scholarly journals Targeting Burkitt Lymphoma By CAR-Extracellular Vesicles: A Tropism-Delivery Platform for CRISPR/Cas9 System

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3487-3487
Author(s):  
Qian Xu ◽  
Zheng Zhang ◽  
Lei Zhao ◽  
Yun Qin ◽  
Yicheng Zhang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Extracellular Vesicles ◽  
Burkitt Lymphoma ◽  
Confocal Microscope ◽  
Scid Mice ◽  
Whole Body ◽  
Raji Cells ◽  
Uptake Efficiency ◽  
Delivery Platform

Abstract Introduction CRISPR/Cas9 system is a highly efficient genomic editing system. However, application of CRISPR/Cas9 system in vivo is somewhat limited for delivery methods and safety concerns. Extracellular vesicles (EVs) are released by exocytosis which results in consistent surface membrane proteins with original cells and capable to deliver several kinds of materials in vivo. However, EVs can be absorbed by other organs or tissues besides tumors, leading to possible off-target effects. Therefore, it is imperative to enhance the targeting property of EVs in vivo. Recently, clinical advances have confirmed that CAR-T cells possess great tropism and multiple choices of target. This may be an effective means to improve EVs' selective accumulation in tumors. In this proof-of-principle study, we exploit the target precision of CAR to establish an EV-derived tropism-delivery platform for CRISPR/Cas9 system as a novel therapeutic strategy for malignant diseases. Methods We established CD19-CAR-293T cell line by CAR lentivirus transfection. Raji cells (Burkitt lymphoma) were chosen as target tumor cells for it's CD19 positive. For the isolation of EVs from CD19-CAR-293T and 293T cells, culture medium was harvested, centrifuged and filtered through 0.45μm filters to remove the dead cells and large microvesicles. Purified EVs were examined by (NTA). The plasmids of CRISPR/Cas9 system targeting human cMyc gene were constructed by standard protocols and electroporated into EVs using Gene Pulser Xcell Electroporation System (BioRad). EVs were labelled with lipophilic fluorescent dye for visualization of confocal images. Uptake of EVs in Raji cells was analyzed using confocal microscope. To observe the biodistribution of EVs, Raji-bearing xenograft NOD/SCID mice were generated. When the tumors reached 200mm3, labelled EVs were intracardially injected to mice. Biofluorescence images were obtained using IVIS imaging system. Results NTA showed that a size distribution of approximately 100nm to 450nm for two types of EVs (Fig. 1A). The modifications didn't affect the physical properties. We used a fixed number ratio of Raji cells to EVs in incubation to compare the uptake efficiency between normal EVs and CD19-CAR-EVs. After one hour of incubation with Raji cells, uptake efficiency of the CD19-CAR-EVs was higher than that of the normal EVs, as indicated by signal intensity in the confocal microscope images (Fig. 1B). To confirm whether CD19-CAR-EVs could target tumors in vivo, we intracardially injected the same number of labelled EVs or CD19-CAR-EVs to Raji xenograft NOD/SCID mice and observed at 0h and 24h post-injection. As results, the CD19-CAR-EVs significantly accumulated in tumor sites compared to liver, kidney and other tissues. In contrast, the normal EVs were distributed throughout the body (Fig. 1C). Ex vivo images exhibited that compared with the EV group, the CD19-CAR-EVs accumulated more in tumors and less in liver, kidney and other tissues (Fig. 1C). The tumor/whole-body fluorescence intensity ratios were calculated in the EV and CD19-CAR-EV groups (Fig. 1D). At 24h post-injected, the ratios in the CD19-CAR-EV group were higher than that in EV group (P<0.05). Twelve days after the subcutaneous xenograft models were generated, CD19-CAR-EVs loaded with CRISPR/Cas9 system targeting human cMyc were injected intracardially or intratumorally (i.t.) to mice with interval of 4 days for 5 times. Mice injected with EVs only, with plasmid of cMyc sgRNA/Cas9 only, and with Cas9 loaded CD19-CAR-EVs without sgRNA were used as control groups. Tumor sizes were compared among groups at 18 days post the first EVs injection. Tumors from intracardially injected CD19-CAR-EVs+cMyc sgRNA/Cas9 group were smaller than those from EVs+cMyc sgRNA/Cas9 group. And the tumors from intratumoral injection of CD19-CAR-EVs+cMyc sgRNA/Cas9 were the smallest ones among groups (Fig. 1E). Conclusion In this study, we take advantages of CAR's affinity to tumor cells and EVs' delivery capacity, and generated CD19-CAR-EVs capable of delivering CRISPR/Cas9 system. This CAR-EV-derived tropism-delivery system can be administered systemically or locally and accumulated selectively in tumor tissues to achieve gene editing in vivo. With evolving repertoires of CARs and multiplex genome engineering abilities of CRISPR/Cas9, this approach is with great potential for biotechnological and therapeutic applications. Disclosures No relevant conflicts of interest to declare.

