Patient-derived Xenotransplantation Models Research Articles

Inhibition of PAK6-Mediated Survival and Cell Cycle Controls Selectively Targets Therapy-Resistant CML Stem/Progenitor Cells

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have recently identified miR-185 as a useful biomarker to predict therapy response in treatment-naïve CML stem/progenitor cells. We also demonstrated that restored miR-185 expression in LSCs impaired survival, sensitizing them to TKIs in vitro and in preclinical patient-derived xenotransplantation models, indicating that miR-185 is a critical regulator mediating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was uncovered as a target gene of miR-185 by RNA-seq and was found to be upregulated in CD34+ TKI-nonresponder cells vs. TKI-responders, but its biological functions in CML are largely unknown. To investigate the biological effects of inhibiting PAK6 activity in TKI-resistant cells, we tested a pre-clinically validated pan-PAK inhibitor (PF-3758309) in vitro. PF-3758309 significantly reduced the growth of IM-resistant cell lines, including K562-resistant cells, BV173 blast cells (IC50 25-70 nM) and CD34+ TKI-nonresponder cells, as assessed by viability and CFC assays, and increased their apoptosis; these effects were significantly enhanced by TKIs (~2-fold, P<0.05). PF-3758309 alone, or in combination with a TKI, did not have obvious inhibitory effects on CD34+ normal bone marrow. These results were further confirmed in IM-resistant cells using a lentiviral knockdown system that specifically inhibits PAK6. Interestingly, PF-3758309 alone, or in combination with a TKI, greatly reduced mitochondrial activity in CD34+ TKI-nonresponder cells, as shown in functional assessments with MitoTracker, a dye that accumulates in active mitochondria, the site of OXPHOS; this was not seen with TKI alone (P<0.002). Similarly, CellROX analysis confirmed a reduction in ROS levels upon PF-3758309 treatment, or a combination of PF-3758309 with TKI, in these cells. In addtion, MDM2, a critical negative regulator of the p53 tumor suppressor, was identified as one of substrates of PAK6, by PhosphoSitePlus analysis. Its expression was found to be correlated with PF-3758309 treatment in CML cells based on CellMinerDB univariate analyses using gene-small-molecule association data from the CTRPv2 database. Indeed, Western blot analysis showed that PAK6 knockdown in K562 and IM-resistant cells led to a reduction in MDM2 protein levels. Furthermore, MDM2 downregulation by PAK6 inhibition corresponded to an increase in p21 levels, suggesting a mechanism of MDM2-mediated p21 regulation independent of p53, as these cells are p53-null. Most interestingly, PAK6 knockdown in IM-resistant cells leads to G2/M phase accumulation and increased senescence levels (2-fold, P<0.05), detected by senescence-associated β-galactosidase staining. PAK6 knockdown induced senescence was further supported by observations of enlarged cell size (p<0.05) and increased granulation, as well as changes in senescence-associated protein markers, including p21, p27, MMP-3 and the DNA damage marker pH2Ax, by Western blot analysis. Hence, our findings indicate that dual targeting of miR-185-PAK6-mediated survival, cell cycle and metabolic pathways, along with BCR-ABL, selectively eradicates drug-resistant CML stem/progenitors. Specifically, PAK6 plays roles in MDM2/p21-mediated apoptosis, senescence and cell cycle controls, offering a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.

Up-Regulation of miR-26a-5p Inhibits E2F7 to Regulate the Progression of Renal Carcinoma Cells.

BackgroundMetastasis is the main cause of renal cell carcinoma (RCC) tumor death, and effective inhibition of RCC metastasis is an essential means to meliorate the prognosis of RCC patients. MicroRNAs (miRs) have been proved to be stable and important biomarkers for several malignancies. This study is therefore set out to explore the metastasis-related miR and its mechanism in RCC.MethodsThe expression of miR-26a-5p in RCC was analyzed using the expression profile in the Cancer Genome Atlas (TCGA). MiR-26a-5p and E2F transcription factor 7 (E2F7) in RCC patients were detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) was adopted to assess cell proliferation, Transwell was utilized to evaluate migration and invasion, and flow cytometry (FC) was used to determine apoptosis. Mouse cell-derived and patient-derived xenotransplantation models were established to evaluate the effect of miR-26a-5p on tumor growth and metastasis in vivo. The molecular mechanism of miR-26a-5p was analyzed by dual-luciferase reporter (DLR) gene analysis, qRT-PCR, and Western blot (WB) both in vivo and in vitro.ResultsMiR-26a-5p was reduced in renal carcinoma cells and may serve as a biomarker for renal cancer metastasis and prognosis. MiR-26a-5p up-regulation inhibited migration and invasion in renal cell lines and tumor metastasis in vivo. Bioinformatics target prediction and RNA-seq results showed that E2F7 was among the targets of miR-26a-5p and was significantly inhibited by miR-26a-5p in vivo and in vitro.ConclusionMiR-26a-5p presents low expression in RCC and promotes RCC cell apoptosis and prevents cells from proliferating and invading by targeting E2F7, which is a promising therapeutic target for RCC.

