Julie R. Beegle

Ultrasound increases nanoparticle delivery by reducing intratumoral pressure and increasing transport in epithelial and epithelial-mesenchymal transition tumors.

Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared with EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was approximately 1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.

Insonation of targeted microbubbles produces regions of reduced blood flow within tumor vasculature.

ObjectivesIn ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects. Materials and MethodsA total of 114 mice carrying either Met-1 or neu deletion mutant (NDL) tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5-MHz color-Doppler pulses, 4 or 2 MPa peak-negative pressure, 8.1-millisecond pulse repetition period, 6-cycle pulse length, and 900-millisecond insonation). Microbubbles conjugated with cyclic-arginine-glycine-aspartic acid (cRGD) or cyclic-aspartic-acid-glycine-tyrosine (3-NO2)-glycine-hydroxyproline-asparagine (LXY-3) peptides or control (no peptide) microbubbles were injected, and contrast pulse sequencing was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects. ResultsAfter the accumulation of targeted microbubbles and high-pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (P < 0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (P < 0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (P < 0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5/18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150 kDa fluorescein-isothiocyanate (FITC)-dextran were observed by histology and confocal microscopy. Preinjection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals. ConclusionHigh-pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within 2 syngeneic mouse tumor models for ∼30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.

Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia

Numerous clinical trials are utilizing mesenchymal stem cells (MSC) to treat critical limb ischemia, primarily for their ability to secrete signals that promote revascularization. These cells have demonstrated clinical safety, but their efficacy has been limited, possibly because these paracrine signals are secreted at subtherapeutic levels. In these studies the combination of cell and gene therapy was evaluated by engineering MSC with a lentivirus to overexpress vascular endothelial growth factor (VEGF). To achieve clinical compliance, the number of viral insertions was limited to 1–2 copies/cell and a constitutive promoter with demonstrated clinical safety was used. MSC/VEGF showed statistically significant increases in blood flow restoration as compared with sham controls, and more consistent improvements as compared with nontransduced MSC. Safety of MSC/VEGF was assessed in terms of genomic stability, rule-out tumorigenicity, and absence of edema or hemangiomas in vivo. In terms of retention, injected MSC/VEGF showed a steady decline over time, with a very small fraction of MSC/VEGF remaining for up to 4.5 months. Additional safety studies completed include absence of replication competent lentivirus, sterility tests, and absence of VSV-G viral envelope coding plasmid. These preclinical studies are directed toward a planned phase 1 clinical trial to treat critical limb ischemia.

Hypoxic pre-conditioning increases the infiltration of endothelial cells into scaffolds for dermal regeneration pre-seeded with mesenchymal stem cells

Many therapies using mesenchymal stem cells (MSC) rely on their ability to produce and release paracrine signals with chemotactic and pro-angiogenic activity. These characteristics, however, are mostly studied under standard in vitro culture conditions. In contrast, various novel cell-based therapies imply pre-seeding MSC into bio-artificial scaffolds. Here we describe human bone marrow-derived MSC seeded in Integra matrices, a common type of scaffold for dermal regeneration (SDR). We show and measured the distribution of MSC within the SDR, where cells clearly establish physical interactions with the scaffold, exhibiting constant metabolic activity for at least 15 days. In the SDR, MSC secrete VEGF and SDF-1α and induce transwell migration of CD34+ hematopoietic/endothelial progenitor cells, which is inhibited in the presence of a CXCR4/SDF-1α antagonist. MSC in SDR respond to hypoxia by altering levels of angiogenic signals such as Angiogenin, Serpin-1, uPA, and IL-8. Finally, we show that MSC-containing SDR that have been pre-incubated in hypoxia show higher infiltration of endothelial cells after implantation into immune deficient mice. Our data show that MSC are fully functional ex vivo when implanted into SDR. In addition, our results strongly support the notion of hypoxic pre-conditioning MSC-containing SDR, in order to promote angiogenesis in the wounds.

Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways

Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%‐97% CD31+ and 78%‐83% VE‐cadherin+) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE‐cadherin+). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil‐acetylated low‐density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC‐derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell‐derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909–919

Reduced blood flow in murine tumors after the destruction of bound, targeted microbubbles

The insonation of circulating microbubbles (MBs) by low frequency (1 MHz) ultrasound (US) pulses has previously been associated with changes in vascular permeability and local changes in blood flow. Here, using a clinical scanner, 5 MHz insonation of bound, targeted MBs is demonstrated to locally alter blood flow in murine tumors. Peptide-targeted MBs were administrated into murine Met-1 and NDL tumor models via tail vein injection (5×107 MBs). Thirty frames of CPS contrast images (Siemens Sequoia 512, 0.09 MI, 10 Hz frame rate) were recorded to assess tumor blood flow. Seven minutes after injection, freely-circulating MBs had cleared from the blood stream leaving bound MBs that had accumulated in the tumor vasculature. At this time, 5 MHz pulses with a peak negative pressure (PNP) of 2 or 4 MPa, a pulse length of 5 cycles and a pulse repetition period of 8.1 ms were transmitted for 0.9 second. Five minutes after the high-pressure pulse sequence, a second dose of MBs was injected and 30 frames of CPS images were acquired. Optical images of systemically-injected FITC-dextran (MW=150,000), pre-administration of an anti-CD41 antibody, and histology were used to investigate the possible mechanism for the vascular changes. After the insonation of bound MBs with a 4 MPa PNP, additional regions of reduced blood flow were observed in 71% of Met-1 tumors (n=28) and 40% of NDL tumors (n = 10). In Met-1 tumors insonified with 4 MPa pulses, the area over which reduced blood flow was observed increased from 22±13% to 63±17% (pμ0.01) of the tumor region of interest. Decreasing the PNP to 2 MPa decreased the percentage of Met-1 tumors with additional regions of reduced blood flow from 71% to 28%. Histological analysis of Met-1 tumors after 4 MPa insonation demonstrated that the mean microvascular diameter in insonified tumors was approximately 17±8 μm, compared to 7±4 μm in control tumors (pμ0.01). Extravasation of FITC-dextran was observed in 4 MPa insonified, but not control, Met-1 tumors. The results suggest that high-pressure insonation of targeted MBs which had accumulated at high concentration, may result in changes in blood flow.

A Preview of Selected Articles ‐ October 2018

Cancer does not arise from just a small group of tumorigenic cells, but involves a complex orchestration of a variety of other cell types and signals [1]. The tumor microenvironment plays a critical role in both suppression and promotion of progression of cancer. Understanding the components of specialized microenvironments, such as the bone marrow (BM) niche, can help us recognize and intervene at points where tumor cells hijack endogenous functions to promote their own survival. Despite a growing body of knowledge of the composition and specific functions of cells that reside in the BM niche, our understanding of the dynamic changes that regulate the niche both during normal physiological processes and in leukemic conditions, remains unclear. In our first featured article, Jeong et al. discuss how their niche stimulation model reveals a remodeling process by which the BM niche shifts toward promoting normal hematopoietic stem cell activity and suppressing the tumor cell-driven proleukemic microenvironment. Determining signals that are key regulators of disease progression can provide specific steps for targeted intervention, particularly when the disease relies on endogenous properties of the native cells. In a related article, Redondo et al. further describes the phenotype of mesenchymal stem cells (MSC) isolated from patients with progressive multiple sclerosis and the negative correlation between the cells’ capacity to regulate natural antioxidants and progression of the disease. Different species exhibit varying abilities to resist this hijacking of native cells’ coordinated maintenance of physiological homeostasis [2]. We can look toward organisms that defy the statistical probability of developing cancer to elucidate evolutionary mechanisms for suppressing the disease, potentially adapting these strategies for improved treatments and preventative strategies in humans. In our second featured article, Mamchur et al. describe the evolutionary adaptations that allow the Middle Eastern blind mole rat, Spalax ehrenbergi, to survive to an unusually old age in an extreme habitat, and hypothesize that these adaptations contribute to the ability of the species to resist cancer. Paradoxically, MSC, including adipose derived stem cells (ADSC), have been shown to play critical roles in both the suppression and growth of tumors [3]. In a related article, Xu et al. examine their ability to take advantage of the upregulation of the inflammatory modulating cytokine angiotensin II in cases of acute lung injury to increase migration of gene modified MSC to the site of injury, thereby increasing their known therapeutic benefit.

