Direct Intratumoral Injection Research Articles

Evaluation of Three Morphologically Distinct Virus-Like Particles as Nanocarriers for Convection-Enhanced Drug Delivery to Glioblastoma.

Glioblastoma is a particularly challenging cancer, as there are currently limited options for treatment. New delivery routes are being explored, including direct intratumoral injection via convection-enhanced delivery (CED). While promising, convection-enhanced delivery of traditional chemotherapeutics such as doxorubicin (DOX) has seen limited success. Several studies have demonstrated that attaching a drug to polymeric nanoscale materials can improve drug delivery efficacy via CED. We therefore set out to evaluate a panel of morphologically distinct protein nanoparticles for their potential as CED drug delivery vehicles for glioblastoma treatment. The panel consisted of three different virus-like particles (VLPs), MS2 spheres, tobacco mosaic virus (TMV) disks and nanophage filamentous rods modified with DOX. While all three VLPs displayed adequate drug delivery and cell uptake in vitro, increased survival rates were only observed for glioma-bearing mice that were treated via CED with TMV disks and MS2 spheres conjugated to doxorubicin, with TMV-treated mice showing the best response. Importantly, these improved survival rates were observed after only a single VLP–DOX CED injection several orders of magnitude smaller than traditional IV doses. Overall, this study underscores the potential of nanoscale chemotherapeutic CED using virus-like particles and illustrates the need for further studies into how the overall morphology of VLPs influences their drug delivery properties.

Theranostic niosomes for direct intratumoral injection: marked enhancement in tumor retention and anticancer efficacy.

For simultaneous bioimaging and drug delivery via direct intratumoral injection, doxorubicin and Ag2S quantum dots co-loaded multifunctional niosomes were prepared and fully characterized. Various theranostic niosomes were prepared and investigated regarding cytotoxicity, in vivo imaging, drug accumulation in breast cancer tumor and antitumor activity. Niosomes composed of Tween-60, Tween-80 or Span 60 produced strong and more durable detectable fluorescence signals. Despite a higher accumulation of Tween-60 niosomes in tumor, the Span 60 formulation showed the highest antitumor efficacy when compared with the free drug (71.7 and 20.3% inhibition in tumor growth, respectively). Direct intratumoral injection of theranostic niosomes with appropriate composition could be a powerful tool for combined multimodal imaging and therapy.

Targeted and Sustained Corelease of Chemotherapeutics and Gene by Injectable Supramolecular Hydrogel for Drug-Resistant Cancer Therapy.

Coadministration of chemotherapeutics as well as therapeutic gene could play a synergistic effect on cancer treatment. It is noteworthy that targeted and sustained codelivery of chemotherapeutic and therapeutic gene was rarely achieved in previous reports, while it might serve as an important platform for treating solid tumor with possible surrounding lesions. Herein, an injectable supramolecular hydrogel formed by α-cyclodextrin (α-CD) and cationic amphiphilic copolymer made of methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene imine) with folic acid targeted group (MPEG-PCL-PEI-FA), is rationally designed to achieve sustained codelivery of chemotherapeutic paclitaxel (PTX) and B-cell lymphoma-2 (Bcl-2) conversion gene Nur77 in the form of nanocomplex up to 7 days, to effectively inhibit the growth of folate receptor overexpressing H460/Bcl-2 therapeutic-resistant tumors (induced by overexpression of anti-apoptotic Bcl-2 protein), with peritumoral injection rather than direct intratumoral injection of hydrogel. To the best of our knowledge, this is a pioneer report on injectable MPEG-PCL-PEI-FA/α-CD supramolecular hydrogel with the ability to codeliver and sustainedly release PTX and Nur77gene to combat Bcl-2 overexpressed therapeutic-resistant tumors in a targeted manner, which might be beneficial for further design in personalized medicine.

Improved Targeting and Tumor Retention of a Newly Synthesized Antineoplaston A10 Derivative by Intratumoral Administration: Molecular Docking, Technetium 99m Radiolabeling, and In Vivo Biodistribution Studies.

