Nonobese Diabetic/severe Combined Immunodeficient Research Articles

Development of Novel CD47-Specific ADCs Possessing High Potency Against Non-Small Cell Lung Cancer in vitro and in vivo.

Targeted therapies hold promise for efficiently and accurately delivering cytotoxic drugs directly to tumor tissue to exert anticancer effects. CD47 is a membrane protein expressed in a variety of malignant tumors and hematopoietic cells, which plays a key role in immune escape and tumor progression. Although CD47 immunocheckpoint therapy has been developed in recent years, many patients cannot benefit from it because of its low efficiency. To strengthen and extend the therapeutic efficacy of anti-CD47 monoclonal antibody (mAb), we used the newly developed 7DC2 and 7DC4 mAbs as the targeting payload adaptor and VCMMAE as the toxin payload to construct novel CD47-specific immunotoxin (7DC-VCMMAE) by engineering cysteine residues. These CD47-specific ADCs have the better cell penetration, excellent DAR, similar payload distribution and good antigen-binding affinity. In vitro, 7DC-VCMMAE treatment induced death of non-small cell lung cancer (NSCLC) cell lines 95D and SPC-A1, but not A549 that express low levels of CD47 on the cell membrane. This finding suggests that 7DC-VCMMAE may possess greater therapeutic effect on NSCLC tumors expressing a high level of CD47 antigen; however, 7DC-VCMMAE treatment also promoted phagocytosis of A549 cells by macrophages. In vivo, 7DC-VCMMAE treatment had remarkable antitumor effects in a NSCLC cell line-derived xenograft (CDX) mouse model based on nonobese diabetic/severe combined immunodeficient (NOD/SCID). In summary, this study combined VCMMAE with anti-CD47 mAbs, emphasizing a novel and promising immunotherapy method for direct killing of NSCLC, which provides a valuable new way to meet the needs of the cancer therapy field.

High cystic fibrosis transmembrane conductance regulator expression in childhood B-cell acute lymphoblastic leukemia acts as a potential therapeutic target.

BackgroundThe role of cystic fibrosis transmembrane conductance regulator (CFTR) in hematopoiesis and adult leukemia has been demonstrated using a zebrafish model and leukemia cell lines in our previous works. Here, we continue to explore the association between CFTR and human childhood B-cell acute lymphoblastic leukemia (B-ALL).MethodsWe continued to collect the peripheral blood and bone marrows of human childhood patients diagnosed with primary B-ALL as well as non-leukemia controls and isolated lymphocytes for analysis using western blotting and quantitative real-time polymerase chain reaction (qPCR) assay. Then, we used immunofluorescence, co-immunoprecipitation, western blotting, luciferase, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays to identify the interaction of CFTR with Wnt signaling in B-ALL. Finally, we established B-ALL xenograft model in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice using SUP-B15 cells, and examined whether the CFTR inhibitor CFTR-inh172 could active against SUP-B15-Dependent B-ALL in vivo.ResultsHighly expressed CFTR protein and mRNA are associated with primary childhood B-ALL patients. Aberrantly upregulated CFTR and Wnt signaling, our previously reported CFTR-Dvl2-β-catenin pathway, is found in human childhood B-ALL patients. Interference with CFTR in B-ALL cell lines induces the downregulation of DVL2/β-catenin and Wnt downstream target accompanied by a reduction of cell proliferation. Furthermore, B-ALL cell lines SUP-B15 cell-transplanted NOD/SCID mice treated with CFTR inhibitor CFTRinh-172 had significantly longer survival and slower leukemia progression compared with mice treated with vehicle dimethyl sulfoxide (DMSO).ConclusionsThese findings demonstrate that highly expressed CFTR is associated with human childhood B-ALL and the potential of CFTR inhibitor CFTR-inh172 for the treatment of human B-ALL.

Poly-Lysine Dendritic Nanocarrier to Target Epidermal Growth Factor Receptor Overexpressed Breast Cancer for Methotrexate Delivery.

