Dual role of E-cadherin in the regulation of invasive collective migration of mammary carcinoma cells.

In this article, we explore a non-canonical form of collective cell migration, displayed by the metastatic murine mammary carcinoma cell line 4T1. We show here that in sparsely plated 4T1 cells, E-cadherin levels are moderately reduced (~50%), leading to the development of collective migration, whereby cells translocate in loose clusters, interconnected by thin membrane tethers. Knocking down E-cadherin blocked tether formation in these cells, leading to enhancement of migration rate and, at the same time, to suppression of lung metastases formation in vivo, and inhibition of infiltration into fibroblast monolayers ex vivo. These findings suggest that the moderate E-cadherin levels present in wild-type 4T1 cells play a key role in promoting cancer invasion and metastasis.

Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure.

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry.

INTRODUCTION: The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry. MATERIALS AND METHODS: Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection. RESULTS: The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung. CONCLUSIONS: Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.

A Novel Intravital Imaging Window for Longitudinal Microscopy of the Mouse Ovary.

The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion.

Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8(+) T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c(hi)CX3CR1(lo)) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c(lo)CX3CR1(hi)) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

Stiffening of rabbit corneas by the bacteriochlorophyll derivative WST11 using near infrared light.

PURPOSE: We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models. METHODS: Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment. RESULTS: WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium. CONCLUSIONS: Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots.

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice.

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in vitro and their recovery in transplanted recipients in vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

In vivo characterization of tumor and tumor vascular network using multi-modal imaging approach.

We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment.

Neuroprotection and progenitor cell renewal in the injured adult murine retina requires healing monocyte-derived macrophages.

The death of retinal ganglion cells (RGCs) is a hallmark of many retinal neuropathies. Neuroprotection, axonal regeneration, and cell renewal are vital for the integrity of the visual system after insult but are scarce in the adult mammalian retina. We hypothesized that monocyte-derived macrophages, known to promote healing in peripheral tissues, are required after an insult to the visual system, where their role has been largely overlooked. We found that after glutamate eye intoxication, monocyte-derived macrophages infiltrated the damaged retina of mice. Inhibition of this infiltration resulted in reduced survival of RGCs and diminished numbers of proliferating retinal progenitor cells (RPCs) in the ciliary body. Enhancement of the circulating monocyte pool led to increased RGC survival and RPC renewal. The infiltrating monocyte-derived macrophages skewed the milieu of the injured retina toward an antiinflammatory and neuroprotective one and down-regulated accumulation of other immune cells, thereby resolving local inflammation. The beneficial effect on RGC survival depended on expression of interleukin 10 and major histocompatibility complex class II molecules by monocyte-derived macrophages. Thus, we attribute to infiltrating monocyte-derived macrophages a novel role in neuroprotection and progenitor cell renewal in the injured retina, with far-reaching potential implications to retinal neuropathies and other neurodegenerative disorders.

Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activity.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Novel design principles enable specific targeting of imaging and therapeutic agents to necrotic domains in breast tumors.

INTRODUCTION: Necrosis at the tumor center is a common feature of aggressive breast cancers and has been associated with poor prognosis. It is commonly identified by means of invasive histopathology, which often correlates with morbidity and potential tumor cell dissemination, and limits the reconstruction of the whole necrotic domain. In this study we hypothesized that non covalent association to serum albumin (SA) and covalent binding to ligands for tumor-abundant cell receptors should synergistically drive selective accumulation and prolonged retention of imaging and therapeutic agents in breast tumor necrotic domains enabling in vivo identification, imaging and possibly treatment of such tumors. METHODS: Cyclo-Arg-Gly-Asp-D-Phe-Lys (c(RGDfK)) were conjugated to bacteriochlorophyll-derivatives (Bchl-Ds), previously developed as photodynamic agents, fluorescent probes and metal chelators in our lab. The c(RGDfK) component drives ligation to alphaVbeta3 integrin receptors over-expressed by tumor cells and neo-vessels, and the Bchl-D component associates to SA in a non-covalent manner. STL-6014, a c(RGDfK)-Bchl-D representative, was i.v. injected to CD-1, nude female mice bearing necrotic and non-necrotic human MDA-MB-231-RFP breast cancer tumors. The fluorescence signals of the Bchl-Ds and RFP were monitored over days after treatment, by quantitative whole body imaging and excised tumor/tissue samples derived thereof. Complementary experiments included competitive inhibition of STL-6014 uptake by free c(RGDfK), comparative pharmacokinetics of nonconjugated c(RGDfK) Bchl-D (STL-7012) and of two human serum albumin (HSA) conjugates: HSA-STL-7012 and HSA-STL-6014. RESULTS: STL-6014 and STL-7012 formed complexes with HSA (HSA/STL-6014, HSA/STL-7012). STL-6014, HSA-STL-7012 and HSA-STL-6014, selectively accumulated at similar rates, in tumor viable regions over the first 8 h post administration. They then migrated into the necrotic tumor domain and presented tumor half lifetimes (T1/2) in the range of days where T1/2 for HSA-STL-6014 > STL-6014 > HSA-STL-7012. No accumulation of STL-7012 was observed. Pre-injection of c(RGDfK) excess, prevented the uptake of STL-6014 in the small, but not in the large tumors. CONCLUSIONS: Non-covalent association to SA and covalent binding to c(RGDfK), synergistically enable the accumulation and prolonged retention of Bchl-Ds in the necrotic regions of tumors. These findings provide novel guidelines and strategy for imaging and treatment of necrotic tumors.