scholarly journals Allogeneic tumor rejection induced by the intravenous injection of Lyt-2+ cytolytic T lymphocyte clones.

1982 ◽  
Vol 156 (4) ◽  
pp. 1280-1285 ◽  
Author(s):  
H D Engers ◽  
A L Glasebrook ◽  
G D Sorenson
Keyword(s):  
T Cell ◽  
Tumor Cells ◽  
Intravenous Injection ◽  
T Lymphocyte ◽  
Tumor Rejection ◽  
Whole Body ◽  
Cell Clones ◽  
Cytolytic T Lymphocyte ◽  
Body Counting

The in vivo activity of murine Lyt-2+ cytolytic T lymphocyte clones was assessed in a tumor allograft model system. Mice that had been sublethally irradiated 16 h previously were injected intraperitoneally with 131I-IUdR-labeled tumor cells. Simultaneously, various doses of four cytolytic T cell clones were injected intravenously and the mice monitored for tumor cell elimination by whole-body counting tecniques. These four clones had been selected on the basis of their ability to proliferate in response to alloantigens in the absence of added T cell growth factor(s). With two of the four clones tested, rapid elimination of tumor cells within the peritoneal cavity was observed, as early as 48 h after intravenous injection of the cloned T cells.

scholarly journals Knockout of MMP3 Weakened Solid Tumor Organoids and Cancer Extracellular Vesicles

2020 ◽  
Author(s):  
Eman A. Taha ◽  
Chiharu Sogawa ◽  
Yuka Okusha ◽  
Hotaka Kawai ◽  
May Wathone Oo ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Tumor Cells ◽  
Extracellular Vesicles ◽  
Fluorescent Proteins ◽  
Three Dimensional ◽  
3D Culture ◽  
Proliferating Cells ◽  
Ki 67 ◽  
Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (300-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 leads to a significant reduction in tumoroid size and to the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. These weakened phenotypes by MMP3 KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated into recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3 resulting in increasing the proliferation and CD9+ tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2114-2114 ◽  
Author(s):  
Haiming Chen ◽  
Eric Sanchez ◽  
Mingjie Li ◽  
Cathy Wang ◽  
Abby Gillespie ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Scid Mice ◽  
M Protein ◽  
Cytotoxic Effects ◽  
Jak2 Inhibitor

Abstract Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV). Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs. Results: In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone. Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals MRI tracking reveals selective accumulation of stem cell-derived magneto-extracellular vesicles in sites of injury

2019 ◽  
Author(s):  
Zheng Han ◽  
Senquan Liu ◽  
Yigang Pei ◽  
Zheng Ding ◽  
Yuguo Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Extracellular Vesicles ◽  
Kidney Injury ◽  
Induced Pluripotent Stem Cell ◽  
Superparamagnetic Iron Oxide ◽  
Whole Body ◽  
Body Distribution ◽  
Magnetic Resonance Imaging Mri ◽  
Induced Pluripotent