A fourth generation poly-lysine dendritic nanocarrier (P4LDN)-based targeted chemotherapy for breast cancer is attempted by incorporating an epidermal growth factor receptor (EGFR)-specific short peptide E2 (ARSHVGYTGAR) and the drug methotrexate (MTX) into a nanocarrier system. The drug is incorporated into the nanocarrier using a cathepsin B cleavable spacer: glycine–phenylalanine–leucine–glycine (GFLG). The in vitro analysis of the time-dependent drug release, binding and internalization ability, and the cytotoxic nature showed that this drug delivery system (DDS) is highly effective. The efficacy analysis using non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice also showed that compared to the control group, the DDS can effectively reduce tumor volume. The mice that received the DDS appeared to gain weight more rapidly than the free drug, which suggests that the dendrimer is more easily tolerated by mice than the free drug.

Fluorine MR Imaging Probes Dynamic Migratory Profiles of Perfluorocarbon-Loaded Dendritic Cells After Streptozotocin-Induced Inflammation.

The pathogenesis of type 1 diabetes (T1D) involves presentation of islet-specific self-antigens by dendritic cells (DCs) to autoreactive T cells, resulting in the destruction of insulin-producing pancreatic beta cells. We aimed to study the dynamic homing of diabetes-prone DCs to the pancreas and nearby organs with and without induction of pancreatic stress in a T1D susceptible model of repeated streptozotocin (STZ) injection. In vitro labeling of activated bone marrow-derived DCs (BMDCs) from NOD (Nonobese diabetes) mice was performed using zonyl perfluoro-15-crown-5-ether nanoparticles (ZPFCE-NPs). Internalization of particles was confirmed by confocal microscopy. Two groups of NOD.SCID (nonobese diabetic/severe combined immunodeficiency) mice with (induced by low dose STZ administration) or without pancreatic stress were compared. Diabetogenic BMDCs loaded with BDC2.5 mimotope were pre-labeled with ZPFCE-NPs and adoptively transferred into mice. Longitudinal in vivo fluorine MRI (19F MRI) was performed 24h, 36h and 48h after transfer of BMDCs. For ex vivo quantification of labeled cells, 19F NMR and flow cytometry were performed on dissected tissues to validate in vivo 19F MRI data. In vitro flow cytometry and confocal microscopy confirmed high uptake of nanoparticles in BMDCs during the process of maturation. Migration/homing of activated and ZPFCE-NP- labeled BMDCs to different organs was monitored and quantified longitudinally, showing highest cell density in pancreas at 48-h time-point. Based on 19F MRI, STZ induced mild inflammation in the pancreatic region, as indicated by high accumulation of ZPFCE-NP-labeled BMDCs in the pancreas when compared to the vehicle group. Pancreatic draining lymph nodes showed elevated homing of labeled BMDCs in the vehicle groups in contrast to the STZ group after 72h. The effect of STZ was confirmed by increased blood glucose levels. We showed the potential of 19F MRI for the non-invasive visualization and quantification of migrating immune cells in models for pancreatic inflammation after STZ administration. Without any intrinsic background signal, 19F MRI serves as a highly specific imaging tool to study the migration of diabetic-prone BMDCs in T1D models in vivo. This approach could particularly be of interest for the longitudinal assessment of established or novel anti-inflammatory therapeutic approaches in preclinical models.

Establishment of Patient-derived Orthotopic Xenografts (PDX) as Models for Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death. The purpose of the present study was to establish a patient-derived orthotopic xenograft model (PDOX) for pancreatic ductal adenocarcinoma (PDAC), thus providing a tumor microenvironment resembling that of the human pancreas to identify novel potential biomarkers and treatment regimens. PDAC tissue samples were received from 35 patients, following informed consent, and three mouse strains were implemented. Successful PDOX engraftment was performed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID gamma (NSG) mice. Nonetheless, we found a higher rate of successful engraftment and tumor growth in NSG compared to NOD/SCID mice, possibly owning to the different level of immunosuppression and more specifically of the natural killer cells presence. Our suggested PDOX model represents a preclinical cancer research model with a high affinity for the patient's tumor microenvironment, thus enabling the acceleration of PDAC research.