Recently, the direct intratumoral (i.t.) injection of anticancer agents has been investigated. A newly synthesized Antineoplaston A10 analog 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) showed an antitumor activity in human breast cancer cell line. Unfortunately, MPD suffered from poor water solubility. Pseudoternary phase diagram of oil (isopropyl myristate), surfactant (Tween 80), cosurfactant (ethanol), and water was plotted. MPD microemulsion (MPDME) was developed and characterized for particle size (PS), polydispersity index (PDI), zeta potential (ZP), and morphology (transmission electron microscopy). MPDME and MPD solution (MPDS) were radiolabeled with technetium 99m (99mTc) using stannous chloride dihydrate (SnCl2.2H2O). Molecular docking of MPD and 99mTc-MPD was performed to study the interaction with DNA. The impacts of intravenous (i.v.) and i.t. injections of 99mTc-MPDME and 99mTc-MPDS on biodistribution were studied. The developed MPDME showed spherical droplets with mean PS (74.00 ± 5.69 nm), PDI (0.25 ± 0.03), and ZP (33.90 ± 0.90 mV). Labeling yield of 99mTc-MPDME and 99mTc-MPDS was 97.00% ± 0.60% and 92.02% ± 0.45%, respectively. MPD and 99mTc-MPD showed almost same binding affinity with DNA binding site. Biodistribution results showed that i.t. injection of 99mTc-MPDME significantly enhanced tumor retention compared to i.v. route. Herein, the authors concluded that microemulsion could be used as i.t. injectable delivery vehicle to improve targeting and tumor retention of MPD.

Optimal method of gold nanoparticle administration in melanoma-bearing mice.

The present study assessed different methods of administering gold nanoparticles (GNPs) using different formulations to determine which of the methods achieved optimal radiosensitization. Cells from the B16F10 mouse melanoma cell line were implanted in the femoral area of mice, assigned to one of the eight following groups: i) Control; ii) intravenous (IV) injection of polyethylene glycol (PEG)-binding GNPs (Peg-GNPs) alone; iii) direct intratumoral (IT) injection of Peg-GNPs alone; iv) radiotherapy (RT)-alone; v) Peg-GNP IV + RT; vi) Peg-GNP IT + RT; vii) naked GNP (N-GNPs) IV + RT; and viii) N-GNP IT + RT. Injection volumes of the Peg-GNPs (particle size, 15 nm; dose, 2.8 mg/ml) and N-GNPs (particle size, 15 nm; dose, 200 mg Au/cc) were 0.3 and 0.2 ml per mouse, respectively, for IV and IT. The femoral area was irradiated with a single dose of 10 Gy. To evaluate the effects of GNPs, the current study measured the changes in the tumor volume ratio to the initial tumor volume over time and observed the survival rate. Administration of GNPs with RT did not improve the suppression of tumor growth or survival to a statistically significant extent. The administration of Peg-GNPs alone indicated a slight tumor suppressing effect at the early stage. The current study was not able to confirm the radiosensitization effect of GNPs in melanoma-bearing mice with tumors that were large in comparison to previous studies. Further research is required to validate the radiosensitizing effect on large tumors.

Direct injection of (188)Re-microspheres in the treatment of hepatocellular carcinoma: compared with traditional percutaneous ethanol injection: an animal study.

The AIM of this study was to compare the therapeutic efficacies of direct intratumoural injection of (188)Re microspheres (DIRM) and direct intratumoural injection of ethanol (DIE) in rabbits bearing liver tumours. Fifteen rabbits bearing liver tumours were divided into three groups: group 1 received DIE, group 2 received DIRM, and group 3 (control) received saline. Tumour size was measured by liver sonography before injection, as well as 2, 4, 8, and 12 weeks after injection. Survival time was calculated from the day of treatment to three months after treatment by Kaplan-Meier survival analysis. The mean survival time was 68 +/- 9.8 days for the rabbits in the DIRM group, 55.8 +/- 11.8 days for the DIE group, and 38.8 +/- 6.2 days for the control group. The rabbits survived longer in the DIRM group than in the DIE group although there is no statistical significance. We believe the DIRM method has a good potential to be an alternative to DIE for the treatment of liver tumours.

Pharmacokinetics and safety of paclitaxel delivery into porcine airway walls by a new endobronchial drug delivery catheter.

