Objective Thrombin mediates the life-threatening cerebral edema that occurs following intracerebral hemorrhage. Therefore, we examined the mechanisms of thrombin-induced injury to the blood-brain barrier (BBB) and subsequent mechanisms of BBB repair. Methods Intracerebroventricular (i.c.v.) injection of thrombin (20 U) was used to model intraventricular hemorrhage in adult rats. Results Thrombin reduced brain microvascular endothelial cell (BMVEC) and peri-vascular astrocyte immunoreactivity –indicating either cell injury or death; and, functionally disrupted the BBB as measured by increased water content and extravasation of sodium fluorescein and Evans blue dyes 24h later. Administration of non-specific src family kinase inhibitor PP2 immediately following thrombin injections blocked brain edema and BBB disruption. At 7 to 14 days after thrombin injections newborn endothelial cells and astrocytes were observed around cerebral vessels at the time when BBB permeability and cerebral water content resolved. Delayed administration of PP2 on days 2 through 6 following thrombin injections prevented resolution of the edema and abnormal BBB permeability. Interpretation Thrombin, via its PAR receptors, is postulated to activate src kinase phosphorylation of molecules that acutely injure the BBB and produce edema. Thus, acute administration of src antagonists blocks edema. In contrast, src blockade for 2-6 days following thrombin injections is postulated to prevent resolution of edema and abnormal BBB permeability in part because src kinase proto-oncogene members stimulate proliferation of newborn BMVECs and peri-vascular astrocytes in the “neurovascular niche” that repair the damaged BBB. Thus, src kinases not only mediate acute BBB injury but also mediate chronic BBB repair after thrombin-induced injury.

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Bortezomib (Velcade®) is a proteasome inhibitor that has been approved for the treatment of multiple myeloma and mantle cell lymphoma. It has been shown to inhibit the expression of cell adhesion molecules, co-stimulatory molecules, and NFĸB activation, to deplete alloreactive T lymphocytes, and to decrease Th1 cytokine production. The anti-inflammatory effects of bortezomib were further investigated in this current set of studies. Systemic treatment with bortezomib was efficacious in the thioglycolate-induced MCP-1 production model, and the dinitrofluorobenzene-induced delayed-type hypersensitivity model. Psoriasis is an autoimmune disease that affects about 2% of the world population. Many treatments have been reported with varying degrees of efficacy. A topical bortezomib formulation was developed to minimize systemic exposure. Its tolerability was investigated in a topical imiquimod (IMQ)-induced psoriasis model. Daily application of IMQ on mouse skin induced inflamed scaly skin lesions resembling plaque-type psoriasis. Fatality was observed in the 1-mg/ml dose group. At 0.1 and 0.01 mg/ml, bortezomib potentiated IMQ-induced erythema, scaling, skin thickening, and caused necrotic lesions. Lower doses had no effect on the clinical observations. Histologically, bortezomib dose-dependently increased parakeratosis, hyperkeratosis, acanthosis, and inflammatory cell infiltration. This study demonstrated that topical bortezomib is not suitable for the treatment of psoriasis.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma affecting children and is often diagnosed with concurrent metastases. Unfortunately, few effective therapies have been discovered that improve the long-term survival rate for children with metastatic disease. Here we determined effectiveness of targeting the receptor tyrosine kinase, EphB4, in both alveolar and embryonal RMS either directly through the inhibitory antibody, VasG3, or indirectly by blocking both forward and reverse signaling of EphB4 binding to EphrinB2, cognate ligand of EphB4. Clinically, EphB4 expression in eRMS was correlated with longer survival. Experimentally, inhibition of EphB4 with VasG3 in both aRMS and eRMS orthotopic xenograft and allograft models failed to alter tumor progression. Inhibition of EphB4 forward signaling using soluble EphB4 protein fused with murine serum albumin failed to affect eRMS model tumor progression, but did moderately slow progression in murine aRMS. We conclude that inhibition of EphB4 signaling with these agents is not a viable monotherapy for rhabdomyosarcoma.

Embryonal rhabdomyosarcoma (eRMS) is one of the most common soft tissue sarcomas in children and adolescents. Parameningeal eRMS is a variant that is often more difficult to treat than eRMS occurring at other sites. A 14-year-old female with persistent headaches and rapid weight loss was diagnosed with parameningeal eRMS. She progressed and died despite chemotherapy with vincristine, actinomycin-D, and cyclophosphamide plus 50.4 Gy radiation therapy to the primary tumor site. Tumor specimens were acquired by rapid autopsy and tumor tissue was transplanted into immunodeficient mice to create a patient-derived xenograft (PDX) animal model. As autopsy specimens had an ALK R1181C mutation, PDX tumor bearing animals were treated with the pan-kinase inhibitor lestaurtinib but demonstrated no decrease in tumor growth, suggesting that single agent kinase inhibitor therapy may be insufficient in similar cases. This unique parameningeal eRMS PDX model is publicly available for preclinical study.