A highly efficient and faithful MDS patient-derived xenotransplantation model for pre-clinical studies

Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. Here we report a MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient “MISTRG” mice that provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients’ dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample’s genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells.

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.

Functional evidence for derivation of systemic histiocytic neoplasms from hematopoietic stem/progenitor cells

AbstractLangerhans cell histiocytosis (LCH) and the non-LCH neoplasm Erdheim-Chester disease (ECD) are heterogeneous neoplastic disorders marked by infiltration of pathologic macrophage-, dendritic cell–, or monocyte-derived cells in tissues driven by recurrent mutations activating MAPK signaling. Although recent data indicate that at least a proportion of LCH and ECD patients have detectable activating kinase mutations in circulating hematopoietic cells and bone marrow–based hematopoietic progenitors, functional evidence of the cell of origin of histiocytosis from actual patient materials has long been elusive. Here, we provide evidence for mutations in MAPK signaling intermediates in CD34+ cells from patients with ECD and LCH/ECD, including detection of shared origin of LCH and acute myelomonocytic leukemia driven by TET2-mutant CD34+ cell progenitors in one patient. We also demonstrate functional self-renewal capacity for CD34+ cells to drive the development of histiocytosis in xenotransplantation assays in vivo. These data indicate that the cell of origin of at least a proportion of patients with systemic histiocytoses resides in hematopoietic progenitor cells prior to committed monocyte/macrophage or dendritic cell differentiation and provide the first example of a patient-derived xenotransplantation model for a human histiocytic neoplasm.

Engraftment of Human MDS Samples in Xenotransplanted Mice Depends on Human Cytokines but Not Mesenchymal Stem Cells (MSC)

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with limited treatment options. Development of a patient derived xenotransplantation model for pre-clinical studies is a priority goal in the field. It has been hypothesized that patient derived mesenchymal stem cells and/or human cytokines are necessary to establish MDS engraftment in immunocompromised mice. In this study we evaluated the ability of mesenchymal stem cells (MSC) to facilitate the engraftment process having as a goal to generate a robust patient-derived xenograft (PDX) mouse model for MDS. We also assessed the contribution of human cytokines as expressed in transgenic mice.Methods: MSC from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-S) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC. MSC were from normal donors, from same patient as MNC (autologous), from other MDS patients or normal donors (allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration at 6-8, 10 weeks and at necropsy at 12-32 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSG-S mice. 1 x 106 human CD45+ cells were passaged without or with 5 x 105 MSC. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD), chronic myelomonocytic leukemia (CMML)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells by means of FACS. Lymphoid, myeloid and erythroid engraftment was assessed similarly. 5 x 105 MSCs from normal donors were transduced with GFP (pELNS.CBR-T2A-GFP) and intrafemorally injected into NSG mice and the presence of human MSC was evaluated by in vivo Imaging (Xenogen Spectrum system and Living Image Version 4.3 software.).Results: 12 out of 12 injected MDS samples showed persistent human cells when assessed at 6-8 weeks. For most samples, engraftment at that time-point was low (<2%) and was not consistently influenced by the presence or absence of MSC. On week 10, 4 out of 12 injected MDS patient samples showed increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSC. Two of these were from patients with RAEB and CMML and showed both myeloid and erythroid engraftment. Molecular analysis showed consistent engraftment of the malignant clone in 2 out of 2 patient samples tested. Overall, only 3 out of 12 patients showed long-term engraftment (> 12 weeks) and these samples were transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC showed variable engraftment levels on week 10 after injections. One of them reached long-term engraftment (>12 weeks), whereas the second gradually lost engraftment after week 10 and the third is ongoing. MSC tracking showed a gradual loss of MSC after intrafemoral injections to the point that no MSC were detected 3 weeks later. Comparison of engraftment in NSG mice to NSG-S mice (the latter producing human cytokines) showed a slight increase in engraftment at week 6 which continued at later time points.Conclusions: Passive transfer of MDS hematopoietic cells as assessed at 6-8 weeks after intrafemoral injection is highly consistent and not dependent on MSC when total MNC fraction is used. The latter may be explained since MSC are lost within the first three weeks after injections. Direct comparison of NSG vs. NSG-S mice showed that the presence of hGM-CSF, hIL-3 and h-SCF in the NSG-S mice increased the engraftment levels. Successful long-term engraftment of MDS cells in xenotransplanted mice was achieved for one patient sample so far, indicating that MDS initiating cells can be maintained in NSG-S mice. However, establishing long-term engraftment can only be achieved with a subset of patient samples. MDS engraftment is influenced by human cytokines but not with MSC cells which may suggest that MDS does not depend on MSC's for long term growth. DisclosuresNunez-Cruz:Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding.