Intratumoral administration of chemotherapeutic agents is a treatment modality that has proven efficacious in reducing the recurrence of tumours and increases specificity of treatment while minimizing systemic side effects. Direct intratumoral injection of malignant airway obstruction has potential therapeutic benefits but tissue drug concentrations and side-effect profiles are poorly understood. Bronchial wall injection of generic paclitaxel (PTX) (102 injections of 0.05, 0.5, 1.5 or 2.5 mg/mL in 10 healthy pigs), saline (14 injections in 2 healthy pigs) or Abraxane (ABX) (24 injections of 0.5 mg/mL in 4 healthy pigs) was performed with a microneedle infusion catheter. Local histopathology, plasma and tissue PTX concentrations were evaluated at 7, 20 or 28 days post-injection. Injection of generic PTX directly into the bronchial wall at doses up to 1.5 mg/mL only caused minimal tissue injury. Dose-limiting tissue reaction was observed at 2.5 mg/mL. Plasma PTX was detectable for up to 5 days but not at 28 days, with area under the curve (AUC)(0-5d) 20- to 50-fold lower than the AUC(0-∞) of 6300 ng h/mL for the approved intravenous dose. At 7 and 28 days post-injection, bronchial PTX tissue concentrations were above a 10-nmol/L cancer therapeutic level. PTX was not found in peripheral tissues. Similar results were observed between ABX and generic PTX. Results of these studies confirm the administration of PTX directly into the bronchial wall is safe and feasible. PTX was detectable in plasma for <7 days but tissue concentrations remained therapeutic throughout the follow-up period.

Oncolytic Viral Therapy for Mesothelioma.

The limited effectiveness of conventional therapy for malignant pleural mesothelioma demands innovative approaches to this difficult disease. Even with aggressive multimodality treatment of surgery, radiation, and/or chemotherapy, the median survival is only 1–2 years depending on stage and histology. Oncolytic viral therapy has emerged in the last several decades as a rapidly advancing field of immunotherapy studied in a wide spectrum of malignancies. Mesothelioma makes an ideal candidate for studying oncolysis given the frequently localized pattern of growth and pleural location providing access to direct intratumoral injection of virus. Therefore, despite being a relatively uncommon disease, the multitude of viral studies for mesothelioma can provide insight for applying such therapy to other malignancies. This article will begin with a review of the general principles of oncolytic therapy focusing on antitumor efficacy, tumor selectivity, and immune system activation. The second half of this review will detail results of preclinical models and human studies for oncolytic virotherapy in mesothelioma.

Pre-operative embolization of hypervascular spinal metastasis using percutaneous direct intra-tumoural injection with Onyx under local anesthesia

Intra-operative blood loss remains a major cause of perioperative morbidity for patients with hypervascular spinal metastasis undergoing surgery. Pre-operative embolization is used to reduce intraoperative blood loss and operative time. This is commonly performed under general anesthesia via a trans-arterial approach, which carries a risk of spinal stroke. We propose an alternative technique for embolization of hypervascular metastases using the Onyx embolic agent via a percutaneous direct intra-tumoural injection under local anesthesia and sedation to reduce embolization risks and procedure time, as well as operative blood loss and operative time. A 74-year-old man presented with thoracic myelopathy with back and radicular pain on background of metastatic renal cell carcinoma. Magnetic resonance imaging (MRI) revealed a 3cm mass centered on the right lamina of T10 with extension into the spinal canal. The patient underwent a percutaneous imaging-guided direct intra-tumoural contrast parenchymogram, and Onyx embolization via a single needle. Initial needle placement and tumour assessment was completed in 30min; embolization time was 15min. Complete devascularization was achieved with no complications. Surgical resection was performed with lower than expected operative blood loss (150ml) and operative time (90min). His pre-operative symptoms improved, and he was discharged home the following day. At 6-month follow-up there was no recurrence of his symptoms. Further evaluation of direct percutaneous intra-tumoural Onyx embolization for hypervascular spinal tumours is warranted.

