NOD-SCID Mice Research Articles

Induction of monoamine oxidase A-mediated oxidative stress and impairment of NRF2-antioxidant defence response by polyphenol-rich fraction of Bergenia ligulata sensitizes prostate cancer cells in vitro and in vivo

Prostate cancer (PCa) is a major cause of mortality and morbidity in men. Available therapies yield limited outcome. We explored anti-PCa activity in a polyphenol-rich fraction of Bergenia ligulata (PFBL), a plant used in Indian traditional and folk medicine for its anti-inflammatory and antineoplastic properties. PFBL constituted of about fifteen different compounds as per LCMS analysis induced apoptotic death in both androgen-dependent LNCaP and androgen-refractory PC3 and DU145 cells with little effect on NKE and WI38 cells. Further investigation revealed that PFBL mediates its function through upregulating ROS production by enhanced catalytic activity of Monoamine oxidase A (MAO-A). Notably, the differential inactivation of NRF2-antioxidant response pathway by PFBL resulted in death in PC3 versus NKE cells involving GSK-3β activity facilitated by AKT inhibition. PFBL efficiently reduced the PC3-tumor xenograft in NOD-SCID mice alone and in synergy with Paclitaxel. Tumor tissues in PFBL-treated mice showed upregulation of similar mechanism of cell death as observed in isolated PC3 cells i.e., elevation of MAO-A catalytic activity, ROS production accompanied by activation of β-TrCP-GSK-3β axis of NRF2 degradation. Blood counts, liver, and splenocyte sensitivity analyses justified the PFBL safety in the healthy mice. To our knowledge this is the first report of an activity that crippled NRF2 activation both in vitro and in vivo in response to MAO-A activation. Results of this study suggest the development of a novel treatment protocol utilizing PFBL to improve therapeutic outcome for patients with aggressive PCa which claims hundreds of thousands of lives each year.

166-OR: Coinfusion of Mesenchymal Stem Cells Improves Human Islet Survival in the Hepatic Islet Transplantation Mouse Model

Islet survival is improved when islets are co-transplanted with mesenchymal stromal cells (MSCs) under the kidney capsule. We assessed whether co-infusion of MSCs with human islets into the liver, as performed in patients, would protect those islets from death post-transplantation (PT) in streptozotocin-induced diabetic NOD-SCID mice (blood glucose, BG >350mg/dl). Blood glucose levels were measured as surrogate markers for islet survival and function. Transplantation of a suboptimal islet number led to normoglycemia (BG<200mg/dl) at 60 days PT in 33% of 6 mice. In contrast, 91% (n=12) of mice receiving islets co-infused with MSCs (Islet+MSC) reached and remained normoglycemic. Mice receiving islet+MSC showed lower non-fasting glucose levels, higher baseline and stimulated c-peptide levels and better function when challenged with high glucose during an intravenous glucose tolerance test. Immunohistochemistry staining of beta cells identified that significantly more islets and islet positive area were observed in the livers of islet+MSC compared to islets alone mice (even in mice with normoglycemia). We show that MSCs led to better islet survival in MSC-co-infused mice after transplantation. By using firefly luciferase-expressing MSCs, we found that co-transplanted luciferase+ (Luc+) MSC was detected up to 7 days in the liver PT. Insulin expressing MSCs were detected at 60 days PT, suggesting MSCs can stay in the liver long-term and may convert into insulin+ cells. Inflammatory-related chemokines, including IL-6, KC, and MCP-1, were also significantly reduced in islet+MSC mice serum at day 1-3 PT. The protective effects of MSC on beta cell survival was confirmed in vitro when human islets were co-cultured with MSCs in a Transwell system. These data suggest that the mechanisms of MSC protection were mediated through suppression of the innate immune response and inflammation-induced β cell death when MSC and islets were co-transplanted into the liver. Disclosure W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536)

Spontaneous Fusion of MSC with Breast Cancer Cells Can Generate Tumor Dormancy.

