Continuous intravenous infusions of bromodeoxyuridine as a clinical radiosensitizer.

1984 ◽  
Vol 2 (10) ◽  
pp. 1144-1150 ◽  
Author(s):  
T J Kinsella ◽  
J B Mitchell ◽  
A Russo ◽  
M Aiken ◽  
G Morstyn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Normal Skin ◽  
Systemic Toxicity ◽  
Constant Infusion ◽  
Local Toxicity ◽  
Arterial Plasma ◽  
Infusion Period

Twelve patients were treated with continuous intravenous (24-hour) infusions of bromodeoxyuridine (BUdR) at 650 or 1,000 mg/m2/d for up to two weeks. Myelosuppression, especially thrombocytopenia, was the major systemic toxicity and limited the infusion period to nine to 14 days. However, bone marrow recovery occurred within seven to ten days, allowing for a second infusion in most patients. Local toxicity (within the radiation field) was minimal, with the exception of one of four patients, who underwent abdominal irradiation. Pharmacology studies revealed a steady-state arterial plasma level of 6 X 10(-7) mol/L and 1 X 10(-6) mol/L during infusion of 650 and 1,000 mg/m2/d, respectively. In vivo BUdR uptake into normal bone marrow was evaluated in two patients by comparison of preinfusion and postinfusion in vitro radiation survival curves of marrow CFUc with enhancement ratios (D0-pre/D0-post) of 1.8 (with 650 mg/m2/d) and 2.5 (with 1,000 mg/m2/d). In vivo BUdR incorporation into normal skin and tumor cells using an anti-BUdR monoclonal antibody and immunohistochemistry was demonstrated in biopsies from three patients revealing substantially less cellular incorporation into normal skin (less than 10%) compared with tumor (up to 50% to 70%). We conclude that local and systemic toxicity of continuous infusion of BUdR at 1,000 mg/m2/d for approximately two weeks is tolerable. The observed normal tissue toxicity is comparable with our previous clinical experience with intermittent (12 hours every day for two weeks) infusions of BUdR. Theoretically, a constant infusion should allow for greater incorporation of BUdR into cycling tumor cells and thus, for further enhancement of radiosensitization.

In Vivo and in Vitro Pharmacologic Purification of Bone Marrow Contaminated with Tumor Cells Using VP16-213 and Nitrogen Mustard

1984 ◽  
pp. 183-196 ◽  
Author(s):  
Patrick J. Stiff ◽  
Thomas P. U. Wustrow ◽  
Alan R. Koester ◽  
Michael F. Derisi ◽  
Bayard D. Clarkson
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Nitrogen Mustard

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2728-2728
Author(s):  
Yong Zhang ◽  
Christopher P. Rombaoa ◽  
Aldo M Roccaro ◽  
Susanna Obad ◽  
Oliver Broom ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Mrna Levels ◽  
Advisory Committees ◽  
Qrt Pcr

Abstract Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

In Vivo Effects Of Ibrutinib On The Migration Of Chronic Lymphocytic Leukemia Cells Differ Between Patients and Reduce The Ability Of The Bone Marrow Microenvironment To Attract The Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 604-604
Author(s):  
Sarah E. M. Herman ◽  
Jade Jones ◽  
Rashida Z. Mustafa ◽  
Mohammed Farooqui ◽  
Adrian Wiestner
Keyword(s):  
Bone Marrow ◽  
Cell Migration ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Cells ◽  
Lymphocytic Leukemia ◽  
Dual Chamber ◽  
Bone Marrow Plasma ◽  
Pre Treatment