Abstract 1609: Oncolytic virotherapy for SCLC using immunocompetent mouse models

Abstract Background: Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer with few treatment advances over the past 3 decades and poor survival. Locally targeted oncolytic virotherapy employs a viral vector that has selective cytotoxicity for tumor cells and non-toxic for normal cells and tissues. The therapeutic benefit is proposed to arise from an initial viral cytotoxic phase following by host immune responses. Myxoma Virus (MYXV) has been widely tested in Australia to control rabbit populations with no toxicity to humans. MYXV selectively infects mouse and human tumors cells with no cytotoxicity to normal tissues and is a promising oncolytic virotherapy agent that has not been tested in human lung cancer or in clinical trials. Methods: To study MYXV infection and viral replication in vitro, we utilized human and mouse SCLC cell lines with MYXV engineered with fluorescent reporters. Using an optimized conditional genetically engineered mouse model (GEMM) (Ade-CRE mediated p53/Rb1/p130 null) we examined the effects of intrapulmonary MYXV treatment on SCLC tumors at 5 and 30 days post-MYXV treatment. Survival analysis was determined following intranasal MYXV 3 months post-Ade-CRE induction. We also tested intratumoral MYXV in patient derived xenografts (PDX) and subcutaneous syngeneic allografts in immunocompetent mice. Results: We optimized a conditional SCLC GEMM using limiting dilutions of intratracheal Ade-CRE to reduce the SCLC foci to simulate human disease and generated mouse SCLC lines from individual clones. We observed efficient MYXV infection, late viral replication, and cytotoxicity in both human and mouse SCLC in vitro. In contrast, we did not detect productive infection nor cytotoxicity in non-tumor cells. Following intranasal MYXV instillation we observed MYXV localized exclusively within lungs at 3 days and no longer detected by 7 days’ post treatment. TUNEL staining of SCLC lesions showed apoptosis and necrosis at 5 days within SCLC and the effect persisted with discrete foci of tumor necrosis 30 days’ post-treatment. There was no toxicity to any mouse tissues. SCLC GEMM treated with intrapulmonary MYXV (n=30) showed a modest but statistically significant prolongation of survival compared to PBS control mice (n=30) (p &amp;lt; 0.05). Direct intratumoral MYXV injections performed on PDX tumors in immunodeficient mice showed efficient infection and late viral replication in all patient samples. We observed extensive tumor necrosis at 7 days, and persisting virus at 10 days’ post treatment. Direct intratumoral MYXV injections performed on allograft tumors in syngeneic immunocompetent mice showed extensive necrosis accompanied by immune cell infiltration into the tumor, and the virus was undetectable 7 days’ post treatment. Conclusions: We show the potential for MYXV as an oncolytic virotherapy for SCLC with enhanced cytotoxicity in immunocompetent mice suggesting a role for concurrent immune checkpoint therapies. Citation Format: Patrick Kellish, Daniil Shabashvili, Masmudur M. Rahman, Mary Reinhard, Grant McFadden, Frederic Kaye, Maria Zajac-Kaye. Oncolytic virotherapy for SCLC using immunocompetent mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2017-1609

Near infrared-assisted Fenton reaction for tumor-specific and mitochondrial DNA-targeted photochemotherapy.

The strong dependence on oxygen level, low ultraviolet/visible (UV/vis) light penetration depth and the extremely short lifetime of reactive oxygen species (ROS) are the major challenges of photodynamic therapy (PDT) for tumors. Fenton reaction can produce abundant ROS such as reactive hydroxyl radicals (OH) with significantly higher oxidation performance than singlet oxygen (1O2), which, however, has been rarely used in biomedical fields due to strict reaction conditions (favorably in pH range of 3-4, mostly under UV/vis light catalysis). Herein we propose and demonstrate a photochemotherapy (PCT) strategy of cancer therapy using near-infrared (NIR)-assisted tumor-specific Fenton reactions. NIR light-upconverted UV/vis light by upconversion nanoparticles (UCNPs) catalyze the intra-mitochondrial Fenton reaction between the delivered Fe2+ and H2O2 species over-expressed in cancer cell's mitochondria to in-situ kill the cancer cells. The intra-mitochondrial ROS generation of enabled by directly targeting the mitochondrial DNA (mtDNA) helix minimized the distance between the ROS and mtDNA molecules, thus the present PCT strategy showed much enhanced and tumor-specific therapeutic efficacy, as demonstrated by the intratumoral-accelerated OH burst and elevated cytotoxicity. Following the direct intratumoral injection, the PCT revealed marked tumor regression effect invivo. This constructed PCT-agent is the first paradigm of NIR-upconversion catalyzed intra-mitochondrial Fenton reaction in response to tumoral microenvironment, establishing a novel photochemotherapy strategy for efficient cancer therapy.