Direct cellular interactions of MDA-MB-231cherry breast cancer cells with GFP-transduced human mesenchymal stroma/stem-like cells (MSCGFP) in a co-culture model resulted in spontaneous cell fusion by the generation of MDA-MSC-hyb5cherry GFP breast cancer hybrid cells. The proliferative capacity of MDA-MSC-hyb5 cells was enhanced about 1.8-fold when compared to the parental MDA-MB-231cherry breast cancer cells. In contrast to a spontaneous MDA-MB-231cherry induced tumor development in vivo within 18.8 days, the MDA-MSC-hyb5 cells initially remained quiescent in a dormancy-like state. At distinct time points after injection, NODscid mice started to develop MDA-MSC-hyb5 cell-induced tumors up to about a half year later. Following tumor initiation, however, tumor growth and formation of metastases in various different organs occurred rapidly within about 10.5 days. Changes in gene expression levels were evaluated by RNA-microarray analysis and revealed certain increase in dormancy-associated transcripts in MDA-MSC-hyb5. Chemotherapeutic responsiveness of MDA-MSC-hyb5 cells was partially enhanced when compared to MDA-MB-231 cells. However, some resistance, e.g., for taxol was detectable in cancer hybrid cells. Moreover, drug response partially changed during the tumor development of MDA-MSC-hyb5 cells; this suggests the presence of unstable in vivo phenotypes of MDA-hyb5 cells with increased tumor heterogeneity.

Upregulation of wild-type p53 by small molecule-induced elevation of NQO1 in non-small cell lung cancer cells.

The tumor suppressor p53 is usually inactivated by somatic mutations in malignant neoplasms, and its reactivation represents an attractive therapeutic strategy for cancers. Here, we reported that a new quinolone compound RYL-687 significantly inhibited non-small cell lung cancer (NSCLC) cells which express wild type (wt) p53, in contract to its much weaker cytotoxicity on cells with mutant p53. RYL-687 upregulated p53 in cells with wt but not mutant p53, and ectopic expression of wt p53 significantly enhanced the anti-NSCLC activity of this compound. RYL-687 induced production of reactive oxygen species (ROS) and upregulation of Nrf2, leading to an elevation of the NAD(P)H:quinoneoxidoreductase-1 (NQO1) that can protect p53 by inhibiting its degradation by 20S proteasome. RYL-687 bound NQO1, facilitating the physical interaction between NQO1 and p53. NQO1 was required for RYL-687-induced p53 accumulation, because silencing of NQO1 by specific siRNA or an NQO1 inhibitor uridine, drastically suppressed RYL-687-induced p53 upregulation. Moreover, a RYL-687-related prodrug significantly inhibited tumor growth in NOD-SCID mice inoculated with NSCLC cells and in a wt p53-NSCLC patient-derived xenograft mouse model. These data indicate that targeting NQO1 is a rational strategy to reactivate p53, and RYL-687 as a p53 stabilizer bears therapeutic potentials in NSCLCs with wt p53.

Weekly osimertinib dosing to prevent EGFR mutant tumor cells destined to home mouse lungs.

e20504 Background: The recent ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR-mutated non-small cell lung cancerpatients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion, to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. Methods: We present an assessment of the homing of PC9-luciferase cells to the lungs of mice injected through mouse tail vein. Thirty NOD-SCID mice were divided into five groups as follows: two groups each for daily and weekly treatment and one group for vehicle control. For all the mice in the respective groups, erlotinib (25 mg/kg) and osimertinib (15 mg/kg) was orally administered. Homing of cells in the lungs of mice was assessed by bioluminescence imaging regularly. Results: In the control group, 100% of the mice showed homing and retention of cells in the lungs of mice 18 days post-injection. In mice receiving erlotinib daily, homing of the cells in lungs was absent in all four mice, except in one mouse where emergence was observed after day eight post-injection. In the case of erlotinib weekly group, out of four only one mouse showed absence of homing of cells in the lungs of mice. Interestingly, 100% of the mice in both the groups receiving osimertinib daily or weekly showed complete absence of homing of cells in the lungs of mice from day 3 onwards post-injection (Table). The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Conclusions: Based on the complete absence of the homing of cells in lungs of osimertinib treated mice, in both weekly and daily regimens, adjuvant osimeritinib may represent a viable treatment option in delaying the onset of disease post resection of early stage tumors or among patients with pre-disposition to EGFR mutant lung cancers harboring germline EGFR mutations. In addition, low-dose once-a-week osimertinib could potentially have several advantages over daily dosing, including lower toxicity, affordability, ease of administration and delaying or preventing acquired resistance that remains to be explored. [Table: see text]

Histone Methyltransferase G9a-Promoted Progression of Hepatocellular Carcinoma Is Targeted by Liver-Specific Hsa-miR-122.