LINC00853 restrains T cell acute lymphoblastic leukemia invasion and infiltration by regulating CCR9/CCL25

BackgroundLeukemia is a group of hematopoietic malignancies characterized by the accumulation and infiltration of abnormal hematopoietic stem cells or early progenitor cells. T cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy occurring in 15 % of pediatric and 25 % of adult ALL cases. Infiltration and metastasis of leukemic cells to specific organs are consequences of disease relapse and dismal prognosis. Long non-coding RNAs (lncRNAs) have been identified to function in the migration, invasion and infiltration of tumors by regulating gene expression. Our previous studies showed that CC chemokine receptor 9 (CCR9), which specifically bind to CC chemokine ligand 25 (CCL25), promotes T-ALL infiltration. MethodsBioinformatic methods were used to screen LINC00853 in gene expression omnibus (GEO) datasets. RT-qPCR, western bolt and flow cytometry were applied to detect the expression of LINC00853 and CCR9. Transwell and martrigel-transwell were employed to assess the cells migration and invasion abilities. Fluorescence microscope was applied to observed the green fluorescence protein positive (GFP+) cells. Lentivirus and adenovirus were packed to construct nc-blank, sh-LINC00853-blank and sh-LINC00853-rescue jurkat cell lines. ResultsIn this study, we found out the negative correlation of LINC00853 and CCR9 expression. LINC00853 was downregulated while CCR9 was upregulated in GEO datasets, T-ALL cell lines and clinical samples. Moreover, LINC00853 suppressed jurkat cells migration and invasion in vitro and restrained infiltration in liver, spleen, kidney, lung, brain, ovary of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. ConclusionsThese findings indicate that LINC00853 restrains T-ALL cell invasion and infiltration by regulating CCR9/CCL25.

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Repurposing Melanoma Chemotherapy to Activate Inflammasomes in the Treatment of BRAF/MAPK Inhibitor Resistant Melanoma

The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested invitro and invivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy.

Chandipura virus induces cell death in cancer cell lines of human origin and promotes tumor regression in vivo

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-β pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future.

Antidiabetic DPP-4 Inhibitors Reprogram Tumor Microenvironment That Facilitates Murine Breast Cancer Metastasis Through Interaction With Cancer Cells via a ROS–NF-кB–NLRP3 Axis

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS–NRF2–HO-1 axis in nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS–NRF2–HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-κB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2–HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS–NRF2–HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive lymphocytes in metastatic sites, but did not significantly alter cell viability, apoptosis, differentiation, and suppressive activation of 4T1-induced splenic MDSCs. Moreover, we revealed that DPP-4i triggered ROS-NF-κB-dependent NLRP3 inflammasome activation in BC cells, leading to increase in inflammation cytokines such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), IL-1β and IL-33, and MDSCs inductors granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF, which play a crucial role in the remodeling of tumor immune-suppressive microenvironment. Thus, our findings suggest that antidiabetic DPP-4i reprograms tumor microenvironment that facilitates murine BC metastasis by interaction with BC cells via a ROS–NRF2–HO-1–NF-κB–NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic ROS–NRF2–HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the improvement of tumor microenvironment to alleviate DPP-4i-induced BC metastasis.

Patient-Derived Xenograft Models in Cervical Cancer: A Systematic Review.

Background: Patient-derived xenograft (PDX) models have been a focus of attention because they closely resemble the tumor features of patients and retain the molecular and histological features of diseases. They are promising tools for translational research. In the current systematic review, we identify publications on PDX models of cervical cancer (CC-PDX) with descriptions of main methodological characteristics and outcomes to identify the most suitable method for CC-PDX. Methods: We searched on PubMed to identify articles reporting CC-PDX. Briefly, the main inclusion criterion for papers was description of PDX created with fragments obtained from human cervical cancer specimens, and the exclusion criterion was the creation of xenograft with established cell lines. Results: After the search process, 10 studies were found and included in the systematic review. Among 98 donor patients, 61 CC-PDX were established, and the overall success rate was 62.2%. The success rate in each article ranged from 0% to 75% and was higher when using severe immunodeficient mice such as severe combined immunodeficient (SCID), nonobese diabetic (NOD) SCID, and NOD SCID gamma (NSG) mice than nude mice. Subrenal capsule implantation led to a higher engraftment rate than orthotopic and subcutaneous implantation. Fragments with a size of 1–3 mm3 were suitable for CC-PDX. No relationship was found between the engraftment rate and characteristics of the tumor and donor patient, including histology, staging, and metastasis. The latency period varied from 10 days to 12 months. Most studies showed a strong similarity in pathological and immunohistochemical features between the original tumor and the PDX model. Conclusion: Severe immunodeficient mice and subrenal capsule implantation led to a higher engraftment rate; however, orthotopic and subcutaneous implantation were alternatives. When using nude mice, subrenal implantation may be better. Fragments with a size of 1–3 mm3 were suitable for CC-PDX. Few reports have been published about CC-PDX; the results were not confirmed because of the small sample size.