AbstractHuman stem-cell-derived extracellular vesicles (EVs) are currently being investigated for cell-free therapy in regenerative medicine applications, but their biodistribution and tropic properties for homing to injured tissues are largely unknown. Here, we labeled EVs with magnetic nanoparticles to create magneto-EVs that can be tracked by magnetic resonance imaging (MRI). Superparamagnetic iron oxide (SPIO) nanoparticles were coated with polyhistidine tags, which enabled purification of labeled EVs by efficiently removing unencapsulated SPIO particles in the solution. The biodistribution of systemically injected human induced pluripotent stem cell (iPSC)-derived magneto-EV was assessed in three different animal models of kidney injury and myocardial ischemia. Magneto-EVs were found to selectively home to the injury sites and conferred substantial protection in a kidney injury model. In vivo MRI tracking of magnetically labeled EVs represents a new powerful method to assess and quantify their whole-body distribution, which may help optimize further development of EV-based cell-free therapy.

The Establishment of Mice Model with Myelodysplastic Syndrome Cell Line MUTZ-1 Cell.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4911-4911
Author(s):  
Jie Lu ◽  
Jie Jin ◽  
Weilai Xu ◽  
Zhimei Chen ◽  
Wei Ding
Keyword(s):  
Latent Period ◽  
Cell Line ◽  
Arsenic Trioxide ◽  
Tumor Cells ◽  
First Generation ◽  
Abdominal Cavity ◽  
Tumor Formation ◽  
Scid Mice ◽  
Mice Model

Abstract Objective To establish myelodysplastic syndrome cell line MUTZ-1 cell mice model. Methods 75 SCID mice and 10 BALB/CA-node mice were studied in this experiment. MDS-REBT cell line MUTZ-1 cells were cultured in vitro and 1 x108 /ml cell were subcutancously implanted in 4~6-week-old First-Generation SCID mice and BALB/CA-node mice respectively. The subcutancous tumor cells from First-Generation MDS-REBT cell line MUTZ1 cells mice model were respectively implanted in Second-Generation SCID mice and BALB/CA-node mice. The latent period and the rate of subcutancous tumor formation was observed, and tumor size was measured by the MTD twice a week. The biological characteristics of the subcutancous tumor cells were checked and evaluated by the methods of cell morphology and pathology and histopathology and immunology by flow cytometer, chromosome analysis and immunohistochemistry stain. It has been experimented tentatively that arsenic trioxide (AS2O3) (with 7.5mg/g mouse/d x 3d) was injected into abdominal cavity in 10 SCID mice to observe the drug effect on tumor formation in vivo. Results The rate of the subcutancous tumor formation was 98.6% (70/71)in SCID mice and 62.5%(5/8)in BALB/CA-nude micerespectively,(P=0.0027). The latent period of the subcutancous tumor formation were 10~17d (median 12d) in 61 SCID mice without injected arsenic trioxide and were 26~31d (median 29d) in 10 SCID mice with arsenic trioxide (with 7.5mg/g mouse/d x 3d) injected into abdominal cavity respectively. The latent period of the subcutancous tumor formation in the group of SCID mice injected by AS2O3 was significantly longer than that of non-injected by AS2O3(Z=5.339, P&lt;0.001), which showed that AS2O3 has probably a inhibition effect on the subcutancous tumor growth of SCID mice model in vivo. The results showed the subcutancous tumor cells of mice model have the biological characteristics of a Hum-MDS-REBT cell line MUTZ1 cell and were anthropo- source. The metastases in liver were found 8 weeks after implanted. HE staining on paraffin sections showed that the tumor cells were microscopic observed on liver and spleen and kidney and medullary cavity of bone and skin. Immunohistochemistry staining showed the subcutancous tumor cells of the mice model have the overexpression of P53 protein, which cued that these tumor cells has the P53 mutation and showed human tumor propert. Conclusion A human myelodysplastic syndromes-REBT cell line MUTZ1 cell-severe combined immunodeficiency (SCID) mice model was successfully established, which could be used for the advanced investigation of drug experiment as an animal model system in vivo.