Humanized mice are being applied widely to study human immune system homeostasis, function, and as a testing platform for cancer immunotherapies. A major limitation for many humanized mouse models is the lack of functional and mature human innate immune cells, which are critical for effective human immune system-tumor interactions. Previous studies have demonstrated that delivery of human FLT3L into immunodeficient mice that lack mouse FLT3, promotes the development of human innate immune cell subsets following engraftment with human hematopoietic stem cells (HSC). Here we describe a NOD-scid IL2rgnull (NSG) mouse that lacks the expression of mouse FLT3 and expresses human FLT3L transgenically (NSG-mFLT3nullTg (HuFLT3L) or NSG-FLT3L. In these studies, NSG-FLT3L and NSG mice were engrafted with human umbilical cord blood (UCB) CD34+ HSC and compared for human immune system development and function. HSC-engrafted NSG-FLT3L and NSG mice show similar levels of total human CD45+ cells in blood over the course of 18 weeks post-engraftment. However, HSC-engrafted NSG-FLT3L mice show significantly higher levels of human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes, CD56+ NK cells and CD3+ T cells in the blood as compared to NSG mice. CD34-FLT3L mice also show increased levels of human immune cell infiltration into the gut mucosa. CD56+ NK cells and CD3+ T cells from HSC-engrafted NSG-FLT3L mice express granzyme A and granzyme B, indicating cytotoxic activity. Following treatment of HSC-engrafted NSG-FLT3L mice with LPS, heightened levels of human cytokines were detected in serum samples, confirming innate immune system function. The growth kinetics of tumor cells from a triple negative breast cancer cell line MDA-MB-231 and a lung PDX model LG1306 in HSC-engrafted NSG-FLT3L mice are work in progress and will be compared with HSG-engrafted NSG mice. Overall these results demonstrate that the NSG-FLT3L model supports enhanced development of functional, human innate immune cells and is a novel tool to study human immuno-oncology. Citation Format: Li-Chin Yao, Shantashri Vaidya, Pali Kaur, James G. Keck, Leonard D. Shultz, Dale L. Greiner, MIchael A. Brehm. Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1640.

Acute myeloid leukemia (AML) is a genetically heterogeneous cancer of progenitor cells in myeloid hematopoiesis. Due to heterogeneity, most of AMLs are not responsive to targeted therapy. In addition, treatment outcome varies greatly depending upon patient age, disease etiology and mutational status. Previously, a large repository of well-characterized patient-derived- xenografts (PDXs) of hematologic malignancies was established (Townsend et al., 2016). This repository was generated by injecting primary bone marrow and blood samples from patients with leukemia into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. An average success rate of 23.2% engraftment was observed in first passage (P0). In this study, we have characterized ten AML PDX models in NSG-SGM3 mice by serial transplantation. NSG-SGM3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) mice have been found to have increased proliferation and survival of engrafted AML cells along with decreased disease latency (Wunderlich et al., 2010). Leukemic engraftment in these models was confirmed by flow analysis for hCD45+ hCD33+ in the peripheral blood, bone marrow and spleen of engrafted mice. Kinetics of engraftment varied from one model to another; and ranged from 10% - 80% of hCD33+ in peripheral blood 8 week post AML cell injection (1X106 cells/mouse). Four models displayed positive response to Cytarabine (Ara-C) treatment with reduction in levels of human CD33+ cells following a 5-day treatment cycle. Data will be presented on model characterization to show distinct engraftment kinetics, correlation with disease mutations and response to AML standard of care drug Cytarabine. These models have varying mutational profiles and treatment histories. Mutations include FLT3, FLT3-ITD, NPM1, TP53, DNMT3A and CUX1 alone or in combination. Treatment phases at the time of collection varied from untreated to relapse post-chemotherapy. These AML PDX models are useful tools for testing new experimental therapeutics. Diversity of these AML PDX models will facilitate focus on specific AML targets, thus providing an invaluable tool set to support drug discovery. Citation Format: Pali Kaur, Victoria Sachs, Amanda L. Christie, David M. Weinstock, James G. Keck. Characterization of new AML PDX models: Engraftment kinetics and mutational profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 700.

NSG mice engraft human PBMC and allow efficacy testing of immunomodulatory drugs in a GvHD setting. Engraftment is progressive and at d5 PBL has 10% human cells with a CD4:CD8 T cell ratio of 3:1. The CD4 cells are 30% CD45RO− CCR7+ naïve (Tn), 40% CD45RO+ CCR7+ central memory (Tcm) and 10% CD45RO+ CCR7− effector memory (Tem). The CD8 cells at d5 contain 9% Tn, 3% Tcm, and 5% Tem. At d18 human cells are 70% with a CD4:CD8 ratio of 1:3, 0% Tn, 10% Tcm and are 22% CD4 Tem and 52% CD8 Tem. Engrafted mice were treated either prophylactically (day −1) or therapeutically (day 5) with Abatacept or an unknown test article. PBL at d10 post engraftment from mice treated at day −1 showed human cells at 14.6%, 2.2%, and 1.6% for vehicle, Abatacept, and test article, respectively. The Abatacept group were primarily CD4 Tn. Abatacept and test article treated mice showed 100% survival, almost no tissue infiltration, normal body weight, and were healthier based on disease activity index (DAI) scores. When dosed therapeutically, human cells at d18 were 67.3%, 16.9%, and 37.9%, respectively. Both treatments suppressed T cell expansion, maintained a 3:1 CD4:CD8 ratio and decreased development of the CD8 Tem pool (CD8 Tem 32.7%, 15.4%, and 4.9%, respectively). 100% of the d5 dosed Abatacept group survived, maintained body weight, and had improved DAI. 70% of the d5 dosed test article group survived and at d20 began losing body weight and accumulated poor DAI scores. The data presented here provide a detailed analysis of activated human T cell pools and progression of GvHD in NSG recipients of human PBMC. The model allows detailed analysis of in vivo modulation of T cell activity and therefore enables rapid evaluation of therapies relevant to not only GvHD, but to autoimmunity and oncology.