Oksana Zavidij

Distinct Types of Tumor-Initiating Cells Form Human Colon Cancer Tumors and Metastases

Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.

Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo

Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.

Ultrasmall Silica-Based Bismuth Gadolinium Nanoparticles for Dual Magnetic Resonance–Computed Tomography Image Guided Radiation Therapy

Selective killing of cancer cells while minimizing damage to healthy tissues is the goal of clinical radiation therapy. This therapeutic ratio can be improved by image-guided radiation delivery and selective radiosensitization of cancer cells. Here, we have designed and tested a novel trimodal theranostic nanoparticle made of bismuth and gadolinium for on-site radiosensitization and image contrast enhancement to improve the efficacy and accuracy of radiation therapy. We demonstrate in vivo magnetic resonance (MR), computed tomography (CT) contrast enhancement, and tumor suppression with prolonged survival in a non-small cell lung carcinoma model during clinical radiation therapy. Histological studies show minimal off-target toxicities due to the nanoparticles or radiation. By mimicking existing clinical workflows, we show that the bismuth-gadolinium nanoparticles are highly compatible with current CT-guided radiation therapy and emerging MR-guided approaches. This study reports the first in vivo proof-of-principle for image-guided radiation therapy with a new class of theranostic nanoparticles.

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady‐state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady‐state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification‐mediated‐polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV‐injected animals for up to 24 months, indicating stable marking of murine steady‐state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long‐term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short‐lived progenitor cells. Myelosuppressive treatment of mice prior to LV‐injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady‐state hematopoiesis. Stem Cells2012;30:1961–1970

Evaluation of the T-REx™ transcription switch for conditional expression and regulation of HSV-1 vectors

Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx™ tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx™ gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.

Hematopoietic activity of human short-term repopulating cells in mobilized peripheral blood cell transplants is restricted to the first 5 months after transplantation

Kinetics of hematopoietic recovery driven by different types of human stem and progenitor cells after transplantation are not fully understood. Short-term repopulating cells (STRCs) dominate early hematopoiesis after transplantation. STRCs are highly enriched in adult mobilized peripheral blood compared with cord blood, but the length of their contribution to hematopoiesis remains unclear. To understand posttransplantation durability and lineage contribution of STRCs, we compared repopulation kinetics of mobilized peripheral blood (high STRC content) with cord blood transplants (low STRC content) in long-lived NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (IL2RG(-/-)) mice. This comparison demonstrates that quantitative contribution of human STRCs to hematopoiesis is restricted to the first 5 months after transplantation. The ratio of STRCs to long-term repopulating cells dramatically changes during ontogeny. This model enables to precisely determine early and late engraftment kinetics of defined human repopulating cell types and to preclinically assess the engraftment kinetics of engineered stem cell transplants.

Blocking IFNRA1 inhibits multiple myeloma-driven Treg expansion and immunosuppression

Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN &agr; and &bgr; receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.

10-Year stability of clinical-grade serum-free γ-retroviral vector-containing medium

More than 10 years ago, we developed an efficient protocol for serum-free retroviral transduction of human hematopoietic stem cells derived from mobilized peripheral blood. After upscaling of the methodology, serum-free retroviral gibbon-ape leukemia virus (GALV) pseudotype PG13/LN vector supernatant produced under strict good manufacturing practice (GMP) conditions was used in the first clinical gene-marking trial in Germany. In this study, we analyzed the titer and transduction efficiency of this serum-free clinical-grade retroviral supernatant 10 years after production to evaluate the long-term stability. Long-term storage and transport on dry ice resulted in modestly decreased titers and levels of transduction efficiency in CD34+ cells ranging from 38.4 to 49.1%. We conclude that the stability of retroviral vectors in serum-free medium allows extended storage and distribution of approved clinical-grade retroviral vector stocks to distant sites in multicenter clinical trials.

Inhibition of microRNA-138 enhances bone formation in multiple myeloma bone marrow niche

Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.

Bortezomib overcomes the negative impact of CXCR4 mutations on survival of Waldenstrom's Macroglobulinemia patients

TO THE EDITOR: Waldenstrom macroglobulinemia (WM) is a rare lymphoplasmacytic lymphoma associated with immunoglobulin M monoclonal gammopathy.[1][1] The majority of patients carry the L265P mutation in MYD88,[2][2] whereas 40% of patients carry mutations in CXCR4.[3][3] Mutation status has an