The Heparanase Inhibitor SST0001 Is a Potent Inhibitor of Myeloma Growth In Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 246-246 ◽  
Author(s):  
Yang Yang ◽  
Joseph P. Ritchie ◽  
Telisha Swain ◽  
Annamaria Naggi ◽  
Giangiacomo Torri ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Heparan Sulfate ◽  
Cell Line ◽  
Tumor Cells ◽  
Subcutaneous Injection ◽  
Scid Mice ◽  
Rpmi 8226

Abstract Heparanase (HPSE) is an enzyme that cleaves heparan sulfate (HS) chains of proteoglycans. Work by us and others has demonstrated that heparanase promotes the growth and metastasis of many types of tumors, including multiple myeloma (MM). Heparanase expression is rare in normal tissue but becomes evident in many human tumors, making it a viable target for cancer therapy. SST0001, a chemically modified heparin that is 100% N-acetylated and 25% glycol-split, dramatically inhibits heparanase activity. SST0001 lacks anticoagulant activity and thus can be administered at relatively high doses in vivo. We previously reported that delivery of SST0001 by Alzet osmotic pumps to SCID mice potently inhibited growth of subcutaneous tumors formed by CAG human myeloma cells. In the present studies, we further tested the effects of SST0001 against additional MM cell lines, using alternative routes of drug delivery in two different animal models. Ten days after subcutaneous injection of either MM.1S or RPMI 8226 tumor cells, mice were treated for 28 days using Alzet pumps delivering 30 mg/kg/day of SST0001. Results showed that, compared to PBS control, MM.1S and RPMI-8226 tumors in SST0001-treated mice were reduced by 50% and 51%, respectively. In a separate experiment, delivery of SST0001 by distant subcutaneous injection inhibited tumor growth by 77% in comparison to controls. In the SCID-hu model, in which CAG cells were implanted directly into human bones engrafted in SCID mice, SST0001 also significantly inhibited tumor growth as measured by human immunoglobulin kappa light chain in murine sera (1055 ± 295 ng/ml in PBS-treated mice vs 155 ± 295 ng/ml in SST0001- treated mice (P &lt;0.003)). These data demonstrate that SST0001 is a strong inhibitor of MM growth in vivo, even when tumors grow within the bone microenvironment and that the effect of SST0001 is not cell-line specific. We did not observe any adverse side effects in animals, even at doses as high as 120 mg/kg/day. To determine the mechanism of action of SST0001, we examined several pharmacodynamic parameters. Immunohistochemistry demonstrated that SST0001 treatment significantly reduced microvessel density of tumors as compared to controls (99% in CAG and 54% in RPMI-8226 tumors). In addition, SST0001 treatment blocked HGF expression (CAG, RPMI 8226 and MM.1S tumors) and inhibited VEGF expression in CAG tumors but not RPMI 8226 and MM.1S tumors. Moreover, a series of in vitro experiments, using the CAG MM cell line and human umbilical vein endothelial cells (HUVEC), were performed. Unlike its strong antitumor effect in vivo, SST0001 only slightly inhibited CAG cell proliferation, cell cycle and growth factor signaling in vitro, suggesting that the compound does not have a direct cytotoxic effect on tumor cells. Since blood vessels are an important element of the tumor microenvironment and angiogenic endothelium in tumors also expresses high levels of heparan sulfate proteoglycans and heparanase, we assessed the effects of SST0001 on HUVEC cells. In contrast with results on CAG MM cells, SST0001 treatment showed a strong inhibition on HUVEC proliferation (46%, MTT assay), dramatically blocked the phosphorylation of ERK stimulated by HS-binding growth factors (HGF, VEGF, HDGF and EGF), blocked the Akt pathway of HGF signaling in HUVECs and inhibited HUVEC tube formation, stimulated by HGF and VEGF. Based on these results, we conclude that SST0001 strongly inhibits the growth of myeloma tumors in vivo by targeting the tumor microenvironment, including a significant inhibition of tumor angiogenesis. Because of its unique target site in the tumor microenvironment, we predict that the combination of SST0001 with conventional tumor cell-targeting chemotherapeutic drugs will greatly improve patient outcome in MM.

