Modeling Spontaneous Bone Metastasis Formation of Solid Human Tumor Xenografts in Mice

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro “metastasis” assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.

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Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽

10.1182/blood.v116.21.1460.1460 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1460-1460

Author(s):

Laura A Paganessi ◽

Lydia Luy Tan ◽

Sucheta Jagan ◽

Robin Frank ◽

Antonio M. Jimenez ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Gm Csf ◽

Cytokine Treatment ◽

Ex Vivo Culture ◽

Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

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Human MAIT cells are devoid of alloreactive potential: prompting their use as universal cells for adoptive immune therapy

10.1101/2021.04.29.21256184 ◽

2021 ◽

Author(s):

Marie Tourret ◽

Nana Talvard-Balland ◽

Marion Lambert ◽

Ghada Ben Youssef ◽

Mathieu F. Chevalier ◽

...

Keyword(s):

T Cells ◽

Ex Vivo ◽

Immune Therapy ◽

Adoptive Therapy ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Mait Cells ◽

Microbial Defense

ABSTRACTBackgroundMucosal associated invariant T (MAIT) cells are semi-invariant T cells that recognize microbial antigens presented by the highly conserved MR1 molecule. MAIT cells are predominantly localized in the liver and barrier tissues and are potent effectors of anti - microbial defense. MAIT cells are very few at birth and accumulate gradually over a period of about 6 years during infancy. The cytotoxic potential of MAIT cells, as well as their newly described regulatory and tissue repair functions, open the possibility of exploiting their properties in adoptive therapy. A prerequisite for their use as “universal” cells would be a lack of alloreactive potential, which remains to be demonstrated.MethodsWe used ex vivo, in vitro and in vivo models to determine if human MAIT cells contribute to allogeneic responses.ResultsWe show that recovery of MAIT cells after allogeneic hematopoietic stem cell transplantation recapitulates their slow physiological expansion in early childhood, independent of recovery of conventional T cells. In vitro, signals provided by allogeneic cells and cytokines do not induce sustained MAIT cell proliferation. In vivo, human MAIT cells do not expand nor accumulate in tissues in a model of T-cell mediated xenogeneic graft-versus-host disease (GVHD) in immunodeficient mice.ConclusionsAltogether, these results provide evidence that MAIT cells are devoid of alloreactive potential and pave the way for harnessing their translational potential in universal adoptive therapy overcoming barriers of HLA disparity.

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Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽

10.1182/blood.v93.3.1097 ◽

1999 ◽

Vol 93 (3) ◽

pp. 1097-1105 ◽

Cited By ~ 111

Author(s):

G. Güenechea ◽

J.C. Segovia ◽

B. Albella ◽

M. Lamana ◽

M. Ramı́rez ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Short Term ◽

Immunodeficient Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

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Initiation of immunotherapy with activated natural killer cells and anti-GD2 antibody dinutuximab prior to resection of primary neuroblastoma prolongs survival in mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001560 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001560

Author(s):

Michael John Zobel ◽

Abigail K Zamora ◽

Hong-wei Wu ◽

Jianping Sun ◽

Danny Lascano ◽

...

Keyword(s):