Permanent occlusion of feeding arteries and draining veins in solid mouse tumors by vascular targeted photodynamic therapy (VTP) with Tookad.

BACKGROUND: Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24-48 h. METHODOLOGY/PRINCIPAL FINDINGS: A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec+/-1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP. CONCLUSION/SIGNIFICANCE: Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas.

Transcriptional regulation of vascular endothelial growth factor C by oxidative and thermal stress is mediated by lens epithelium-derived growth factor/p75.

Vascular endothelial growth factor C (VEGF-C) plays a critical role in tumor lymphangiogenesis and lymph node metastasis. We report here that VEGF-C expression is regulated by microenvironmental stress including hyperthermia and oxidative stress. Furthermore, we show that this stress response is mediated by transcriptional activation mediated by lens epithelium-derived growth factor (LEDGF/p75). Ectopic expression of LEDGF/p75 in C6 rat glioma and in H1299 human non-small cell lung carcinoma induced VEGF-C expression in vitro, whereas in subcutaneous mouse tumor xenografts, LEDGF/p75 stimulated VEGF-C expression and augmented angiogenesis and lymphangiogenesis. Conversely, overexpression of a LEDGF/p75 native antisense or LEDGF/p75-targeted short interfering RNA downmodulated VEGF-C expression. LEDGF seemed to conferred this activity on binding to a conserved stress response element (STRE) located in the VEGF-C gene because mutating the STRE was sufficient for the suppression of basal and stress-induced activations of the VEGF-C promoter. Thus, the study reported here identified a role for LEDGF/p75 in stress-regulated transcriptional control of VEGF-C expression. These results provide a possible link for LEDGF/p75 in tumor lymphangiogenesis and cancer metastasis. Hence, our data suggest the LEDGF-VEGF-C axis as a putative biomarker for the detection of stress-induced lymphangiogenesis and LEDGF as a potential target for antimetastatic therapy.

Harnessing competing endocytic pathways for overcoming the tumor-blood barrier: magnetic resonance imaging and near-infrared imaging of bifunctional contrast media.

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.

Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity.

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease.

Uterine DCs are crucial for decidua formation during embryo implantation in mice.

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-beta1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

Tumorigenic potential and disease manifestations of malignant B-cell variants differing in their fibronectin adhesiveness.

OBJECTIVE: Microenvironmental interactions of malignant B cells can modulate various in vitro physiological responses, including proliferation, migration, apoptosis, and drug resistance. Disease manifestations of human malignant B-cell variants, isolated based on their differential interactions with fibronectin, were examined in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. MATERIALS AND METHODS: Disease manifestations were assessed by pathological examinations and skeletal imaging of NOD/SCID mice injected with malignant B-cell variants. Dissemination patterns were analyzed by whole-body real-time imaging of mice injected with fluorescence-labeled malignant cells. RESULTS: Initial dissemination patterns and dynamics of both high (type A) and low (type F)-adherent variants, following intravenous inoculation, were similar. Both cell types reached the spleen and liver within 30 minutes after injection, then increasingly accumulated within the bone marrow. Mice injected with type-A cells developed multiple myeloma-like disease within the bone marrow, with multiple lytic bone lesions. In contrast, type-F cells displayed low tumorigenic capacity in spite of their efficient homing to the bone marrow niche. In addition, type-A cells grew as extramedullary tumors in some of the intravenous-inoculated mice, and formed solid tumors following subcutaneous injection. Both cell variants retained their characteristics surface markers following in vivo outgrowth as tumors, indicating that at least some of their properties are relatively stable. CONCLUSION: Data suggest that the differential tumorigenicity of B-cell adhesive variants is attributable to the capacity of type-A cells to survive and proliferate within the bone marrow, rather than to different initial dissemination of the two cell populations.

A daidzein-daunomycin conjugate improves the therapeutic response in an animal model of ovarian carcinoma.

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.