New Therapeutic Approach for the Treatment of B-Cell Disorders Using Chlorambucil/Hydroxychloroquine-Loaded AntiCD20 Nanoparticles

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 158-158
Author(s):  
Gabriele Pozzato ◽  
Nelly Mezzaroba ◽  
Sonia Zorzet ◽  
Erika Secco ◽  
Stefania Biffi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Tumor Cells ◽  
Burkitt Lymphoma ◽  
Intraperitoneal Injection ◽  
Therapeutic Approach ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
Multi Drug Resistance ◽  
High Dose

Abstract Abstract 158 B-cell disorders show highly variable clinical courses, ranging between indolent diseases like the chronic lymphocytic leukemia (CLL) and highly aggressive lymphoproliferative disorders like Burkitt Lymphoma. The treatments of these disorders have been characterized by the development of new approaches, including dose-intensive chemotherapy regimens and immunotherapy via monoclonal antibodies (Ab). Despite the promising survival rates, these multi-agent treatments are flawed by a high degree of toxicity and a significant fraction of patients do not respond. The use of core shell nanoparticles design with specific Ab-coating represents a new strategy to target only tumor cells with high-dose chemotherapy. We characterized, both in vitro and in vivo, the effects of a new kind of biodegradable nanoparticles (BNP) coated with an anti-CD20 antibody and loaded with Chlorambucil (CLB) and Hydroxychloroquine (HCQ). BJAB and MEC1 are two well characterized Burkitt lymphoma and CLL cell line, respectively; both are mutated in P53, express different amount of CD20 and result quite resistant to CLB, but more susceptible to HCQ. HCQ/CLB-loaded antiCD20 BNP can release enough amounts of drugs inside the cancer cells and overcome multi-drug resistance mechanisms, which are over-expressed in many B-cell disorders. The presence of antiCD20 antibody targets BNP on cells expressing this antigen, inducing a selective penetration in these cells, as demonstrated by electron microscopy studies; the cytotoxic effect was evident analyzing apoptosis and after 24–48 hours all the cancer B-cells resulted destroyed. Data indicate that BNP were able to kill also cells expressing very low amount of CD20, where Rituximab was almost ineffective. These results were confirmed using primary cells isolated from more than 40 patients with untreated chronic lymphocytic leukemia. These data were obtained incubating fresh isolated blood with HCQ/CLB-loaded antiCD20 BNP and analyzing residual viable or apoptotic cells. Endovenous or intraperitoneal injection of HCQ/CLB-loaded antiCD20 BNP in mice did not induced tissue damages, weight loss or death, while the same amount of free cytotoxic agent cause death of all the animals. Biodistribution analysis demonstrated a selective targeting of the antiCD20 BNP to tumor cells, with a peak 48 hours after injection; BNP elimination was mainly documented in liver and intestine but not by spleen, kidney, lungs or other organs. The intraperitoneal injection of BJAB cells in SCID mice determined the formation of a tumor mass visible at the site of injection followed by early multi-organ involvement and death of all animals. Eight injections of antiCD20 BNP containing CLB/HCQ (400 micrograms each) in 17 days were able to increase survival in 100% of mice and 90% of animals were cured. The same amount of CLB+HCQ cannot be used as free drugs because of their toxicity able to kill all the animals in about a week. The intravenous injection of MEC1 cells in SCID mice allowed the development a new human/mouse model of leukemia. The treatment with antiCD20 BNP containing CLB/HCQ showed comparable therapeutic efficacy, while free drugs were almost uneffective. In conclusion, the results of the present study demonstrate that antiCD20 BNP containing HCQ/CLB can be effective as a single agent in controlling both aggressive and indolent models of disseminated B-cell disorders and provide a rationale for adopting this new therapeutic approach in clinical trials. Furthermore, the results demonstrate that nanoparticles are effective via different administration methods. Disclosures: No relevant conflicts of interest to declare.