Abstract The Bruton’s tyrosine kinase inhibitor ibrutinib has recently been shown to be well tolerated, and to induce objective clinical responses in the majority of patients, irrespective of adverse prognostic markers (Byrd et al., NEJM 2013). Despite the demonstrated clinical activity, ibrutinib also leads to a transient lymphocytosis that is thought to reflect a redistribution of cells from tissue compartments into the peripheral blood. The mechanisms contributing to this lymphocytosis are not well understood. To date, two groups have demonstrated that in vitro treatment with ibrutinib inhibits migration of CLL cells in chemokine gradients (de Rooji et al., Blood 2012 and Ponader et al., Blood 2012). Here we sought to assess the in vivo effect of ibrutinib on cellular migration. To validate our assay we first treated CLL cells with 1uM ibrutinib for 1 hour in vitro and measured migration of CLL cells to a mix of SDF-1 (at 200ng/mL) and CCL19 (at 100ng/mL), two chemo-attractants known to induce migration of CLL cells. Migration was assayed in a dual chamber system separated by a membrane with 5µm pores after 3 hours of incubation. Confirming published data we found a significant reduction in the migration index (ratio of migration to chemokines divided by migration to media alone) of ibrutinib treated cells compared to untreated cells (mean reduction 24%; P = 0.04). Next, we analyzed the migration of CLL cells obtained from patients (n = 9) enrolled on a clinical trial with single agent ibrutinib that were sampled pre-treatment and after 4 weeks on drug. We observed highly variable responses; in about half of the patients treated cells showed increased migration, while in the other half there was decreased migration to the SDF-1/CCL19 mix. Interestingly, patients showing a decrease in migration on treatment often had del17p but there was no difference in regards to IGHV mutation status and no correlation to the degree of lymphocytosis observed in the patient. T-cell migration was not affected by ibrutinib. In order to extend the analysis to a mix of chemo-attractants that the tumor cells may encounter in vivo we used the supernatant harvested from bone marrow aspirates and found that it efficiently induced migration of CLL cells in the dual chamber assay (mean fold increase 5.2 compared to control). Comparing CLL cells from patients sampled pre-treatment to those obtained on treatment day 28 we again found the same mixed effects of ibrutinib on the ability of CLL cells to migrate to bone marrow plasma as we had observed with the SDF-1/CCL19 mix. Thus, direct inhibition of CLL cell migration can account for only a subset of patients with treatment-induced lymphocytosis. Given reports that ibrutinib can inhibit cytokine and chemokine secretion from CLL cells and T-cells (Ponader et al., Blood, 2012; Herman et al., Blood, 2011), we hypothesized that ibrutinib treatment might change the content of chemo-attractants in the bone marrow We therefore compared the ability of the bone marrow plasma obtained pre-treatment and after 2 months on ibrutinib to attract CLL cells (these cells were obtained from the peripheral blood pre-treatment from the same patient donating the marrow). We found that in 4/4 patients evaluated there was a significant reduction in the migration of CLL cells to the on-treatment bone marrow plasma compared to the matching pre-treatment sample (mean decrease 20%; P < 0.05). In conclusion, migration of CLL cells from patients on ibrutinib can be inhibited or increased, with most del17p patients showing decreased migration. Intriguingly, these patients tend to have slower resolution of the treatment induced lymphocytosis, raising the question whether inhibition of homing to tissue sites could affect the time to resolution of the lymphocytosis. In addition, we provide evidence that bone marrow plasma on ibrutinib therapy has a reduced capacity to attract CLL cells, suggesting that ibrutinib may alter the composition of the bone marrow microenvironment This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

scholarly journals 3373 Modulation of Hedgehog Signaling Alters Immune Infiltration in Pancreatic Cancer

2019 ◽  
Vol 3 (s1) ◽  
pp. 16-16
Author(s):  
Nina Steele ◽  
Valerie Irizarry-Negron ◽  
Veerin Sirihorachai ◽  
Samantha Kemp ◽  
Eileen Carpenter ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
Tumor Cells ◽  
Pancreatic Tumors ◽  
Hh Signaling ◽  
Immune Profiling

OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.

scholarly journals Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.

1989 ◽  
Vol 170 (1) ◽  
pp. 191-202 ◽  
Author(s):  
C L Sentman ◽  
J Hackett ◽  
V Kumar ◽  
M Bennett
Keyword(s):  
Bone Marrow ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Immune Functions ◽  
Infected Cells

NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.

scholarly journals Estradiol Augments the Production and Actions of Polymorphonuclear Cells to Promote Lymphangioleiomyomatosis (LAM) Progression

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1017-A1017
Author(s):  
Briaunna M N Minor ◽  
Stephen R Hammes
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Polymorphonuclear Cells ◽  
Null Cell ◽  
Independent Manner ◽  
Potent Inducer ◽  
Cystic Lung

