Rationale: Efficient labeling methods for mesenchymal stem cells (MSCs) are crucial for tracking and understanding their behavior in regenerative medicine applications, particularly in cartilage defects. MegaPro nanoparticles have emerged as a potential alternative to ferumoxytol nanoparticles for this purpose.Methods: In this study, we employed mechanoporation to develop an efficient labeling method for MSCs using MegaPro nanoparticles and compared their effectiveness with ferumoxytol nanoparticles in tracking MSCs and chondrogenic pellets. Pig MSCs were labeled with both nanoparticles using a custom-made microfluidic device, and their characteristics were analyzed using various imaging and spectroscopy techniques. The viability and differentiation capacity of labeled MSCs were also assessed. Labeled MSCs and chondrogenic pellets were implanted into pig knee joints and monitored using MRI and histological analysis.Results: MegaPro-labeled MSCs demonstrated shorter T2 relaxation times, higher iron content, and greater nanoparticle uptake compared to ferumoxytol-labeled MSCs, without significantly affecting their viability and differentiation capacity. Post-implantation, MegaPro-labeled MSCs and chondrogenic pellets displayed a strong hypointense signal on MRI with considerably shorter T2* relaxation times compared to adjacent cartilage. The hypointense signal of both MegaPro- and ferumoxytol-labeled chondrogenic pellets decreased over time. Histological evaluations showed regenerated defect areas and proteoglycan formation with no significant differences between the labeled groups.Conclusion: Our study demonstrates that mechanoporation with MegaPro nanoparticles enables efficient MSC labeling without affecting viability or differentiation. MegaPro-labeled cells show enhanced MRI tracking compared to ferumoxytol-labeled cells, emphasizing their potential in clinical stem cell therapies for cartilage defects.

Rationale: As a cancer, Glioblastoma (GBM) is a highly lethal and difficult-to-treat. With the aim of improving therapies to GBM, we developed novel and target-specific theranostic nanoparticles (TNPs) that can be selectively cleaved by cathepsin B (Cat B) to release the potent toxin monomethyl auristatin E (MMAE). Methods: We synthesized TNPs composed of a ferumoxytol-based nanoparticle carrier and a peptide prodrug with a Cat-B-responsive linker and the tubulin inhibitor MMAE. We hypothesized that intratumoral Cat B can cleave our TNPs and release MMAE to kill GBM cells. The ferumoxytol core enables in vivo drug tracking with magnetic resonance imaging (MRI). We incubated U87-MG GBM cells with TNPs or ferumoxytol and evaluated the TNP content in the cells with transmission electron microscopy and Prussian blue staining. In addition, we stereotaxically implanted 6- to 8-week-old nude mice with U87-MG with U87-MG GBM cells that express a fusion protein of Green Fluorescence Protein and firefly Luciferase (U87-MG/GFP-fLuc). We then treated the animals with an intravenous dose of TNPs (25 mg/kg of ferumoxytol, 0.3 mg/kg of MMAE) or control. We also evaluated the combination of TNP treatment with radiation therapy. We performed MRI before and after TNP injection. We compared the results for tumor and normal brain tissue between the TNP and control groups. We also monitored tumor growth for a period of 21 days. Results: We successfully synthesized TNPs with a hydrodynamic size of 41 ± 5 nm and a zeta potential of 6 ± 3 mV. TNP-treated cells demonstrated a significantly higher iron content than ferumoxytol-treated cells (98 ± 1% vs. 3 ± 1% of cells were iron-positive, respectively). We also found significantly fewer live attached cells in the TNP-treated group (3.8 ± 2.0 px2) than in the ferumoxytol-treated group (80.0 ± 14.5 px2, p < 0001). In vivo MRI studies demonstrated a decline in the tumor signal after TNP (T2= 28 ms) but not control (T2= 32 ms) injections. When TNP injection was combined with radiation therapy, the tumor signals dropped further (T2 = 24 ms). The combination therapy of radiation therapy and TNPs extended the median survival from 14.5 days for the control group to 45 days for the combination therapy group. Conclusion: The new cleavable TNPs reported in this work accumulate in GBM, cause tumor cell death, and have synergistic effects with radiation therapy.