Simple SummaryTargeting epigenetic alterations in hepatocellular carcinoma (HCC) provides therapeutic options in addition to traditional treatments. The aim of our study was to evaluate the potential of targeting histone methyltransferase G9a in the development of a therapeutic target. We confirmed the prognostic values of mRNA and protein levels of G9a expression in HCC respectively from public database and tissue microarray. We also confirmed the aggressive phenotypes supported by G9a in both HBV+ and HBV− HCC cells. The identification of a regulation axis between liver-specific tumor suppressor miR-122 and G9a further supported the important roles of G9a during the tumorigenesis and progression of HCC. Combination of lower miR-122 and higher G9a levels may provide prognostic potential for poor clinical outcomes and therapeutic potential for epigenetic targeting therapies.Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers, which is the second most lethal tumor worldwide. Epigenetic deregulation is a common trait observed in HCC. Recently, increasing evidence suggested that the G9a histone methyltransferase might be a novel regulator of HCC development. However, several HCC cell lines were recently noted to have HeLa cell contamination or to have been derived from non-hepatocellular origin, suggesting that functional validation of G9a in proper HCC models is still required. Herein, we first confirmed that higher G9a messenger RNA and protein expression levels were correlated with poor overall survival (OS) and disease-free survival (DFS) rates of HCC patients from The Cancer Genome Atlas (TCGA) dataset and our recruited HCC cohort. In an in vitro functional evaluation of HCC cells, HCC36 (hepatitis B virus-positive (HBV+) and Mahlavu (HBV−)) cells showed that G9a participated in promoting cell proliferation, colony formation, and migration/invasion abilities. Moreover, orthotopic inoculation of G9a-depleted Mahlavu cells in NOD-SCID mice also resulted in a significantly decreased tumor burden compared to the control group. Furthermore, after surveying microRNA (miRNA; miR) prediction databases, we identified the liver-specific miR-122 as a G9a-targeting miRNA. In various HCC cell lines, we observed that miR-122 expression levels tended to be inversely correlated to G9a expression levels. In clinical HCC specimens, a significant inverse correlation of miR-122 and G9a mRNA expression levels was also observed. Functionally, the colony formation and invasive ability were attenuated in miR-122-overexpressing HCC cells. HCC patients with low miR-122 and high G9a expression levels had the worst OS and DFS rates compared to others. Together, our results confirmed the importance of altered G9a expression during HCC progression and discovered that a novel liver-specific miR-122-G9a regulatory axis exists.

Transferrin Modified GSH Sensitive Hyaluronic Acid Derivative Micelle to Deliver HSP90 Inhibitors to Enhance the Therapeutic Efficacy of Brain Cancers.

Simple SummaryHeat shock protein 90 (HSP90) is a key element of a multi-chaperone complex involved in the stabilizing of many client proteins, oncoproteins, which play essential roles in tumorigenesis. As the result, HSP90 has been taken as a promising target for anticancer therapies. AUY922 has good antitumor activity by inhibiting the ATPase activity of HSP90, while it has certain limitations, including poor water solubility and lack of selectivity, which have incited the development of a novel targeted nanoformulation. In this study, we have successfully synthesized and characterized a GSH-sensitive micelle that can encapsulate hydrophobic AUY922 in its core region to enhance its therapeutic efficacy against brain cancers. All in vitro and in vivo experimental results showed nanoformulated AUY922 has a better therapeutic efficacy against brain cancer in comparison to the free AUY922.Herein, GSH-sensitive hyaluronic acid-poly(lactic-co-glycolic acid) (HA-SS-PLGA) was synthesized. Surface modification of PLGA with hyaluronic acid produced a highly stable micelle at physiological pH while a micelle was destabilized at a higher GSH level. Fluorescence microscopy results showed that rhodamine-encapsulated micelle was taken up by brain cancer cells, while competitive inhibition was observed in the presence of free HA and free transferrin. In vitro cytotoxicity results revealed that transferrin-targeted nanoformulated AUY922 (TF-NP-AUY922) shows higher cytotoxicity than either free AUY922 or non-targeted AUY922-loaded micelles (NP-AUY922). In comparison to the control groups, free AUY922, TF-NP-AUY922 or NP-AUY922 treatment revealed the upregulation of HSP70, while the expression of HSP90 client proteins was simultaneously depleted. In addition, the treatment group induced caspase-dependent PARP cleavage and the upregulation of p53 expression, which plays a key role in apoptosis of brain cancer cells. In vivo and ex vivo biodistribution studies showed that cypate-loaded micelle was taken up and accumulated in the tumor regions. Furthermore, in vivo therapeutic efficacy studies revealed that the AUY922-loaded micelle significantly suppressed tumor growth in comparison to the free AUY922, or control groups using tumor-bearing NOD-SCID mice. Moreover, biochemical index and histological analysis revealed synthesized micelle does not show any significant cytotoxicity to the selected major organs. Overall, a synthesized micelle is the best carrier for AUY922 to enhance the therapeutic efficiency of brain cancer.