Cystathionine β-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis

BackgroundHepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine β-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of...

Creation of a vascular inducing device using mesenchymal stem cells to induce angiogenesis

Conventional treatments of peripheral vascular disease and coronary artery disease have partial success but are still limited. Methods to deliver angiogenic factors into ischemic areas using gene, protein and cell therapies are faced with difficult issues such a delivery, effective concentration and duration of action. Tissue engineering offers the possibility of creating a functional self-contained three-dimensional (3D) unit that works as a coordinated biological pump that can secrete a whole range of angiogenic factors. We report a tissue engineering approach using decellularized micro-fragments and mesenchymal stem cells (MSCs) to create a vascular inducing device (VID). Proteomic analysis of the decellularized micro-fragments and of the VIDs reveals a large number of extracellular-matrix (ECM) proteins. Moreover, the VIDs were found to transcribe and secrete a whole repertoire of angiogenic factors in a sustained manner. Furthermore, preliminary results of implantation VIDs into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice indicate formation of vascular network at the site within a week. We propose that those VIDs could serve as a safe, localized, simple and powerful method for the treatment of certain types of vascular diseases.

Depletion of CD56+CD3+ invariant natural killer T cells prevents allergen-induced inflammation in humanized mice

CD56-expressing natural killer (NK) cells as well as invariant NK T (iNKT) cells have been shown to either promote or inhibit allergic immune responses. The aim of the present study was to investigate the impact of these cells in a recently developed humanized mouse model of allergen-induced IgE-dependent gut and lung inflammation. Nonobese diabetic-severe combined immunodeficiency γ-chain knockout mice were injected intraperitoneally with human PBMCs or CD56-depleted (CD56neg) PBMCs from highly sensitized donors with birch or grass pollen allergy together with the respective allergen or with NaCl as a control. Three weeks later, the mice were challenged with the allergen rectally and gut inflammation was monitored by video miniendoscopy and by histology. Furthermore, airway inflammation was measured after an additional intranasal allergen challenge. Allergen-specific human IgE in mouse sera, detectable only after coinjection of the respective allergen, was reduced in mice being injected with CD56neg PBMCs compared with in mice receiving nondepleted PBMCs. Consequently, allergen-induced IgE-dependent colitis, airway hyperreactivity, and mucus-producing goblet cells were significantly inhibited in these mice. Interestingly, reconstitution of CD56neg PBMCs with nondepleted CD56+ cells and with CD56+CD3+ iNKT cells restored gut as well as lung inflammation, whereas addition of CD3-depleted CD56+ cells did not. These results demonstrate that allergen-specific gut and lung inflammation in PBMC-engrafted humanized mice is promoted by CD56+CD3+ iNKT cells, which opens new possibilities of therapeutic intervention in allergic diseases.

Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration.

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair.

Mechanisms of Resistance to Prostate-Specific Membrane Antigen-Targeted Radioligand Therapy in a Mouse Model of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) is effective against prostate cancer (PCa), but all patients relapse eventually. Poor understanding of the underlying resistance mechanisms represents a key barrier to development of more effective RLT. We investigate the proteome and phosphoproteome in a mouse model of PCa to identify signaling adaptations triggered by PSMA RLT. Methods: Therapeutic efficacy of PSMA RLT was assessed by tumor volume measurements, time to progression, and survival in C4-2 or C4-2 TP53-/- tumor-bearing nonobese diabetic scid γ-mice. Two days after RLT, the proteome and phosphoproteome were analyzed by mass spectrometry. Results: PSMA RLT significantly improved disease control in a dose-dependent manner. Proteome and phosphoproteome datasets revealed activation of genotoxic stress response pathways, including deregulation of DNA damage/replication stress response, TP53, androgen receptor, phosphatidylinositol-3-kinase/AKT, and MYC signaling. C4-2 TP53-/- tumors were less sensitive to PSMA RLT than were parental counterparts, supporting a role for TP53 in mediating RLT responsiveness. Conclusion: We identified signaling alterations that may mediate resistance to PSMA RLT in a PCa mouse model. Our data enable the development of rational synergistic RLT-combination therapies to improve outcomes for PCa patients.