The purpose of our study was to investigate if vascular injury in immature epiphyses affects cartilage repair outcomes of matrix-associated stem cell implants (MASI). Porcine bone marrow mesenchymal stromal stem cells (BMSCs) suspended in a fibrin glue scaffold were implanted into 24 full-thickness cartilage defects (5 mm ø) of the bilateral distal femur of six Göttingen minipigs (n = 12 defects in 6 knee joints of 3 immature pigs; age 3.5–4 months; n = 12 defects in 6 knee joints of 3 mature control pigs; age, 21–28 months). All pigs underwent magnetic resonance imaging (MRI) at 2, 4, 12 (n = 24 defects), and 24 weeks (n = 12 defects). After the last imaging study, pigs were sacrificed, joints explanted and evaluated with VEGF, H&E, van Gieson, Mallory, and Safranin O stains. Results of mature and immature cartilage groups were compared using the Wilcoxon signed-rank test. Quantitative scores for subchondral edema at 2 weeks were correlated with quantitative scores for cartilage repair (MOCART score and ICRS score) at 12 weeks as well as Pineda scores at end of the study, using linear regression analysis. On serial MRIs, mature joints demonstrated progressive healing of cartilage defects while immature joints demonstrated incomplete healing and damage of the subchondral bone. The MOCART score at 12 weeks was significantly higher for mature joints (79.583 ± 7.216) compared to immature joints (30.416 ± 10.543, p = 0.002). Immature cartilage demonstrated abundant microvessels while mature cartilage did not contain microvessels. Accordingly, cartilage defects in immature joints showed a significantly higher number of disrupted microvessels, subchondral edema, and angiogenesis compared to mature cartilage. Quantitative scores for subchondral edema at 2 weeks were negatively correlated with MOCART scores (r = − 0.861) and ICRS scores (r = − 0.901) at 12 weeks and positively correlated with Pineda scores at the end of the study (r = 0.782). Injury of epiphyseal blood vessels in immature joints leads to subchondral bone defects and limits cartilage repair after MASI.

Objectives Iron oxide nanoparticles have been used to track the accumulation of chimeric antigen receptor (CAR) T cells with magnetic resonance imaging (MRI). However, the only nanoparticle available for clinical applications to date, ferumoxytol, has caused rare but severe anaphylactic reactions. MegaPro nanoparticles (MegaPro-NPs) provide an improved safety profile. We evaluated whether MegaPro-NPs can be applied for in vivo tracking of CAR T cells in a mouse model of glioblastoma multiforme. Materials and Methods We labeled tumor-targeted CD70CAR (8R-70CAR) T cells and non–tumor-targeted controls with MegaPro-NPs, followed by inductively coupled plasma optical emission spectroscopy, Prussian blue staining, and cell viability assays. Next, we treated 42 NRG mice bearing U87-MG/eGFP-fLuc glioblastoma multiforme xenografts with MegaPro-NP-labeled/unlabeled CAR T cells or labeled untargeted T cells and performed serial MRI, magnetic particle imaging, and histology studies. The Kruskal-Wallis test was conducted to evaluate overall group differences, and the Mann-Whitney U test was applied to compare the pairs of groups. Results MegaPro-NP-labeled CAR T cells demonstrated significantly increased iron uptake compared with unlabeled controls ( P < 0.01). Cell viability, activation, and exhaustion markers were not significantly different between the 2 groups ( P > 0.05). In vivo, tumor T2* relaxation times were significantly lower after treatment with MegaPro-NP-labeled CAR T cells compared with untargeted T cells ( P < 0.01). There is no significant difference in tumor growth inhibition between mice injected with labeled and unlabeled CAR T cells. Conclusions MegaPro-NPs can be used for in vivo tracking of CAR T cells. Because MegaPro-NPs recently completed phase II clinical trial investigation as an MRI contrast agent, MegaPro-NP is expected to be applied to track CAR T cells in cancer immunotherapy trials in the near future.

Adenovirus (Ad)-based vectors have shown considerable promise for gene therapy. However, Ad requires the coxsackievirus and adenovirus receptor (CAR) to enter cells efficiently and low CAR expression is found in many human cancers, which hinder adenoviral gene therapies. Here, cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-folate liposomes (Df) encapsulating replication-deficient Ad were synthesized, which showed improved transfection efficiency in various CAR-deficient cell lines, including epithelial and hematopoietic cell types. When encapsulating replication-competent oncolytic Ad (TAV255) in DOTAP-folate liposome (TAV255-Df), the adenoviral structural protein, hexon, was readily produced in CAR-deficient cells, and the tumor cell killing ability was 5× higher than that of the non-encapsulated Ad. In CAR-deficient CT26 colon carcinoma murine models, replication-competent TAV255-Df treatment of subcutaneous tumors by intratumoral injection resulted in 67% full tumor remission, prolonged survival, and anti-cancer immunity when mice were rechallenged with cancer cells with no further treatment. The preclinical data shows that DOTAP-folate liposomes could significantly enhance the transfection efficiency of Ad in CAR-deficient cells and, therefore, could be a feasible strategy for applications in cancer treatment.