Intrapulmonary distal airway stem cell transplantation repairs lung injury in chronic obstructive pulmonary disease.

ObjectivesChronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage including chronic bronchitis and emphysema, which could further develop into respiratory failure. Many studies have revealed a potential regenerative function of the distal airway stem/progenitor cells (DASCs) after lung injury.Materials and MethodsMouse and human DASCs were expanded, analysed, and engrafted into injured mouse lungs. Single‐cell analyses were performed to reveal the differentiation path of the engrafted cells. Finally, human DASCs were transplanted into COPD mice induced by porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS) administration.ResultsWe showed that isolated mouse and human DASCs could be indefinitely expanded and were able to further differentiate into mature alveolar structures in vitro. Single‐cell analysis indicated that the engrafted cells expressed typical cellular markers of type I alveolar cells as well as the specific secreted proteins. Interestingly, transplantation of human DASCs derived from COPD patients into the lungs of NOD‐SCID mice with COPD injury repaired the tissue damage and improved the pulmonary function.ConclusionsThe findings demonstrated that functional lung structure could be reconstituted by intrapulmonary transplantation of DASCs, suggesting a potential therapeutic role of DASCs transplantation in treatment for chronic obstructive pulmonary disease.

Inhibition of TLR7 and TLR9 Reduces Human Cholangiocarcinoma Cell Proliferation and Tumor Development

Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumordevelopment were also examined in a NOD-SCID mouse xenograft model of human ICC. TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC.

Development and Characterization of the Ultra‐Immunodeficient NOD.CB17‐ Prkdc scid IL2rg tm1 /Bcgen (B‐NDG) Mouse Model

The “B-NDG” (NOD.CB17-Prkdcscid IL2rgtm1/Bcgen) mouse was designed and generated by Biocytogen. IL2rg (common gamma) gene was deleted in NOD-scid mice resulting in a mouse that lacks mature B cells, T cells, NK cells and has a deficiency in cytokine signaling. This level of immunodeficiency makes this model ideal for engraftment with human immune cells and human tumor cells or tissues. Herein we describe the development and characterization of the B-NDG model, which includes growth curve, complete blood count, serum chemistry, flow cytometry, tumor growth and humanization. A cohort of B-NDG mice (N=50 males and 50 females) were weighed weekly from 3 to 9 weeks of age. A cohort of six male B-NDG mice were analyzed to determine baseline levels for complete blood cell count and serum chemistry. Splenocytes of C57BL/6, NOD-scid and B-NDG mice were isolated and examined for total T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and regulatory T cells using flow cytometry. Data showed complete lack of all T cells, B cells and NK cells in the B-NDG modelTwo different methods of humanization were examined in the B-NDG model. Human peripheral blood mononuclear cells (hPBMCs) were injected at 5x106 into the caudal vein of three B-NDG mice. After 24 days, the hCD45+ and mCD45+ cells were examined and mice had 30-80% hCD45+ cells. Of those hCD45+ cells, over 99% were identified as T cells. The second humanization method used was injection of 1x105 human CD34+ hematopoietic stem cells after irradiation. The percentage of human CD45+ cell in the peripheral blood was over 20% by eight weeks post injection in over 70% engrafted mice. At 16 weeks post injection, of the total number of human lymphocytes in the humanized mice, 60% were B cells, 2% were NK cells and over 10% were T cells. The percentage of human T cells increases steadily with the extended observation window. We have demonstrated that B-NDG mice have several features that translate many benefits as compared to other immunodeficient models. This model has demonstrated better tumor growth than the NOD-scid and C.B17 scid mice for cell lines as well as PDX lines. We have also shown that this model is extremely useful for studies that require humanization of an immunodeficient mouse model.:

Inflammation targets CDK11 in autoimmune diabetes as a protective mechanism against beta cell apoptosis.