Abstract Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. LAM tumors are caused by mutations in either TSC1 or TSC2 genes that induces defective inhibition of the mTORC1 pathway, leading to increased mTORC1 activity and augmented cell proliferation. We have previously reported that estrogen ablation in our uterine-specific Tsc2 knockout mouse, which grows tumors with characteristic LAM features and lung colonization potential, effects notable regression of tumors. Thus, estrogen is required for to maintain heightened mTORC1 activity and LAM-like tumor progression. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere—in mTORC1 independent manner—in vivo to promote LAM progression. Flow cytometry revealed large numbers of Ly-6Cint Ly-6Ghigh myeloid cells—polymorphonuclear cells or PMNs—in the blood and myometrial tumors of our uterine-specific Tsc2-null mice. Accordingly, we found that Tsc2-null tumors required PMNs for normal disease progression, as Gr-1 (Ly-6C/Ly-6G) depletion or inhibition of PMN recruitment reduced tumor growth. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Using bone marrow cultures, we found that estradiol is indeed a potent inducer of PMN production. This effect occurs equally in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a PMN fate for myeloid progenitors. Additionally, we have evidence implicating estrogen in the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Overall, these data suggest that estradiol maybe facilitating crosstalk in LAM tumors, directly stimulating tumor cells while also promoting the production and actions of PMNs, which in turn promote tumor growth.

L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 887-887
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Phong Quang ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Confocal Microscopy ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vivo Confocal Microscopy ◽  
Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

Osteoclasts in Myeloma Are Derived from Gr-1+CD11b+ Myeloid Immune Suppressor Cells of the Bone Marrow Niche in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 736-736 ◽  
Author(s):  
Junling Zhuang ◽  
Li Yang ◽  
Seint T Lwin ◽  
Claire M. Edwards ◽  
James R Edwards ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Suppressor Cells ◽  
Bone Lesions ◽  
Vicious Cycle ◽  
Tumor Bearing ◽  
Immune Suppressor Cells ◽  
Immune Suppressor

Abstract When cancer cells are resident in bone, they initiate a vicious cycle with osteoclasts (OCs) which perpetuates their growth and aggressive behavior. OCs are critical for the maintenance of the vicious cycle, since they control not only bone destruction associated with cancer, but also the aggressive behavior of tumor cells. It has recently been recognized that tumor cells grow in distant sites because they induce non-malignant cells to establish a “pre-metastatic niche” for tumor cells to later engraft. But nothing is yet known for bone. Primitive bone marrow progenitor cells, called myeloid immune suppressor cells (MISCs), which suppress immune reactivity, are important niche components. MISCs belong to the myelomonocytic lineage with surface markers of Gr-1 and CD11b. We hypothesize that MISCs are precursors of OCs recruited by tumors to assist in the establishment of the vicious cycle. To test this hypothesis, we used the well-characterized 5TGM1 murine myeloma model. 5TGM1-GFP tagged myeloma cells were inoculated via tail vein. The proportion of Gr-1+CD11b+ cells in bone marrow and spleen were assessed by FACS. On week 4 after tumor cell inoculation, %Gr-1+CD11b+ cells were significantly greater in tumor-bearing mice compared with controls (60.9±7.8% vs 37.7±8.6% p<0.05 in marrow; 21.1±4.84% vs 2.4±0.85% p<0.05 in spleen) and paralleled the myeloma burden in bone and spleen. We sorted the Gr-1+CD11b+ cells from the spleens by using magnetic microbeads. MISCs formed multinucleated TRAP positive OCs in medium containing M-CSF (25ng/ml) and RANKL (50ng/ml). The number of OCs derived from tumor MISCs was dramatically greater than those from control mice after 14 day culture (13.4±2.1 vs 1±0.7 per 100x field). MISCs expressed the αv chain of the vitronectin receptor (CD51) and the calcitonin receptor which are specific markers for OCs as they differentiated into multinucleated TRAP+ cells. Only MISCs from tumor-bearing mice and not MISCs from control mice caused resorption pits on dentine discs, demonstrating they were functional OCs. To study the in vivo differentiation of MISCs, we bred lacZ generalized C57 B6 mice with Rag2−/− immune compromised mice to generate lacZ+/− Rag2−/− mice. MISCs were sorted from lacZ+/− Rag2−/− myeloma mice. lacZ positive MISCs were co-injected with 5TGM1 cells to Rag2−/− mice. On day 10 after injection, lacZ+ multinucleated cells could be seen on endosteal surface beneath growth plate by X-gal staining. Those lacZ+ cells were also TRAP+, indicating they were OCs. Treatment of mice with zoledronic acid 100ug/kg s.c. 2/week for 4 weeks reduced % MISCs in tumor bearing mice and impaired the capacity of MISCs to form OCs in vitro (OC# 42.4±4.0 vs 25.6±3.5 per 100x field). Our data suggest that MISCs are increased significantly in marrow and spleen of myeloma-bearing mice and parallel the appearance of lytic bone lesions. These MISCs differentiate avidly and rapidly into functional OCs in vitro as well as in vivo. Zoledronic acid impairs both MISCs, the OC precursors, and mature OCs. These results have a number of implications, including the possibility of reducing bone lesions in myeloma and other malignancies by depleting specific subpopulations of osteoclast precursors.