High Risk ◽

Mouse Model ◽

Natural Killer ◽

Primary Tumor ◽

Ex Vivo ◽

Clinical Investigation ◽

Cell Infiltration ◽

Primary Tumors

BackgroundImmunotherapy with anti-disialoganglioside dinutuximab has improved survival for children with high-risk neuroblastoma (NB) when given after induction chemotherapy and surgery. However, disease recurrence and resistance persist. Dinutuximab efficacy has not been evaluated when initiated before primary tumor removal. Using a surgical mouse model of human NB, we examined if initiating dinutuximab plus ex vivo-activated natural killer (aNK) cells before resection of the primary tumor improves survival.MethodsIn vitro, human NB cells (SMS-KCNR-Fluc, CHLA-255-Fluc) were treated with dinutuximab and/or aNK cells and cytotoxicity was measured. In vivo, NB cells (SMS-KCNR-Fluc, CHLA-255-Fluc, or COG-N-415x PDX) were injected into the kidney of NOD-scid gamma mice. Mice received eight intravenous infusions of aNK cells plus dinutuximab beginning either 12 days before or 2 days after resection of primary tumors. Tumors in control mice were treated by resection alone or with immunotherapy alone. Disease was quantified by bioluminescent imaging and survival was monitored. aNK cell infiltration into primary tumors was quantified by flow cytometry and immunohistochemistry at varying timepoints.ResultsIn vitro, aNK cells and dinutuximab were more cytotoxic than either treatment alone. In vivo, treatment with aNK cells plus dinutuximab prior to resection of the primary tumor was most effective in limiting metastatic disease and prolonging survival. aNK cell infiltration into xenograft tumors was observed after 1 day and peaked at 5 days following injection.ConclusionDinutuximab plus aNK cell immunotherapy initiated before resection of primary tumors decreases disease burden and prolongs survival in an experimental mouse model of NB. These findings support the clinical investigation of this treatment strategy during induction therapy in patients with high-risk NB.

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Ex-Vivo Expansion of Marrow Repopulating Cells by Chromatin Modifying Agents Is Associated with Modulation of Expression of Genes Implicated in HSC Self-Renewal.

Blood ◽

10.1182/blood.v108.11.1670.1670 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1670-1670

Author(s):

Hiroto Araki ◽

Kazumi Yoshinaga ◽

Ronald Hoffman ◽

Piernicola Boccuni ◽

Nadim Mahmud

Keyword(s):

Ex Vivo ◽

Trichostatin A ◽

Donor Cell ◽

Characteristic Functional ◽

Self Renewal ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Cell Divisions

Abstract Human hematopoietic stem cells (HSCs) exposed to cytokine combinations in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program which determines the properties of HSC. In order to expand the number of HSC present in a single unit of cord blood (CB) ex vivo, self-renewal type of HSC division must occur. We hypothesize that in vitro culture conditions result in the silencing of genes crucial for HSC maintenance and that silencing of these genes can be circumvented by addition of chromatin modifying agents. We have attempted to reverse the silencing of the genes crucial for HSC self-renewal which apparently occurs during the ex vivo culture by treatment of CD34+ cells with the chromatin modifying agents, 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA). In our current studies, we have investigated the mechanism of expansion of SRC following treatment with chromatin modifying agents in the culture. We demonstrate that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to cytokines alone. CD34+CD90+ cells exposed to the chromatin modifying agents are capable of producing greater numbers of primitive multipotential progenitors and also form cobblestone areas. When CD34+CD90+ cells that had undergone extensive number of cell divisions (5–10) in vitro in the presence of cytokines alone were re-isolated by FACS and transplanted into immunodeficient mice, donor cell chimerism was not detectable (0 of 5 mice). By contrast, 5azaD/TSA treated cells that had undergone similar numbers of cell divisions retain their marrow repopulating potential (3 of 6 mice). To test whether chromatin modifying agents treated cells following culture possess long-term in vivo repopulation potential, we have performed secondary NOD/SCID assay. Five of six secondary NOD/SCID mice receiving bone marrow from primary mice engrafted with cells treated with 5azaD/TSA resulted in human cell engraftment, indicating that these cells are capable of secondary reconstitution. To understand the molecular mechanism responsible for the expansion of HSC observed following 5azaD/TSA treatment, we examined transcription levels of several genes and their products (i.e., HOXB4, Bmi-1 and P21) implicated in self-renewal of HSC using real-time quantitative PCR and Western blot. The expression of these genes and their products were up-regulated in CB cells treated with 5azaD/TSA. We have also compared the efficacy of an additional HDAC inhibitor valproic acid (VPA) in order to determine its ability to expand HSC ex vivo. VPA was capable of dramatic expansion of CD34+CD90+ cells as well as progenitor cells but was unable to expand SRC. However, unlike the culture exposed to cytokines alone VPA treatment resulted in maintenance of SRC numbers. Currently, we are investigating key candidate genes accountable for the expansion of SRC using a global microarray approach analyzing cells exposed to various chromatin modifying agents in conjunction with their in vivo functional potential. In summary, our data suggest that the loss of SRC can be circumvented by the use of chromatin modifying agents in the culture which results in a slower rate of cell division and is associated with higher expression of a group of HSC regulatory genes.