Abstract Autoimmune diabetes (T1D) is caused by the immune-mediated destruction of insulin-producing beta cells in the pancreas leading to hyperglycemia. Apoptosis is the main mechanism responsible for beta cell demise, including Fas-receptor engagement and Perforin release as essential death executers. However, the chronic inflammatory milieu pervading pancreatic islets prior to diabetes onset, alters beta cells function and primes beta cells for apoptosis long before the debut of the disease. To identify those genes that are targeted by inflammation and are causally related to beta cell fitness and viability we have used the microarray technology. We have identified several candidate genes being downregulated by inflammation, one of this genes is Cdk11, a cyclin-dependent kinase which is involved in transcription (CDK11p130), mitosis and apoptosis (CDK11p58). L-type cyclins are the partners for Cdk11p130, and Cyclin D3 is the partner of CDK11p58. The Cdk11 gene, when is transcribed, generates a single mRNA form, that can be translated either into CDK11p130 or CDK11p58 by a differential IRES (Internal Ribosome Entry Site) usage, being CDK11p58 only expressed during mitosis, while CDK11p130is expressed during all cell cycle phases. We have addressed the role of CDK11 in autoimmune diabetes using the T1D-prone NOD mouse model hemideficient in CDK11, since the CDK11 null mutation is embryonically lethal. First of all, we have confirmed the effect of inflammation on Cdk11 mRNA expression in islet cells by submitting NODSCID mice to adoptive transfer of increasing amounts of diabetogenic NOD splenocytes, and, observed that Cdk11 expression in islet endocrine cells is downregulated by inflammation in a dose-response dependent manner.

Jinfukang inhibits lung cancer metastasis by upregulating CX3CL1 to recruit NK cells to kill CTCs

Ethnopharmacological relevanceCirculating tumor cells (CTCs) play an important role in tumor metastasis and may be a target for metastasis prevention. The traditional Chinese medicine Jinfukang functions to improve immunity, prevent metastasis, and prolong lung cancer patient survival periods. Yet, whether Jinfukang prevents metastasis by regulating immune cells to clearance CTCs is still unknown. Aim of the studyTo explore the anti-metastasis mechanism of Jinfukang from the perspective of regulating NK cells to clear CTCs. Materials and methodsCTC-TJH-01 cell was treated with Jinfukang. Cytokine chip was used to detect cytokines in cell culture supernatant. Lymphocyte recruitment assay was detected by Transwell and flow cytometry. Protein expression was analysis by Western blot. LDH kit was used to detect cytotoxicity. NOD-SCID mice used for tail vein injection to study lung metastasis. ResultsJinfukang could promote the expression and secretion of the chemokine CX3CL1 by CTCs. In addition, Jinfukang could promote the recruitment of natural killer (NK) cells by CTCs and significantly increase the cytotoxic effect of NK cells on CTCs. Moreover, Jinfukang could upregulate the expression of FasL and promote the secretion of TNF-α by NK cells and that NK cells could induce the apoptosis of CTCs through the Fas/FasL signaling pathway. Finally, we confirmed that Jinfukang could promote NK cells to kill CTCs and then inhibit lung cancer metastasis in vivo. The above effects of Jinfukang could be partially reversed by an anti-CX3CL1 mAb. ConclusionsThese results suggest that Jinfukang may prevent lung cancer metastasis by enhancing the clearance of CTCs in the peripheral blood by NK cells, providing evidence for the anti-metastasis effect of Jinfukang.