Targeting Acute Myeloid Leukemia with a New CXCR4 Antagonist IgG1 Antibody (PF-06747143)in NOD/SCID Mice

Chemoresistance represents a considerable barrier to improving outcomes for patients with acute myeloid leukemia (AML) and therapeutic approaches using multiple lines of therapy have been unsuccessful as cancer cells acquire resistance to the chemotherapeutic agents to which they are exposed. This vulnerable patient group needs individualizing therapy through careful selection of appropriate agents based on specific signaling pathways. The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow microenvironment, is highly expressed in 25-30% of patients with AML and its expression is correlated with poor prognosis and drug resistance. The purpose of this study was to investigate a new humanized monoclonal IgG1 antibody to CXCR4 (PF-06747143) and its effects as a monotherapy in AML primary patient samples and in chemotherapy resistant patient-derived xenotransplantation (PDX) models. This antibody was previously shown to be able to induce cell death through its effector function (CDC and ADCC) and to be efficacious in cell-based xenograft models of AML, NHL, CLL and MM.Here we have shown that PF-06747143 binds strongly and specifically to AML cell lines and to AML primary cells, by flow cytometry. Of 16 samples evaluated, 7 displayed low CXCR4 expression (CXCR4neg/low) whereas 9 displayed high expression (CXCR4high). A good correlation was observed between 12G5, a commercially available CXCR4 Ab, and PF-06747143 staining, indicating that PF-06747143 can be used to stratify AML patients. Chemotaxis in response to CXCL12 was significantly inhibited in all AML patient primary samples analyzed. Administration of PF-06747143 to mice engrafted with AML patient cells (PDX models) induced rapid malignant cell mobilization into the peripheral blood at 4 hrs after a single antibody dose, with mobilized cell levels going back down to baseline at 24 hrs post-dose. This is in line with the ability of the antibody to block malignant cell homing to the bone marrow, inducing cell mobilization, as well as induction of cell death through effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different models: 1) P15 model characterized by high CXCR4 expression, inducing aggressive disease, with rapid progression of leukemia and widespread dissemination and chemoresistance; 2) P17 model characterized by a low CXCR4 expression, a less aggressive disease and limited dissemination. Weekly administration of PF-06747143 to leukemic mice previously engrafted with P17 or P15 malignant cells induced a sharp reduction of leukemia cells in the bone marrow, spleen and blood leading to increased survival of leukemic mice in both models. Activity of the antibody as monotherapy was superior to daunorubicin in the P15 chemoresistant model. Secondary transplantation of bone marrow cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143 treatment, with slower tumor growth in mice transplanted with PF-06747143-treated cells. In summary, PF-06747143, a CXCR4 IgG1 antibody, is significantly efficacious as a monotherapy and superior to daunorubicin in AML chemoresistant PDX models. These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Patient Derived Xenotransplantation Model of Atypical Chronic Myeloid Leukemia (aCML)