A phase 1 dose-escalation trial of intratumoral TTI-621, a novel immune checkpoint inhibitor targeting CD47, in subjects with relapsed or refractory percutaneously-accessible solid tumors and mycosis fungoides.

TPS3101 Background: CD47 is an immune checkpoint that binds to signal regulatory protein alpha (SIRPα) and delivers a "do not eat" signal to suppress macrophage phagocytosis. Tumor cells frequently overexpress CD47 and exploit this pathway to evade macrophage-mediated destruction. CD47 blockade promotes both innate (macrophage phagocytosis) and adaptive immunity (T cell responses). TTI-621 (SIRPαFc) is an immune checkpoint inhibitor designed to bind human CD47 and block the “do not eat” signal. The IgG1 region of TTI-621 engages Fcγ receptors on macrophages, converting the inhibitory signal to one that activates, thereby enhancing phagocytosis, and antitumor activity. A Phase 1 study is ongoing to evaluate the safety/tolerability and preliminary efficacy of IV administered TTI-621 in subjects with relapsed/refractory hematologic malignancies. It is hypothesized that employing direct intratumoral injections will result in very high local target engagement, promoting the development of innate and adaptive immune responses. Methods: A Phase 1 multicenter, open-label study was initiated to characterize the safety and tolerability of delivering TTI-621 directly into cancer lesions to achieve high local CD47 engagement to increase phagocytosis of tumor cells (NCT02890368). Subjects are being enrolled in sequential cohorts that gradually increase in dose and dosing frequency to characterize the feasibility of intratumoral TTI-621 injections and their safety, PK, pharmacodynamics, and preliminary antitumor activity. Eligible subjects are adults with relapsed or refractory percutaneously-accessible solid tumors or mycosis fungoides (MF), which have progressed on standard anticancer therapy or for which no other approved therapy exists. TTI-621 is delivered by intratumoral injection at protocol-defined doses and dosing regimens starting at 1 mg/injection. Serial biopsies are being collected to characterize local anti-tumor responses and assess the impact of TTI-621 on the tumor microenvironment. Clinical trial information: NCT02890368.

Intratumoral injection of radioactive holmium ( 166 Ho) microspheres for treatment of oral squamous cell carcinoma in cats.

A "microbrachytherapy" was developed as treatment option for inoperable tumours by direct intratumoral injection of radioactive holmium-166 ( 166 Ho) microspheres (MS). 166 Ho emits β-radiation which potentially enables a high, ablative, radioactive-absorbed dose on the tumour tissue while sparing surrounding tissues. Safety and efficacy of 166 Ho microbrachytherapy were evaluated in a prospective cohort study of 13 cats with inoperable oral squamous cell carcinoma without evidence of distant metastasis. Local response rate was 55%, including complete response or partial response (downstaging) enabling subsequent marginal resection. Median survival time was 113 days overall, and 296 days for patients with local response. Side effects were minimal. Tumour volume was a significant predictor of response. Response rate may be further improved by optimizing the intratumoral spatial distribution of 166 Ho MS. 166 Ho microbrachytherapy has potential as a minimally invasive, single procedure radio-ablation treatment of unresectable tumours with minimal morbidity.

Heterogeneous intratumoral distribution of gadolinium nanoparticles within U87 human glioblastoma xenografts unveiled by micro-PIXE imaging

Metallic nanoparticles have great potential in cancer radiotherapy as theranostic drugs since, they serve simultaneously as contrast agents for medical imaging and as radio-therapy sensitizers. As with other anticancer drugs, intratumoral diffusion is one of the main limiting factors for therapeutic efficiency. To date, a few reports have investigated the intratumoral distribution of metallic nanoparticles. The aim of this study was to determine the quantitative distribution of gadolinium (Gd) nanoparticles after direct intratumoral injection within U87 human glioblastoma tumors grafted in mice, using micro-PIXE (Particle Induced X-ray Emission) imaging. AGuIX (Activation and Guiding of Irradiation by X-ray) 3 nm particles composed of a polysiloxane network surrounded by gadolinium chelates were used. PIXE results indicate that the direct injection of Gd nanoparticles in tumors results in their heterogeneous diffusion, probably related to variations in tumor density. All tumor regions contain Gd, but with markedly different concentrations, with a more than 250-fold difference. Also Gd can diffuse to the healthy adjacent tissue. This study highlights the usefulness of mapping the distribution of metallic nanoparticles at the intratumoral level, and proposes PIXE as an imaging modality to probe the quantitative distribution of metallic nanoparticles in tumors from experimental animal models with micrometer resolution.