Ublituximab (TGTX-1101), A Novel Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Activity in Rituximab-Sensitive and Rituximab–resistant B Non-Hodgkin Lymphoma (B-NHL) Pre-Clinical In vitro and in Vivo models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2756-2756 ◽  
Author(s):  
John Miller ◽  
Matthew J. Barth ◽  
Cory Mavis ◽  
Ping-Chiao Tsai ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Primary Tumor ◽  
Scid Mice ◽  
In Vivo Experiments ◽  
Non Hodgkin Lymphoma ◽  
Tail Vein Injection ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 2756 The addition of rituximab to chemotherapy regimens utilized in treating B-cell non-Hodgkin lymphoma (B-NHL) has resulted in significant improvement in treatment response and clinical outcomes. On the other hand, the use of rituximab is changing the biology and response to second-line therapy in patients with relapsed/refractory disease. Novel anti-CD20 mAbs continue to be developed that may offer additional treatment options for relapsed/refractory rituximab-pre-treated patients. Ublituximab (TGTX-1101) is a novel, chimeric mAb targeting a unique epitope on the CD20 antigen. Ublituximab has been glycoengineered to enhance affinity for all variants of FcγRIIIa receptors. To further characterize the activity of ublituximab, we evaluated its anti-tumor activity in a panel of rituximab-sensitive (RSCL), rituximab–resistant (RRCL) cell lines, primary tumor cells isolated from patients with B-NHL by negative selection using magnetic beads, and in lymphoma SCID mice xenograft models. RSCL (Raji, RL, U2932, Granta, HBL-2, Jeko-1, Mino, Rec1 and Z-138), RRCL (Raji-2R, Raji 4RH, RL-4RH, and U2932-4RH); and cytarabine-resistant (AraCR) mantle cell lymphoma cell (MCL) lines (Granta-AraCR, HBL-2-AraCR, Jeko-AraCR, Mino-AraCR and Rec1-AraCR) were labeled with 51Cr. Subsequently, cells were exposed to ublituximab, rituximab or isotope control and human serum (25%) for complement dependent cytotoxicity (CDC) assays or to effector cells isolated from healthy volunteers (effector:target ratio 40:1) for antibody dependent cellular cytotoxicity (ADCC) assays, respectively. Antibody-induced direct anti-proliferative effects and induction of apoptosis were determined by alamar blue reduction assay and Annexin-V and propidium iodide staining, respectively. Primary tumor cells (n=11) were exposed to ublituximab, rituximab or isotype control +/− pooled human serum for 48 hr. Changes in ATP content were determined using the CellTiterGlo assay. For in vivo studies, 6–8 week old SCID mice were inoculated via tail vein injection with 1×106 Raji cells on day 0 and assigned to rituximab (10mg/kg/dose), ublituximab (10mg/kg/dose) or control group. MAb was given via tail vein injection on days +3, +7, +10 and +14. Differences in survival were analyzed by Kaplan-Meier curves and p values calculated using log rank test. Ublituximab induced significantly higher ADCC when compared to rituximab in 13 out of 17 cell lines tested (including all RRCL and cytarabine resistant MCL cells): (Raji 44.4% vs. 19.8%; Raji 4RH 17.5% vs. 8.3%; Raji 2R 28.2% vs. 12%; RL 40.9% vs. 17.8%; RL-4RH 33.5% vs. 17.2%; U2932 46.9% vs. 28.8%; U2932-4RH 40.2% vs. 22.1%; HBL-2AraCR 30.7% vs. 16.6%; Jeko 34.8% vs. 18.4; Jeko-AraCR 23.8% vs. 9.6; Mino 47.4% vs. 11.6%; Mino-AraCR 32.5% vs. 15.5; Rec1 30.9% vs. 0%; p-values <0.05). There was no significant difference between ublituximab and rituximab in terms of CMC (including studies performed in primary tumor cells) or direct signaling (i.e. apoptosis or cell proliferation). While ublituximab therapy prolonged the survival of lymphoma-bearing SCID mice when compared to controls, the anti-tumor activity in vivo was similar to rituximab. Our results suggest that ublituximab exhibits higher ADCC than rituximab in vitro, including in RRCL and elicits similar CDC and direct anti-tumor effects. Despite this enhanced ADCC activity, initial in vivo experiments did not result in improved survival compared to rituximab, however additional in vivo experiments investigating the activity of ublituximab in RRCL and MCL mouse models, testing alternative dose/schedule regimens and/or in combination with other anti-lymphoma agents are planned. Updated research results will be presented at the annual meeting. A Phase I/II trial of ublituximab in patients with relapsed/refractory NHL is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1260 ◽  
Author(s):  
Eman Taha ◽  
Chiharu Sogawa ◽  
Yuka Okusha ◽  
Hotaka Kawai ◽  
May Oo ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Tumor Cells ◽  
Extracellular Vesicles ◽  
Fluorescent Proteins ◽  
Three Dimensional ◽  
3D Culture ◽  
Proliferating Cells ◽  
Ki 67 ◽  
Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