ONCOLYSIS MEDIATED BY INFB AND ARF GENE TRANSFER ACTIVATES KEY COMPONENTS OF THE IMMUNE SYSTEM

<h3>Background</h3> The importance of the immune system for controlling neoplasms was highlighted with the success of checkpoint blockade therapy. New cancer therapeutics seek to break the immune tolerance inherent in tumor progression. To this end, some virus-mediate gene therapy approaches have been shown to induce oncolysis accompanied by emission of immunogenic cell death (ICD) factors, thus attracting and activating cells of the immune system. <h3>Objective</h3> To use combined adenovirus-mediated gene transfer of p14ARF and human interferon-β (IFNβ) to human melanoma cells to induce oncolysis, ICD and subsequent activation of immune cells. <h3>Methods</h3> Cell death was measured by PI and/or AnnexinV staining. Detection of calreticulin was performed by cytometry, while ATP and IFNβ were detected by ELISA. DC and lymphocytes from healthy donors were cultivated ex vivo. <h3>Results</h3> IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. However, detection of critical markers of ICD) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. In situ gene therapy for s.c. SK-MEL-147 tumors in Nod-Scid mice controlled tumor growth and increased animal survival in response to IFNβ alone or in combination with p14ARF. However, the immunocompromised animal model cannot demonstrate the expected immune activation. Thus, we co-cultured SK-MEL-147 transduced with our combination of adenoviruses with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors. These Mo-DCs showed increased levels of activation markers HLA-DR, CD80, and CD86. Next, we used these activated Mo-DCs to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. T cells primed in this way were co-cultured with fresh non-transduced SK-MEL-147, resulting in tumor cell killing. Currently we are expanding these studies to include three-dimensional cultures of melanoma cell lines and also the use of human melanoma samples in patient derived organoid (PDO) models. We expect to reveal the influence of oncolysis on tumor infiltrating cells, including DCs, macrophages, NK and T cells. <h3>Conclusion</h3> Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

NSG Mice Facilitate ex vivo Characterization of Ewing Sarcoma Lung Metastasis Using the PuMA Model.

Ewing sarcoma (EwS) is a highly malignant bone and soft tissue tumor primarily affecting children and young adults. While most patients initially respond well to conventional front-line therapy, frequent metastasis results in poor 5-year overall survival rates for this disease. Accordingly, there is a critical need to develop better models to understand EwS metastasis. We and others previously used the ex vivo pulmonary metastasis assay (PuMA) to study lung metastasis in solid tumors including osteosarcoma (OS), but this technique has to date not been achievable for EwS. PuMA involves tail vein injection of fluorescent tumor cells into NOD-SCID mice, followed by their visualization in long-term cultures of tumor-bearing lung explants. Here we demonstrate successful implementation of PuMA for EwS cells using NOD-SCID-IL2 receptor gamma null (NSG) immunocompromised mice, which demonstrated high engraftment of EwS cell lines compared to NOD-SCID mice. This may be linked to immune permissiveness required by EwS cells, as increased basal cytotoxicity of EwS cells was observed in NOD-SCID compared to NSG lung sections, possibly due to the absence of natural killer (NK) cell activity in the latter. Together, our data demonstrate the utility of NSG mice for PuMA modeling of EwS lung metastasis.

Sex difference feeding behaviour of NOD SCID mice in a pharmacological model of type 1 diabetes.

This study aimed to assess the sex differences in the feeding behaviour of non-obese diabetic severe combined immunodeficient (NOD SCID) mice in a pharmacological model of type 1 diabetes mellitus (T1Dm). In our study, we chose NOD SCID mice of both sexes and assessed their feeding behaviour, body weight, body fat and water content under identical experimental conditions and diets. After 1month of diabetes mellitus in mice in the experimental group, males and females did not show any increase in body weight, and they weighed significantly less than the control group. However, compared with the control group, in females with a background of T1Dm, there was a significant decrease in body fat. The amount of water consumed in the experimental groups was higher than that in the control groups. The amount of food consumed by males increased when they increased their water consumption, whereas food consumption in females decreased significantly with an increase in water consumption. Thus, we discovered sex differences in the feeding behaviour, body weight and body fat and water content in the pharmacological model of T1Dm after 1month in NOD SCID mice.

Application of germ-free NOD-scid IL2rgnull mice as a humanized model for tumor microbiome precision medicine.

Application of germ-free NOD-scid IL2rgnull mice as a humanized model for tumor microbiome precision medicine.