AML Cells Alter Bone Homeostasis By Promoting the Formation of Immature Bone and Resorption of Mature Bone That Supports Leukemia Progression

Background: Genetic alterations in the osteoprogenitor cells has been shown to induce acute myeloid leukemia (AML) in several mouse models. Moreover, we have recently reported that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSC) to gain growth advantage in the bone marrow (BM; Battula et al., JCI Insight, 2017). However, the effect of AML cells on bone homeostasis/turnover and its impact on AML progression is unknown. Here, we hypothesize that AML cells expand osteoprogenitor cells and alter the balance between bone formation and resorption. Methods: To investigate the effect of AML cells on osteoprogenitor cells and mature osteoblasts, we used triple transgenic reporter mice with the genotype Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. Murine AML cells with MLL-ENL translocation were implanted into these transgenic mice, and Osteoprogenitor (Osx+) cells and mature osteoblasts (Ocn+) in femurs were measured by confocal microscopy. To investigate the effect of human AML cells on bone composition, patient-derived xenograft (PDX) cells were implanted into non-obese diabetic scid interleukin-2Rγnull (NSG) mice and bone histomorphometry was performed using H&E and Goldner's trichrome staining. Computed tomography (micro-CT) was used to measure bone volume (BV) and mineral density (BMD) in mice. Tartrate-resistant acidic phosphatase (TRAP) staining was performed to measure osteoclast number/activation. Finally, bone density on the vertebral bone (T12) was measured in 263 de-novo AML patients and 23 normal individuals by CT imaging. Results: In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osterix+, but not Osteocalcin+ cells, suggesting AML cells expand osteoprogenitor cells in the BM during short term exposure (2-3 weeks). To investigate the effects of AML on bone during a long term exposure, we implanted 10 different AML-PDX models in NSG mice (3 mice per model) and analyzed femurs by micro-CT and bone histomorphometry analysis. Interestingly, we observed a dramatic increase in new web-like medullary bone formation in 5/10 of the PDX models tested. Moreover, higher bone volume is associated with less aggressive PDX models (which takes 4-6 months to reach 90% circulating leukemia), but not aggressive PDX models (only 4-8 weeks to reach 90% circulating cells). These findings were also confirmed by micro-CT of mouse femurs. Interestingly, in some PDX models, CT images revealed large cavities in cortical bones close to epiphysis and metaphysis areas in the femur and tibia of mice with AML suggesting bone resorption. To validate bone resorption, we performed TRAP staining and found a significant increase in osteoclast activity on the endosteal surface and massive bone resorption in AML bone compared to normal bone. These data indicate high bone turnover in mice with AML compared to control mice. Next, we measured bone densities in AML patients and normal individuals by chest CT imaging. We found bone densities were gradually decrease with age in both healthy individuals and AML patients. However, compared to healthy individuals, bone densities are significantly higher in majority of AML patients (~70%) (p<0.01). Interestingly, survival analysis revealed that higher bone density is associated with good prognosis in AML patients (p<0.01), suggesting high bone turnover alters patient outcomes. Conclusion: Our data suggest that AML cells expand osteoprogenitor-rich niche and alter BM microenvironment by high bone turnover. New bone induction by less aggressive AML-PDX models coupled with AML patient CT data suggests that high bone density and volume are associated with favorable prognostic factors in AML. Mechanisms underlying this observation are under investigation. Disclosures Andreeff: Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Konopleva:Ablynx: Research Funding; AstraZeneca: Research Funding; Kisoji: Consultancy; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; Agios: Research Funding; Sanofi: Research Funding; Cellectis: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding. Battula:Leukemia Lymphoma Society: Research Funding; Tolero Pharmaceuticals: Research Funding; Golfers Against Cancer: Research Funding.