Effective Combination of Innate and Adaptive Immunotherapeutic Approaches in a Mouse Melanoma Model.

Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti-CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2+ B78 melanoma model, we show that anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Anti-CD40/CpG + IC/anti-CTLA-4 synergistically induced regression of advanced s.c. tumors, resulting in cure of some mice and development of immunological memory against B78 and wild type B16 tumors. Although the antitumor effect of anti-CD40/CpG did not require T cells, the antitumor effect of IC/anti-CTLA-4 was dependent on T cells. The combined treatment with anti-CD40/CpG + IC/anti-CTLA-4 reduced T regulatory cells in the tumors and was effective against distant solid tumors and lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be developed for clinical testing as a potentially effective novel immunotherapy strategy.

Plasmonic Photothermal Therapy of Transplanted Tumors in Rats at Multiple Intravenous Injection of Gold Nanorods

The aim of this study was to evaluate the morphological changes in tumor tissue in rats with transplanted liver cancer PC-1 after repeated intravenous (IV) administration of gold nanorods (GNRs) and plasmonic photothermal therapy (PPT). GNRs with aspect ratio 4.1 and plasmonic peak at 810 nm were functionalized with thiolated polyethylene glycol and IV administered at a single dose 0.4 mg of Au and by repeated injection of the same dose for 2 and 3 days (the total doses were 0.8 and 1.2 mg of Au, respectively). One day after the last IV injection of GNRs, the tumors were irradiated by an 808-nm NIR diode laser at a power density 2.3 W/cm2 during 15 min. The withdrawal of the animals from the experiment and sampling of the tissues for morphological study and GNR distribution were performed 24 h after the PPT. Repeated IV administration of GNRs in tumor-bearing rats resulted in the highest accumulation of nanoparticles in both liver and spleen tissues. After triplicate IV injection of GNRs, they accumulated in the tumor and related PPT effects were comparable with those observed after direct intratumoral injection of the single GNR dose.

Abstract 3549: The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer

Abstract Introduction. Oncolytic viruses preferentially replicate within and kill cancer cells and are a promising novel cancer treatment. However, the mechanisms by which oncolytic adenoviruses induce death in cancer remains unclear. It was long thought that DNA viruses trigger classical apoptosis but there is now evidence that cell death induced by both adenovirus and vaccinia displays features of necrosis-like programmed cell death. Methods and Results. In order to investigate the role of necrosis in cell death following oncolytic adenovirus infection, a panel of ovarian cancer cells with varying sensitivities was infected with the E1A CR2-deleted adenoviral mutant dl922-947. By electron microscopy, dl922-947 infection induces key morphological features of necrotic death, including membrane rupture, nuclear swelling and cytoplasmic vacuolation. Using specific necrosis inhibitors (necrostatin-1, necrosulfonamide, GSK’840B, GSK’872B and GSK’843A) as well as RNAi-mediated knockdown of receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), we show that adenovirus-infected ovarian cancer cells undergo RIPK3-dependent necrosis and that blockage of the downstream effector MLKL significantly attenuates cell death. Co-immunoprecipitation of caspase 8 shows formation of a complex containing RIPK1, FADD and caspase 8 during adenovirus infection. However, unlike Tumour Necrosis Factor-α (TNF-α)-induced programmed necrosis, which relies on the RHIM-dependent interaction of RIPK1 and RIPK3, RIPK1 appears redundant in adenovirus-induced death. Furthermore, the addition of a TNF-α blocking antibody to virus-infected cells has no effect on either cell death or overall cell survival. In a RIPK3 overexpression model, we show a direct correlation between adenovirus-induced cell death and the extent of RIPK3 expression. Moreover, RIPK3 expression has no effect on infectivity, viral protein expression or infectious virus production. Finally, expression of RIPK3 increases necrosis induction in vivo following direct intra-tumoural dl922-947 injection, and significantly improves anti-tumour efficacy. Conclusions. Our data suggest that cell death induced by oncolytic adenoviruses differs from TNF-induced programmed necrosis but still relies on the kinase RIPK3 and its downstream component MLKL, making these two proteins possible targets for future oncolytic virotherapies. Citation Format: Melanie Weigert, Alexander Binks, Stephen Tait, Iain McNeish. The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3549.