A Novel Activating Mutation Of CXCR4 Plays a Crucial Role In Waldenstrom Macroglobulinemia Biology

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 272-272 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Cristina Jimenez ◽  
Patricia Maiso ◽  
Michele Moschetta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Advisory Board ◽  
Beta Catenin ◽  
Activating Mutation

Abstract Background The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating the biology of B-cell lymphoproliferative disorders. Recent whole genome sequencing studies have identified unique CXCR4 mutations in 29% of the 55 evaluated patients with Waldenstrom Macroglobulinemia (WM). In this study, we sought to better define the mutation status of CXCR4 in B-cell malignancies and define the functional role of this mutation in the progression of WM in vivo. Methods Allele-specific(AS) PCR has been performed on bone marrow (BM)-derived tumor cells of patients with WM (n: 131); IgM monoclonal gammopathy of undetermined significance (MGUS; n: 40); as well as in patients with diffuse large cell lymphomas (DLBCL; n: 75), splenic marginal zone lymphoma (SMZL; n: 14), B-chronic lymphocytic leukemia (B-CLL; n: 37), hairy cell leukemia (HCL; n: 35), multiple myeloma (MM; n: 36), IgA/IgG MGUS (n: 22), lymphoplasmacytic lymphoma without WM criteria (n: 13), and amyloidosis (n: 6). CXCR4-loss and -gain of function studies have been performed on WM cells stably expressing either shRNA-CXCR4, CXCR4-ORF-GFP-tagged or scramble-RFP-tagged (generated via lentivirus-based infection). A mutagenesis kit has been used to generate the C1013GCXCR4 mutant protein (C1013GCXCR4) in WM cells, via lentivirus-based infection. CXCR4-knock-in or C1013GCXCR4-mutated cells and the corresponding controls have been injected i.v. into SCID/Bg mice and tumor dissemination has been evaluated ex vivo by immunohistochemistry IHC (human-CD20; -CXCR4). C1013GCXCR4-mutated cells have been characterized at mRNA levels (U133 plus2) using GSEA. A novel human anti-CXCR4 mAb (BMS-936564/MDX-1338; Bristol Myers Squibb, NY) has been tested in vitro (cell proliferation, MTT, adhesion and migration to primary WM BM mesenchymal stromal cells) and in vivo (10mg/kg i.p. x3-4/week). Tumor growth has been evaluated by IHC ex vivo (hCD20; hCXCR4) and by immunofluorescence. Results We examined the mutational status of C1013GCXCR4 and confirmed the presence of this specific mutation in 28% of the 131 cases evaluated. The mutation was also detected at the stage of IgM-MGUS (20%); while it was present in a minority of patients with DLBCL (1%) and SMZL (7%). Remarkably, it was absent in all MM (n=36) and IgA/IgG MGUS patients (n=22), and it was not detected in healthy subjects (n=32). The functional relevance of the C1013G-CXCR4 variant was next examined in vivo. Mice injected with C1013GCXCR4-cells presented with a significant dissemination of tumor cells, demonstrating involvement of liver, bone marrow, lymph nodes, kidney and lung. IHC showed the presence of CXCR4+ and CD20+ cells in all the tissues examined; and quantification of CXCR4 and CD20 positivity was higher in C1013GCXCR4-cells-, compared to parental(p)-WM cell-injected mice (NIS Elements software, Nikon, Melville, NY; P<0.05). In addition, C1013GCXCR4-cells were further characterized in vitro, showing increased adhesion and cell proliferation in the presence of primary WM BM-MSCs. These findings were also confirmed using CXCR4-overexpressing cells. In contrast CXCR4-knock-down cells presented the opposite behavior, where reduced adhesion and proliferation in presence of primary WM BM-MSCs were observed. By performing GSEA we demonstrated that genes related to invasiveness, cell proliferation, anti-apoptosis, and oncogenesis were all enriched in C1013GCXCR4-cells compared to the parental-WM cells. These findings let us hypothesize that C1013GCXCR4 may act as an activating mutation in WM cells. Indeed, in a different mouse model, CXCR4 over-expressing cells and scramble infected cells were injected into mice, showing similar phenotype to the one observed upon C1013GCXCR4-WM cell-injected-mice. Finally, the novel antibody BMS-936564/MDX-1338 exerted anti-WM activity both in vitro and in vivo, with anti-tumor effects observed also against the mutated variant. This was supported by inhibition of pro-survival pathways (p-ERK; pAKT); induction of pro-apototic proteins (cleaved-PARP and -caspase-9); up-regulation of p-GSK3beta, p-beta catenin and relative beta catenin degradation. Conclusion These findings demonstrate that C1013GCXCR4 acts as an activating mutation in WM; and it is targetable by using MDX936564/1338 thus providing the basis for translating these observations into clinical trials for WM patients. Disclosures: Kuhne: BMS: Employment. Cardarelli:BMS: Employment. Ghobrial:BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Noxxon: Research Funding; Sanofi: Research Funding.

