose-related manner. Moreover, melanin synthesis and tyrosinase activity were inhibited in these melanoma cells in a concentration-dependent way. The anti-tumor potential of oligoesculin fraction was also assessed in vivo. Our results showed that intraperitoneal administration of oligoesculin fraction, at 50mg/kg body weight (b.w.) for 21days, reduced tumor size and weight with percentages of inhibition of 94 and 87%, respectively. Oligoesculin fraction was effective in promoting lysosomal activity and nitric oxide (NO) production by peritoneal macrophages in tumor-implanted mice. In addition, the activities of natural killer (NK), cytotoxic T lymphocytes, and macrophages were significantly enhanced by oligoesculin fraction. These findings suggested that this polymer with its anti-tumor and immunomodulatory properties could be used for the treatment of melanoma.