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KLK4 Induces Anti-Tumor Effects in Human Xenograft Mouse Models of Orthotopic and Metastatic Prostate Cancer

Cancers ◽

10.3390/cancers12123501 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3501

Author(s):

Brian W.-C. Tse ◽

Thomas Kryza ◽

Mei-Chun Yeh ◽

Ying Dong ◽

Kamil A. Sokolowski ◽

...

Keyword(s):

Prostate Cancer ◽

Xenograft Model ◽

In Vivo Studies ◽

Cautionary Tale ◽

Prostate Cancer Cells ◽

Primary Tumors ◽

Intracardiac Injection

Recent reports have suggested the role of kallikrein-related peptidase 4 (KLK4) to be that of remodeling the tumor microenvironment in many cancers, including prostate cancer. Notably, these studies have suggested a pro-tumorigenic role for KLK4, especially in prostate cancer. However, these have been primarily in vitro studies, with limited in vivo studies performed to date. Herein, we employed an orthotopic inoculation xenograft model to mimic the growth of primary tumors, and an intracardiac injection to induce metastatic dissemination to determine the in vivo tumorigenic effects of KLK4 overexpressed in PC3 prostate cancer cells. Notably, we found that these KLK4-expressing cells gave rise to smaller localized tumors and decreased metastases than the parent PC-3 cells. To our knowledge, this is the first report of an anti-tumorigenic effect of KLK4, particularly in prostate cancer. These findings also provide a cautionary tale of the need for in vivo analyses to substantiate in vitro experimental data.

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CMET-26. IN-VITRO & IN-VIVO CULTURE OF PATIENT (PT) DERIVED CSF-CTCS IN LEPTOMENINGEAL DISEASE (LMDZ) FROM MELANOMA TO IDENTIFY NOVEL TREATMENT STRATEGIES

Neuro-Oncology ◽

10.1093/neuonc/noz175.227 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi57-vi57

Author(s):

Vincent Law ◽

Brittany Evernden ◽

John Puskas ◽

Gisela Caceres ◽

Elena Ryzhova ◽

...

Keyword(s):

Single Cell Analysis ◽

Ex Vivo ◽

Treatment Strategies ◽

Braf V600e ◽

Leptomeningeal Disease ◽

Immunodeficient Mice ◽

In Vivo Culture ◽

In Vivo Testing

Abstract BACKGROUND Approx. 5% of melanoma pts develop LMDz. There are essentially no models of LMDz available for therapeutic development. Here we report, the in-vitro & in-vivo culturing of CSF-CTCs. METHODS CSF-CTCs were detected by the Veridex CellSearch® System. Cell-free DNA and cell-associated DNA were extracted, sequenced and profiled. Expanded ex-vivo CSF-CTCs were grown in-vitro and tested for drug sensitivity. CSF-CTCs were grown successfully in-vivo from 1 pt; labeled human Braf V600E WM164 cells were injected IT in as a control. RESULTS CSF-CTCs: 12 LMDz pts and 8 melanoma pts without LMDz were studied. All but 1 LMDz pts (92%) had CSF-CTCs (avg: 2148.6; range 23 - 3055 CTCs/ml). In contrast, 3/8 (37%) melanoma Brain Mets pts without LMDz had CSF-CTCs but fewer of them (avg: 0.31; range 0.13 - 0.6 CTCs/ml CSF). CSF-CTCs Profile: These had BrafV600E (83%), and GNAQ Q209P & NRAS Q61R in 1 pt each. Ex-vivoculture of CSF-CTCs and PDX model: After lengthy optimization of conditions we successfully expanded CSF-CTCs in vitro(~25% of pts), and in-vivo in immunodeficient mice from 1 pt (~10% of samples). Ceritinib, used as a FAK inhibitor, with MEKi was effective in-vitro (p=3.17e-6) and prolonged survival in-vivo in LMDz (median survival: >32 days vs control: 18 days; p=7.81e-5). CONCLUSIONS Though the sample size is small, this is the first report of the successful in-vitro & in-vivo culture of CSF-CTCs from pts with LMDz. Single cell analysis to determine how representative these models are and further in-vivo testing are in progress.