Abstract PR12: Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation

Abstract Introduction: Glioblastoma (GBM) is an aggressive and highly malignant form of brain cancer with a devastatingly poor clinical prognosis. Despite years of research, there is a surprising lack of effective therapies and novel approaches for treatment. There exists a great, unmet need for strategies enabling efficient and targeted modulation of gene expression in established tumors. RNAi-mediated gene silencing is a promising strategy for straightforward modulation of gene expression, and siRNA-based therapies could result in more effective and less toxic treatment for GBM. The Khvorova laboratory has developed a fully chemically stabilized, self-delivering siRNA platform supporting efficient intratumoral uptake and target gene silencing in a GBM orthotopic mouse model. Methods: For all in vitro and in vivo studies, a patient-derived GBM8 cell line was used. Uptake in GBM8 cells was done by treating cells with 0.75 µM of fully chemically stabilized, Cy3-conjugated, hydrophobically-modified siRNA (hsiRNA) targeting the huntingtin mRNA (Cy3-FM-hsiRNAHTT). At different time points (0 to 5.5 hours) GBM8 cells were harvested by cytospin, fixed in 4% formaldehyde, and then imaged by confocal microscopy (40X oil). Next, levels of mRNA knockdown in GBM8 cells were determined by plating 50,000 cells/well in a 96-well plate and treating with increasing doses of FM-hsiRNAHTT (0.01 to 3 µM). After 72 hours, cells were lysed and mRNA was quantified using QuantiGene (Affymetrix) assay. In vivo distribution was done by unilaterally injecting Cy3-FM-hsiRNAHTT into the striatum (2 nmol) or the ventricle of nude mice (5 nmol), 4 weeks after GBM8 tumor implantation. 24 hours after injection, mice were sacrificed, perfused with 4% formaldehyde, and brains were sectioned into 4 µm sections. Knockout in nude mice following GBM8 tumor implantation was also conducted. Mice were treated with FM-hsiRNAHTT (5 nmoles, n=5), a non-targeting control hsiRNA (NTC, 5 nmoles, n=5), or vehicle (5 µL, n=5) and sacrificed after 5 days. 3 biopsies per animal were collected from the striatum of the tumor-bearing side. The levels of huntingtin mRNA expression were determined by QuantiGene® and normalized to a housekeeping gene and reported as percent of CSF. Results: We observed significant uptake in GBM8 cells treated with 0.75 µM Cy3-FM-hsiRNAHTT starting at 2 hours. In addition, huntingtin mRNA was reduced by 50% at 0.3 µM and was reduced by 80% when cells were treated with 1 µM. Following a direct intratumoral injection of Cy3-FM-hsiRNAHTT, we saw widespread distribution of Cy3-fluorescent co-localizing with the tumor. Intratumoral administration of 5 nmoles of FM-hsiRNAHTT resulted in 50% target silencing and was statistically significant when compared to the CSF or NTC treated groups. However, a single injection into the ventricle only resulted in 25% silencing and was only statistically significant relative to a non-targeting hsiRNA control. Summary/Conclusion: Here, we demonstrate that a single intratumoral injection of hydrophobically modified, metabolically stable siRNA (hsiRNA) results in robust and long-lasting silencing of targets in established tumors. Due to the high potency, long duration of effect, and predictable pharmacokinetics of hsiRNA, it is possible that medicine based on this technology can be tailored to a highly diverse patient population. As is, it is believed to be an ideal drug platform for treatment of cancer, as long as efficient delivery to the tumors and metastasis is achieved. Thus, this technology allows immediate modulation of expression in orthotropic tumor models and may contribute to our understating of tumor biology. In addition, this approach can, in the future, serve as a foundation for new therapeutic interventions for this devastating disease. This abstract is also presented as Poster B44. Citation Format: Andrew H. Coles, Maire F. Osborn, Diane Golebowski, Matthew Hassler, Miguel S. Esteves, Anastasia Khvorova. Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR12.