A clear cancer cell line (150057) derived from human endometrial carcinoma harbors two novel mutations

BackgroundCell lines are extremely useful for both basic and clinical research. Thus, establishing endometrial cancer cell lines with malignant histology is important. This study aimed to extensively characterize an endometrial clear cell carcinoma cell line.MethodsThis cell line, named 150,057, was derived from the endometrial clear cell cancer of a 63-year-old woman. The morphology, chromosomes, chemosensitivity, tumor markers, xenotransplantation characteristics, and cancer-related genes of the cell line were characterized.ResultsThis cell line exhibited adequate growth, being passaged more than 70 times. The morphology of the cells was polygonal with a cobblestone-like appearance. Karyotyping of the cell line revealed a hypodiploid chromosomal number. 150057 cells expressed CA19–9 and CA125. The cell line was sensitive to doxorubicin, paclitaxel, carboplatin, and cisplatin. After the cells were transplanted into the subcutaneous region of non-obese diabetic-severe combined immunodeficiency mice, they generated xenograft tumors with similar histology as the original tumor. A total of 59 somatic nucleotide mutations were identified in 25 of the 53 examined tumor suppressor genes and oncogenes. Two novel mutations were found in FGFR3 and ARID1A.ConclusionWe established and characterized an endometrial clear cell carcinoma cell line that may be useful in carcinogenesis and treatment research for endometrial cancer.

Effects of Phthalate Esters on Human Myometrial and Fibroid Cells: Cell Culture and NOD-SCID Mouse Data.

Evidence is growing that phthalate esters play an important role in the pathogenesis of estrogen-dependent gynecologic diseases, especially uterine fibroids. We aimed to investigate whether in vitro treatment with di-(2-ethylhexyl)-phthalate (DEHP) affects angiogenesis, proliferation, and apoptosis in uterine fibroids. To ascertain this, we evaluated vascular endothelial growth factor (VEGF) expression and AKT/ERT phosphorylation and compared the fibroid volume between nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice fed with and without DEHP. VEGF expression was measured using enzyme-linked immunosorbent assay, and AKT/ERK phosphorylation was analyzed by western blot analysis in human myometrial and fibroid cells. The volume of the fibroid tissues implanted to NOD/SCID mice was measured, and the expression of collagen type I protein, Ki-67, proliferating cell nuclear antigen, and B cell lymphoma 2 were analyzed using immunohistochemistry. We could see significant increases in VEGF expression and AKT phosphorylation in human myometrial and fibroid cells treated with DEHP. The volume of the fibroid tissues was significantly increased in NOD/SCID mice fed with DEHP, which was accompanied by increased expression of collagen type I and AKT phosphorylation. Taken together, these results suggest that exposure to phthalate esters may influence uterine fibroid pathogenesis by increasing VEGF and collagen expression and upregulating AKT phosphorylation.

Good Manufacturing Practice-Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform.

Ex vivo (EV)‐derived megakaryocytes (MKs) have shown great promise as a substitute for platelets in transfusion medicine to alleviate a severe shortage of donor‐platelets. Challenges remain that include poor efficiency, a limited scale of production, and undefined short‐term storage conditions of EV‐derived MKs. This study aims to develop a high‐efficiency system for large‐scale production of Good Manufacturing Practice (GMP)‐grade MKs and determine the short‐term storage condition for the MKs. A roller‐bottle culture system was introduced to produce GMP‐grade MKs from small‐molecule/cytokine cocktail expanded hematopoietic stem cells. Various buffer systems and temperatures for the short‐term storage of MKs were assessed by cell viability, biomarker expression, and DNA ploidy levels. MKs stored for 24 hours were transplanted into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm their platelet‐releasing and tissue‐homing ability in vivo. A yield of ~ 2.5 × 104 CD41a+/CD42b+ MKs with purity of ~ 80% was achieved from one original cord blood CD34+ cell. Compared with the static culture, the roller‐bottle culture system significantly enhanced megakaryopoiesis, as shown by the cell size, DNA ploidy, and megakaryopoiesis‐related gene expression. The optimal storage condition for the MKs was defined as normal saline with 10% human serum albumin at 22℃. Stored MKs were capable of rapidly producing functional platelets and largely distributing in the lungs of NOD/SCID mice. The novel development of efficient production and storage system for GMP‐grade MKs represents a significant step toward application of these MKs in the clinic.