32. Safety Studies Towards a Combined Cell and Gene Therapy To Treat Critical Limb Ischemia

Over 240 clinical trials are in progress worldwide using mesenchymal stem cells (MSCs). Some critical advantages of MSCs include simple isolation and expansion, and ease for allogeneic applications due to their immune evading properties. MSCs produce many paracrine signals that promote tissue regeneration, though at sub-therapeutic levels. We have therefore developed a product that combines cell and gene therapy, by engineering MSCs to overexpress vascular endothelial growth factor (VEGF). Here we show our preclinical data that not only confirms the efficacy of our product (MSC-VEGF), but addresses multiple safety aspects. Here, MSCs are transduced with a GMP-grade lentiviral vector to obtain an average of one copy per cell (ranging from 0.8 to 1.3), minimizing occurrence of insertional mutagenesis. The secretion of VEGF is optimized by using a previously clinically evaluated constitutive promoter (MNDU3) and an enhancer element acting in cis (WPRE). Safety studies at this level of secretion (approx. 80pg/ml VEGF secreted per 1000 cells per 24 hours) did not cause edema or hemangiomas in immune deficient NOD/SCID IL-2R-gnull/null (NSG) mice. The genomic stability of MSC-VEGF was addressed by karyotyping analysis, where no abnormalities were found in MSCs transduced with increasing viral loads, and by PCR to evaluate potential genomic rearrangement of the insert. To rule out tumorigenicity of MSC-VEGF, cells transduced with increased viral load were in injected into NSG mice. In contrast to 3 distinct positive controls used, no tumors were detected in over 50 mice treated with MSC-VEGF up to 6 months after injection. Additional safety studies included confirmation by flow cytometry of homogeneity of the transduction level of MSC-VEGF, absence of replication competent lentivirus, freedom of endotoxin, mycoplasma and absence VSV-G viral envelope coding plasmid. Luciferase-expressing MSC-VEGF consistently decreased to undetectable levels by 28 days after injection and PCR confirmed the absence of human DNA 6 months after injection. Pilot efficacy studies using MSC-VEGF in an immune deficient mouse model of hind limb ischemia (HLI) have been completed. MSC-VEGF were injected IM the day after HLI surgery into the ischemic limbs of NSG mice and blood flow was monitored weekly, demonstrating that MSC-VEGF treatment leads to faster revascularization than control treatment. Furthermore, we show that a small percentage of injected MSC-VEGF take a pericyte position on blood vessels. While future assays are intended as IND-enabling safety studies, these pre-clinical safety and efficacy studies are directed towards our planned Phase I clinical trial.

A fast ultrasound molecular imaging method and its 3D visualization in vivo

Using targeted microbubbles (MBs), ultrasound molecular imaging can be used to selectively and specifically visualize upregulated vascular receptors. In order to acquire bound MB echoes, a delay of ~7-15 minutes is commonly required for the clearance of freely circulating MBs. Here, we test whether echoes from MBs can be distinguished from the surrounding tissue, based on the transmission of pulses at low (1.5 MHz) and reception at high (5.5 MHz) frequencies (TLRH), without the requirement for destructive pulses. Pulses with a peak negative pressure of 230 kPa were transmitted (10 fps) and a 7th order IIR pulse-to-pulse filter was applied to the TLRH radiofrequency (RF) data to distinguish the signature of bound MBs from that of flowing MBs. 3D images of the accumulation of intravenously-administrated integrin-targeted MBs in a Met-1 mouse tumor model were acquired. An in vitro study demonstrated that the T2R15 contrast imaging technique has a ~2-fold resolution improvement over 2MHz contrast pulse sequencing (CPS) imaging. By applying the 7th order IIR filter to the TLRH RF data acquired at 2 minutes, echoes from flowing MBs in the surrounding tissue region were suppressed by 26±2 dB, while the signal intensity within the tumor was suppressed by 4±1 dB. The targeted images correctly represented the distribution of bound MBs. After the filter, the signal intensity resulting from cyclic RGD bearing MBs was 25±2 dB higher than that after the injection of non-targeted MBs.