Abstract B35: Enhanced antitumor efficacy by combining oncolytic herpes simplex virus and Aurora A kinase inhibition in models of neuroblastoma and malignant peripheral nerve sheath tumor

Abstract We previously reported xenograft models of neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) are responsive to direct intratumoral injection of oncolytic Herpes Simplex Viruses (Parikh et al., Ped Blood Can 44:469, 2005; Mahller et al., Ped Blood Can 46:745, 2006; Mahller et al., Can Res 68:1170, 2008). We have confirmed these findings using an oncolytic HSV, Seprehvir (HSV1716), which is in clinical trials that include pediatric patients (NCT00931931, NCT02031965). The models showed wide variability, however, with some models having maintained complete responses and others showing only delayed growth. Because DNA viruses are highly dependent on normal nuclear machinery for DNA synthesis/replication and virus packaging/assembly, we postulated that increased transit time in mitosis may be beneficial to an oncolytic DNA virus. Aurora A Kinase is an enzyme critical for formation of centrioles and mitotic spindles during mitosis and is often dysregulated in cancer. The selective AAK inhibitor, Alisertib (MLN8237), arrests cells in G2/M and has significant activity in models of neuroblastoma (data from the Pediatric Preclinical Testing Program) and in MPNST (data from our prior study: Patel et al., Clin Can Res 18:5020, 2012). We found tumor responses in xenograft models using the combination of Seprehvir and Alisertib was more than additive. Immunohistochemical staining for HSV antigens revealed a marked difference in the extent and spread of virus as early as 24 hrs after virus injection. Our preliminary data assessing kinetics of virus infection suggest that Alisertib pretreatment enhances virus uptake and spread, accounting for the combinatorial effects. These data strongly suggest alisertib pretreatment “primes” tumor cells, making them highly susceptible to virus infection and spread and greatly accelerates the virolytic effects. Overall, our findings support clinical testing of Alisertib combined with Seprehvir in patients with refractory neurogenic tumors such as neuroblastoma and MPNST. Citation Format: Mark Currier, Tilat Rizvi, Brooke Nartker, Duo Chen, Chun-Yu Chen, Pin-Yi Wang, Les Sprague, Meghan Franczek, Ami Patel, Katherine Chaney, Keri Streby, Jeff Ecsedy, Joe Conner, Nancy Ratner, Timothy Cripe. Enhanced antitumor efficacy by combining oncolytic herpes simplex virus and Aurora A kinase inhibition in models of neuroblastoma and malignant peripheral nerve sheath tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B35.

Preparation of a paclitaxel-loaded cationic nanoemulsome and its biodistribution via direct intratumoral injection

In this study, a nano-preparation based on nanoemulsome (NES) modified with cetyltrimethylammonium bromide (CTAB) loading paclitaxel (PTX) was designed, and its biodistribution were explored after intratumoral (i.t.) administration on Heps tumor-bearing mice. The PTX-loaded nanoemulsome (PTX-NES) was prepared by using a solvent evaporation method and CTAB, chosen as a cationic material, was absorbed onto the surface of the NES via electrostatic interaction to yield paclitaxel-loaded cationic nanoemulsome (PTX-CTAB-NES). The MTT results exhibited that PTX-CTAB-NES (IC50: 0.50±0.035μg/mL in MCF-7 cells and 0.13±0.048μg/mL in SMMC-7721 cells) had the strongest cytotoxicity compared to Taxol (IC50: 0.88±0.054μg/mL in MCF-7 and 0.15±0.011μg/mL in SMMC-7721) and PTX-NES (IC50: 1.93±0.062μg/mL in MCF-7 and 0.32±0.027μg/mL in SMMC-7721). Body distribution of PTX revealed that the percent of PTX retained in the tumor after i.t. administration of PTX-CTAB-NES (approximately 92.99% at 0.167h and 15.35% at 48h) was higher when compared to that after i.t. injection of Taxol (approximately 58.94% at 0.167h and 0.83% at 48h) or PTX-NES (approximately 83.63% at 0.167h and 6.52% at 48h). Moreover, less PTX accumulated in liver, spleen, kidney, lung and heart after i.t. administration of PTX-CTAB-NES when compared with that after i.v. administration of PTX-CTAB-NES. In conclusion, PTX-CTAB-NES was a prospective in-situ delivery system for the therapy of tumor.