Background:Atypical chronic myeloid leukemia (aCML) is a rare disorder classified as one of the MPN/MDS overlap syndromes. aCML usually presents like CML but lacks the pathognomonic BCR-ABL fusion found in CML. Most patients progress to acute myeloid leukemia (AML) with a median time to AML of 11.2 months and have a median overall survival of only 12.4 months (Wang et al. Blood 2014). Recurrently mutated genes found in aCML patients include SETBP1 , CSF3R, NRAS, EZH2, ASXL1, ETNK1, and U2AF1.The pathogenesis of aCML is poorly understood and neither specific nor effective treatments besides hematopoietic stem cell transplantation are available. We therefore aimed at developing a patient derived xenotransplantation model that allows serial transplantation and expansion of human leukemic cells and evaluation of novel treatments and drugs in vivo.Patient and Methods:Bone marrow cells were harvested from a patient diagnosed with atypical CML based on persistent leukocytosis, immature circulating myeloid precursors (16% metamyelocytes, 8% myelocytes, 9% blasts), marked dysgranulopoiesis, minimal monocytosis and basophilia, hypercellular bone marrow with high myeloid/erythroid ratio and 6% myeloid blasts, dysplasia in megakaryocytes and erythroid progenitors, and absence of BCR-ABL and mutated JAK2. The patient had moderate anemia and normal platelet counts and cytogenetic analysis showed a normal karyotype.Eight hundred thousand bone marrow cells were injected intravenously in NOD/SCID IL-2 receptor γ (NSG) deficient mice. We monitored these mice for human cell engraftment by regular eye bleeds every 4 weeks. Bone marrow and spleen cells from engrafted mice were retransplanted in secondary and tertiary mice of the NSG strain transgenic for SCF, IL3 and GM-CSF (NSGS). Patient cells were analyzed for mutations in fifty four genes by next generation sequencing and mutations were confirmed by Sanger sequencing in primary patient cells and cells from tertiary mice.Results: Human CD45+ cells from the aCML patient showed increasing engraftment over time in the NSG mouse reaching 16% in peripheral blood and 35% in spleen at 26 weeks after transplantation. In secondary (n=2) and tertiary (n=4) mice we used NSGS recipient mice and observed considerably accelerated engraftment kinetics leading to 19, 21 and 73% human cells in peripheral blood, spleen and bone marrow, respectively, between 12 and 15 weeks after transplantation. The myeloid marker CD33 was expressed in 86% of human bone marrow cells, while lymphoid markers CD3 and CD19 were absent. The stem and progenitor phenotype CD34+CD38- was found in 11% of human cells. The progenitor marker CD123 was expressed in 42% of cells, while the myeloid marker CD14 was expressed in 6% of cells. Hemoglobin levels and platelet counts were considerably lower in secondary and tertiary recipients of aCML cells compared to control animals. Spleens were enlarged at time of sacrifice with an average spleen weight of 150 mg. Morphological evaluation of bone marrow cells in tertiary recipients revealed a characteristic picture for aCML with 39% neutrophils, 8% blasts and 53% myeloid progenitors and monocytes. Molecular analysis identified mutations in ASXL1, RUNX1 and EZH2 with variant allele frequencies of 49, 48 and 46 percent, respectively that were confirmed in human cells from tertiary recipient mice. Thus, we show that primary aCML cells can be expanded and serially transplanted in immunodeficient mice and suggest clonal stability of this model.Conclusion:We provide the first patient derived xenotransplantation model for atypical CML, which preserves the phenotypic and molecular characteristics of the primary disease and allows serial transplantation and expansion of aCML cells. This model will serve to better understand the pathogenesis of aCML and to test urgently needed novel treatment approaches. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Efficacy of PI3K Pathway Signaling Inhibition for Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia

The Philadelphia chromosome (Ph)-like subtype of B-precursor acute lymphoblastic leukemia (ALL) comprises approximately 15% of high-risk ALL, has a kinase-activated gene expression profile similar to that of BCR-ABL1-rearranged ALL, and is associated with a variety of mutations and gene fusions known or predicted to activate oncogenic signal transduction. Children and adults with Ph-like ALL have a very high risk of relapse and poor survival when treated with conventional chemotherapy. Others and we have previously observed constitutive activation of cytokine receptor signaling in Ph-like ALL, particularly of the JAK/STAT and PI3K/Akt/mTOR pathways (Tasian et al., Blood 2012). Preclinical and early clinical studies of JAK inhibition in childhood ALL are in progress. However, the functional role of aberrant PI3K pathway signaling has not been previously investigated in Ph-like ALL. The clinical efficacy of the mTOR inhibitor rapamycin and its analogues has proven suboptimal in various solid and hematologic malignancies, at least in part due to upregulation of Akt signaling, a known sequela of signal transduction inhibitor (STI) monotherapy and a common resistance mechanism. We hypothesized that newer-generation STIs that target multiple PI3K pathway signaling proteins or that selectively inhibit PI3K isoforms may result in superior inhibition of leukemia proliferation and minimize upregulation of alternate signaling pathways. We used patient-derived xenotransplantation models to determine the effects of PI3K pathway STIs upon NOD-SCID-γ-null (NSG) mice well-engrafted with de novo (n = 3) or relapsed (n = 1) childhood Ph-like ALL specimens with JAK2 mutations and/or CRLF2 alterations (Maude et al., Blood 2012). Specifically, we tested the PI3Kα inhibitor BYL719 (30 mg/kg/day), the PI3Kδ inhibitor CAL101 (idelalisib; 30 mg/kg/day), the PI3K/mTOR inhibitor PKI587 (10 mg/kg/day), and the TORC1/TORC2 inhibitor AZD2014 (20 mg/kg/day) to identify the most efficacious PI3K pathway inhibitor(s). Initial pharmacodynamic studies demonstrated that mice treated with each of the four STIs for 72 hours demonstrated potent in vivo inhibition of relevant phosphoproteins in comparison to vehicle-treated mice as measured by phosphoflow cytometric analyses of gated human ALL cells within murine spleens. In particular, both BYL719 and CAL101 treatments resulted in marked inhibition of phosphorylated (p) PI3K, mTOR, S6, and AktS473 via comparison of median fluorescent intensities for STI- vs. vehicle-treated groups with the Mann-Whitney test (p <0.01 for all phosphoproteins). Increased phosphorylation of other measured proteins was not observed, suggesting that proximal inhibition effectively abrogated aberrant PI3K pathway signal transduction with minimal compensatory signaling upregulation. PKI587 treatment robustly inhibited pS6 and p4EBP1 in comparison to vehicle-treated mice (p = 0.001 and 0.003, respectively), but, surprisingly, had minimal effects upon upstream phosphoproteins. AZD2014 inhibited pS6, p4EBP1, and pAktS473, as well as pERK (p < 0.05 for all phosphoproteins). In longer-term therapeutic trials, TORC1/TORC2 inhibition induced stable disease in xenografted mice treated for 4 weeks in comparison to vehicle controls (p < 0.005), while PI3K/mTOR inhibition robustly decreased leukemic burden below pre-treatment levels in blood, bone marrow, and spleen versus vehicle-treated mice (p < 0.001). These studies demonstrate that PI3K pathway inhibition is an effective and biochemically relevant therapeutic strategy for Ph-like ALL. Pharmacodynamic studies and therapeutic trials of the four PI3K pathway STIs are currently ongoing in additional xenograft models of ALLs expressing various defined genetic lesions to delineate the potential therapeutic range of these compounds. Results from these studies will help to improve our understanding of the critical biologic mechanisms involved in Ph-like ALL and to inform our development of clinical trials to test STI-based therapies in patients with these high-risk leukemias. Disclosures:No relevant conflicts of interest to declare.

Establishment of patient-derived xenotransplantation models for non-small cell lung cancer in immune deficient mice

Targeted therapies have become a valuable therapeutic option for cancer. Establishment of different animal tumor models has become necessary. This study was to establish xenotransplantation models for patient-derived non-small cell lung cancer (NSCLC) in immune deficient mice. Immune deficient mice, BALB/C-nu, NOD/scid and SCID, 16 in each strain, were used. Sixteen tumor specimens were obtained from patients with NSCLC. Each specimen was subcutaneously transplanted into one mouse from each of the three strains. The tumor formation rate, time to tumor engraftment, tumor volume doubling time were recorded and compared among the three strains of mice. Histology of xenograft tumors was examined. The total tumor formation rate was 75% (12/16). The tumor formation rate was the highest in SCID mice (56.25%). Only four tumors were engrafted in SCID mice, and two in BALB/C-nu mice. The tumor formation rate, time to tumor engraftment, and tumor volume doubling time were not significantly different among the three strains of mice. The incidence of tumor size over 1cm in the upper flanks of the mice (56.25%) was significantly higher than that in the lower flanks (25%) (P=0.037). Haematoxylin Eosin staining revealed a high degree of histological similarity between all xenograft and the parental tumors. We have established xenotransplantation models for patient-derived NSCLC with a success rate of 75% in BALB/C-nu and SCID mice. The xenograft tumors have the same histological features to those of their parental tumors.

Convulsions during Anaesthesia: Atropine in Childhood

Abstract Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. We developed a novel MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient mice that for the first time provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients9 dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample9s genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.