Heparanase Promotes the Osteolytic Phenotype in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 841-841
Author(s):  
Yang Yang ◽  
Joseph P. Ritchie ◽  
Larry J. Suva ◽  
Ralph D. Sanderson
Keyword(s):  
Multiple Myeloma ◽  
Tumor Growth ◽  
Conditioned Medium ◽  
Tumor Cells ◽  
Bone Destruction ◽  
Scid Mice ◽  
Myeloma Cells ◽  
Marrow Cavity

Abstract Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in many human tumors including multiple myeloma. We have shown previously using animal models that heparanase promotes robust myeloma tumor growth and spontaneous metastasis to bone. In the present study, the role of heparanase in promoting myeloma bone disease was investigated. CAG human myeloma cells expressing either high or low levels of heparanase (heparanase-high or heparanase-low cells) were directly injected into the marrow cavity of human fetal long bones implanted subcutaneously in SCID mice (SCID-hu model). A second, non-injected human fetal bone was implanted on the contralateral side. Seven weeks after injection of myeloma cells into the primary bone, mice were euthanized and the osteolytic disease of both implanted bones was evaluated. Both X-ray and microCT analysis revealed marked osteolysis in the primary bones injected with heparanase-high cells, with little osteolytic disease detected in the bones injected with heparanase-low cells. Surprisingly, the non-injected, contralateral bones of the animals bearing heparanase-high tumors were also extensively degraded. Immunohistolochemical analysis of these contralateral bones revealed that osteolysis occurred in the absence of detectable tumor cells in the bone. Consistent with this osteolytic phenotype, TRAP staining of the primary and contralateral human bones harvested from mice bearing heparanase-high tumors showed a significant increase in osteoclast numbers, as compared to bones harvested from animals bearing heparanase-low tumors. In a second approach using heparanase-high or heparanase-low cells injected into the tibia of SCID mice, heparanase again enhanced osteolysis at the site of tumor injection as well as at distal sites, in the absence of resident tumor cells. These findings parallel our previously published observation that heparanase expressing breast cancer cells implanted in the mammary fat pad induced an increase in bone resorption in the absence of tumor cells within bone. The evidence in vivo suggested the release from heparanase-high cells of factor(s) that increase osteoclast formation. To test this idea, in vitro osteoclastogenesis assays were used to test the conditioned medium from heparanase-high cells. The conditioned medium from heparanase-high cells significantly enhanced osteoclastogenesis compared to conditioned medium from heparanase-low cells. Interestingly, conditioned medium derived from CAG cells expressing heparanase mutants lacking enzymatic activity failed to enhance osteoclastogenesis. Together, these data demonstrate for the first time that expression of heparanase is a major determinant of the osteolytic phenotype in myeloma. Increased osteolysis is the result of increased osteoclastogenesis that requires active heparanase enzyme and can occur in bones distal to the primary tumor prior to any subsequent metastasis. Thus, we hypothesize that therapies designed to block heparanase function will not only inhibit tumor growth, but may also protect bone from tumor-related bone destruction and possibly disrupt the metastasis of tumor to bone.

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