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Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo

Cancers ◽

10.3390/cancers12030690 ◽

2020 ◽

Vol 12 (3) ◽

pp. 690 ◽

Cited By ~ 1

Author(s):

Nicholas R. Hum ◽

Aimy Sebastian ◽

Sean F. Gilmore ◽

Wei He ◽

Kelly A. Martin ◽

...

Keyword(s):

Cancer Cell ◽

Ex Vivo ◽

Significant Proportion ◽

3D Culture ◽

Cell Phenotype ◽

Primary Tumors ◽

Altered Expression ◽

Mesenchymal Transition

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

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HGG-12. HUMAN IPSC-DERIVED H3.3K27M NEUROSPHERES: A NOVEL MODEL FOR INVESTIGATING DIPG PATHOGENESIS AND DRUG RESPONSE

Neuro-Oncology ◽

10.1093/neuonc/noab090.078 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i19-i20

Author(s):

Kasey Skinner ◽

Tomoyuki Koga ◽

Shunichiro Miki ◽

Robert F Gruener ◽

R Stephanie Huang ◽

...

Keyword(s):

Principal Component ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Genetically Engineered ◽

High Grade ◽

Primary Tumors ◽

Secondary Tumor ◽

Immunodeficient Mice

Abstract Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). Existing DIPG models have provided insight into the role of H3.3K27M but have limitations: genetically engineered murine models often rely on overexpression of the mutant histone to form tumors; patient-derived xenografts (PDX) are more genetically faithful but preclude examination of the effect of individual mutations on pathogenesis. To address these shortcomings and better recapitulate the genetics of human tumors, we designed a novel DIPG model based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express heterozygous H3.3K27M. Edited iPSC were chemically differentiated into neural progenitor cells, which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Further, neurospheres cultured from primary tumors formed secondary tumors upon reimplantation with more diffuse invasion, suggesting in vivo evolution. To validate this model’s relevance to DIPG transcriptionally, we performed RNA-sequencing on a cohort of primary and secondary tumor neurospheres (termed primary and secondary iDIPG) and compared them to published RNA-seq data from pediatric PDX and patient tumor samples. Hierarchical clustering and principal component analysis on differentially expressed genes (P<0.05) showed that H3.3K27M iDIPG cluster with H3.3K27M PDX and patient tumors. Further, ssGSEA showed that H3.3K27M iDIPG are enriched for astrocytic and mesenchymal signature genes, a defining feature of H3.3K27M DIPG. Finally, we found that primary H3.3K27M iDIPG neurospheres are sensitive to panobinostat, an HDAC inhibitor shown to be effective against H3.3K27M DIPG cells in vitro. Overall, these data suggest that H3.3K27M iDIPG are a promising tool for investigating DIPG biology and new therapeutic strategies.

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Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽

10.1182/blood.v93.3.1097.403k04_1097_1105 ◽

1999 ◽

Vol 93 (3) ◽

pp. 1097-1105 ◽

Cited By ~ 4

Author(s):

G. Güenechea ◽

J.C. Segovia ◽

B. Albella ◽

M. Lamana ◽

M. Ramı́rez ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Short Term ◽

Immunodeficient Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

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