TAMI-51. HORIZONTAL MITOCHONDRIAL TRANSFER FROM THE TUMOR MICROENVIRONMENT TO GLIOBLASTOMA INCREASES TUMORIGENICITY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi208-vi209
Author(s):  
Dionysios Watson ◽  
Defne Bayik ◽  
Justin Lathia
Keyword(s):  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Treatment Strategies ◽  
Tumor Initiating Cells ◽  
Mitochondrial Transfer ◽  
Mitochondria Transfer

Abstract Communication between glioblastoma (GBM) and its microenvironment facilitates tumor growth and therapeutic resistance, and is facilitated through a variety of mechanisms. Organelle transfer between cells was recently observed, including mitochondria transfer from astrocytes to neurons after ischemic stroke. Given the dependence of GBM on microenvironmental interactions, we hypothesized that mitochondria transfer from tumor microenvironment to GBM cells could occur and affect metabolism and tumorigenicity. We interrogated this in vivo by establishing intracranial GBM tumors in mito::mKate2 mice (with trackable fluorescent mitochondria) using syngeneic GFP-expressing tumor cells (SB28 and GL261 models). We also cultured stromal cell types from mito::mKate2 mice with tumor cells, enabling sorting of tumor cells with and without exogenous mitochondria. Confocal microscopy revealed horizontal transfer of mKate2+ mitochondria from mouse cells to implanted GBM cells in vivo and was confirmed by flow cytometry where 20-40% of GBM cells acquired exogenous mitochondria. Transfer was negligible in wildtype mice transplanted with mito::mKate2 bone marrow cells, suggesting that brain-resident cells were the main donors. In vitro, astrocytes and microglia exhibited 5 to 10-fold higher mitochondrial transfer rate than bone-marrow derived macrophages. Seahorse metabolic profiling revealed that GBM cells with mKate2+ mitochondria had 40% lower respiratory reserve compared to cells without exogenous mitochondria. Median survival of mice implanted with SB28 that acquired mitochondria was significantly shorter and in vivo limiting dilution confirmed the frequency of tumor-initiating cells was 3-fold higher in SB28 cells with exogenous mitochondria. Our data indicate that horizontal mitochondrial transfer from brain-resident glia to mouse GBM tumors alters tumor cell metabolism and increases their tumorigenicity. Ongoing studies are assessing gene expression in GBM cells acquiring exogenous mitochondria; validating findings in human specimens; and screening for transfer inhibitor drugs. Horizontal mitochondrial transfer represents a foundational tumor microenvironment interaction contributing to glioblastoma plasticity, and is likely to inform next-generation treatment strategies.

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