Purpose Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model. Procedures TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections. Results The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%). Conclusions We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Marking colon tumors for surgery is normally done with the use of India ink. However, non-fluorescent dyes such as India ink cannot be imaged below the tissue surface and there is evidence for physiological complications such as abscess, intestinal perforation and inconsistency of dye injection. A novel infrared marker was developed using FDA approved indocyanine green (ICG) dye and ultrathin hollow silica nanoshells (ICG/HSS). Using a positively charged amine linker, ICG was non-covalently adsorbed onto the nanoparticle surface. For ultra-thin wall 100 nm diameter silica shells, a bimodal ICG layer of < 3 nm is was formed. Conversely, for thicker walls on 2 μm diameter silica shells, the ICG layer was only bound to the outer surface and was 6 nm thick. In vitro testing of fluorescent emission showed the particles with the thinner coating were considerably more efficient, which is consistent with self-quenching reducing emission shown in the thicker ICG coatings. Ex-vivo testing showed that ICG bound to the 100 nm hollow silica shells was visible even under 1.5 cm of tissue. In vivo experiments showed that there was no diffusion of the ICG/nanoparticle marker in tissue and it remained imageable for as long as 12 days.

Silica particles are convenient ultrasound imaging contrast agents because of their long imaging time and ease of modification; however, they require a relatively high insonation power for imaging and have low biodegradability. In this study, 2 µm ultrathin asymmetric hollow silica particles doped with iron (III) (Fe(III)‐SiO 2 ) are synthesized to produce biodegradable hard shelled particles with a low acoustic power threshold comparable with commercial soft microbubble contrast agents (Definity) yet with much longer in vivo ultrasound imaging time. Furthermore, high intensity focused ultrasound ablation enhancement with these particles shows a 2.5‐fold higher temperature elevation than with Definity at the same applied power. The low power visualization improves utilization of the silica shells as an adjuvant in localized immunotherapy. The data are consistent with asymmetric engineering of hard particle properties that improve functionality of hard versus soft particles.

Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1β release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1β. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.

While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas. We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be surgically administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipililumab. In an in vivomurine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10–100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-γ (IFN-γ) in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (p< 0.001) and IFN- γ levels (p=0.001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU. Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy that can be surgically administered.

A key challenge in immunotherapy for glioblastomas, the most common form of primary adult brain cancer, involves the paucity of immune-stimulatory cells in its "cold" immune-microenvironment. Herein, mechanical acoustic ablation focused by perfluorocarbon (PFC) liquid filled silica microshells is applied to induce immunogenicity via in situ ultrasonic lysis. The inert PFC filled ultra-thin walled silica microshells promote mechanical ablation while aiding in ultrasound guidance. In the presence of programmed cell death protein 1 (PD-1) blockade, tumor injury sites exhibit an increase in tumor infiltrating lymphocytes and interferon-gamma (IFN-gamma) by 1-2 orders of magnitude. At least 75% of mice grafted with the advanced murine glioblastoma tumors achieve remission when treated with a combination of microshell enhanced ablation and PD-1 blockade, which indicates a synergistic effect. In contrast, none of the mice treated with single therapies achieve durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (p < 0.001) and IFN-. levels (p = 0.001). This study demonstrates a PFC filled ultrathin walled microshell enhanced ablation strategy that induces a "hot" immune-microenvironment and augments efficacy of immune checkpoint blockade against advanced tumors.

While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high-duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipilimumab In an in Vivo murine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10 to 100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-gamma in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (P < .001) and IFN-gamma levels (P = .001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy and provide a paradigm for conceptualizing the immune effects of laser thermal ablation

Induced or circumstantial immunosuppression present opportunities for cancer development, and progression. Recently, multiple therapies have been developed to harness the immune system's ability to selectively eradicate cancer cells. These approaches have primarily targeted features of the adaptive immune response such as checkpoint blockade and vaccination of tumorigenic peptides. Toll‐Like receptors (TLRs) are innate immune receptors that recognize molecules broadly shared by various pathogens. TLR pathways are increasingly targeted in cancer immunotherapy approaches seeking to overcome immune‐evasion by cancer cells. Here, we hypothesize that activation of TLR7, an endosomal receptor which recognizes pathogen associated nucleic acids, can mitigate tumor immunosuppression. TLR7 agonist (TLR7‐A) conjugated to silica nanoparticles will be used as an adjuvant in a mouse colon adenocarcinoma (CT26 cell line derived) in vivo tumor model. Transcriptional targets of adaptive immune cells (CD3, CD4, CD8, Granzyme B and Perforin), antigen presenting cells (MHCII, CD80, CD86 and CD40) and inflammatory cytokine response (TNFα, IL‐6, IP‐10, MCP1, MIP1α and IFNγ) were measured by qPCR in tumor tissue following 21 days of treatment. We found that treatment with TLR7‐A unconjugated and conjugated increased localized transcript levels of inflammatory cytokines and chemokines: IL‐6, IP‐10, MCP1, MIP1α and IFNγ. This project will provide valuable insights on the distinct transcriptional immune interplay in response to TLR7‐A treatment (localized vs systemic), support the decision‐making process of subsequent experimental approaches and evaluate TLR7‐A conjugate's potential for treatment.Support or Funding InformationThis research was supported by the National Cancer Institute of the National Institutes of Health under award numbers:U54CA132384 & U54CA132379This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.