Abstract PO047: AVB-500, a receptor tyrosine kinase AXL inhibitor, improves response to olaparib in uterine serous cancer

Abstract Objectives: We determined whether inhibition of GAS6/AXL with AVB-500 in combination with olaparib could improve response in HR-proficient (HRP) uterine serous cancer (USC). Methods: Two USC cell lines (ARK1 &amp; ARK4) were radiated with 10Gy, and RAD51 foci were identified by immunofluorescence (IF). These were treated with AVB-500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB-500 alone, olaparib alone or combination treatment (olaparib + AVB-500). Colonies were stained and absorbance was obtained to calculate relative cell survival using Graph Pad Prism. In vivo studies were performed using NOD-SCID mice injected with 1 × 107 ARK1 cells intraperitoneally followed by treatment q3 days for 14-day treatment period. Treatment groups were vehicle control, AVB-500 alone, olaparib alone and olaparib with AVB-500. Results: ARK1 and ARK4 cells were found to have an increase in gamma-H2AX and RAD51 foci after radiation that was consistent with HRP. In clonogenic assays, colonies were stained and absorbance was obtained for each experimental arm. Olaparib + AVB-500 had significantly less absorbance than the olaparib only group for ARK1s (0.417nm vs 0.756nm, p=0.001) as well as in ARK4s (0.186nm vs 0.641nm, p=0.003). In an intraperitoneal model with ARK1 tumors, the olaparib + AVB-500 treated group had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p=0.002) and AVB-500 alone (0.008g vs 0.145g, p=0.0006) after a 14-day treatment period. Conclusions: AVB-500 in combination with olaparib demonstrates an improved response than olaparib alone with a greater decrease in tumor burden. This was demonstrated in two HRP cell lines. Additional therapeutic and mechanistic experiments are ongoing. Citation Format: Michael D. Toboni, Mary Mullen, Jo'an Tankou, Hollie Noia, Alyssa Oplt, Daniel Wilke, Dineo Khabele, Lindsay Kuroki, Andrea Hagemann, Carolyn McCourt, Premal Thaker, David Mutch, Matthew Powell, Katherine Fuh. AVB-500, a receptor tyrosine kinase AXL inhibitor, improves response to olaparib in uterine serous cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO047.

Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing

We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

MO01.46 Tepotinib Activity in Brain Metastases (BM): Preclinical Models and Clinical Data from MET Exon 14 (METex14) Skipping NSCLC

BM occur in 20–40% of NSCLC harboring METex14 skipping. We investigated the activity of the MET inhibitor tepotinib in BM in preclinical models and patients from the VISION study (NCT02864992). Penetration of the blood–brain barrier was assessed in Wistar rats (n=3) at 3.66 mg/kg/h intravenous tepotinib by determining the unbound brain (fu br)-to-plasma (fu pl) concentration or exposure ratio (Kp u,u). Efficacy was assessed in two lung cancer patient-derived xenografts (PDXs) from BM harboring high MET amplification (gain in copy number: LU5349 = 11, LU5406 = 24) grown in NOD-SCID mice. Subcutaneous PDXs (n=5/group) or PDXs orthotopically implanted into the brain (n=10/group) were treated with tepotinib 125 mg/kg or vehicle control orally once daily. Intracranial tumor growth was monitored by gadolinium-based MRI. In VISION Cohort A, patients with METex14 skipping NSCLC received tepotinib 500 mg once daily. Systemic objective response per RECIST v1.1 by independent review committee (IRC) was a preplanned analysis in patients with baseline BM identified by IRC (BM-IRC) or investigator assessment (BM-INV). Preclinical data indicated high binding of tepotinib in the brain, with unbound tepotinib in brain tissue being lower than in plasma (fu br = 0.4%, fu pl = 4%). Unbound tepotinib concentrations in the brain were 25% of plasma (Kp u,u = 0.25). Tepotinib treatment resulted in tumor regression in both PDXs (mean % tumor volume: –84% in LU5349, –63% in LU5406). As of 1 Jan 2020, 22/152 patients enrolled in Cohort A had baseline BM, with similar characteristics and comparable systemic response data (table) to the overall population. Tepotinib administration resulted in tumor regression in MET-driven lung cancer BM PDX models. Clinical activity in patients with NSCLC harboring METex14 skipping with baseline BM was consistent with the overall population in VISION. Cohort C aims to assess intracranial response. Previously presented at ESMO Congress 2020, “FNP:1286P”, “Santiago Viteri et al.” - Reused with permission