Subcutaneous Melanoma Research Articles

Prime-boost using separate oncolytic viruses in combination with checkpoint blockade improves anti-tumour therapy

The anti-tumor effects associated with oncolytic virus therapy are mediated significantly through immune-mediated mechanisms which depends both on the type of virus and the route of delivery. Here, we show that intra-tumoral (i.t.) oncolysis by Reovirus induced the priming of a CD8+, Th1-type anti-tumor response. In contrast, systemically delivered VSV expressing a cDNA library of melanoma antigens (VSV-ASMEL) promoted a potent anti-tumor CD4+ Th17 response. Therefore, we hypothesised that combining the Reovirus-induced CD8+ T cell response, with the VSV-ASMEL CD4+ Th17 helper response, would produce enhanced anti-tumor activity. Consistent with this, priming with i.t. Reovirus, followed by an intra-venous VSV-ASMEL Th17 boost, significantly improved survival of mice bearing established subcutaneous (s.c.) B16 melanoma tumors. We also show that combination of either therapy alone with anti-PD-1 immune checkpoint blockade augmented both the Th1 response induced by systemically delivered Reovirus in combination with GM-CSF, and also the Th17 response induced by VSV-ASMEL. Significantly, anti-PD-1 also uncovered an anti-tumor Th1 response following VSV-ASMEL treatment that was not seen in the absence of checkpoint blockade. Finally, the combination of all three treatments (priming with systemically delivered Reovirus, followed by double boosting with systemic VSV-ASMEL and anti-PD-1) significantly enhanced survival, with long-term cures, compared to any individual, or double, combination therapies, associated with strong Th1 and Th17 responses to tumor antigens. Our data show that it is possible to generate fully systemic, highly effective anti-tumor immunovirotherapy by combining oncolytic viruses, along with immune checkpoint blockade, to induce complimentary mechanisms of anti-tumor immune responses.

Effect of the autonomic nervous system on cancer progression depends on the type of tumor: solid are more affected then ascitic tumors.

A number of recently published studies have shown that the sympathetic nervous system may influence cancer progression. There are, however, some ambiguities about the role of the parasympathetic nerves in the modulation of growth of different tumor types. Moreover, tumor models used for investigation of the autonomic neurotransmission role in the processes related to the cancer growth and progression are mainly of the solid nature. The knowledge about the nervous system involvement in the modulation of the development and progression of malignant ascites is only fragmental. Therefore, the aim of the present article was to summarize the results of our experimental studies focused on the elucidation of the role of the autonomic nervous system in the modulation of tumor growth in animals. We are summarizing data from studies, in which not only different experimental approaches in order to influence the autonomic neurotransmission, but also different tumor models have been used. Three different types of tumor models, namely solid rat intra-abdominal fibrosarcoma, solid murine subcutaneous melanoma, and rat ascites hepatoma, and three types of interventions have been used in order to modulate the autonomic neurotransmission, specifically chemical sympathectomy, subdiaphragmatic vagotomy, or the electric stimulation of the vagus nerve. We have proved a strong stimulatory effect of the sympathetic nerves on the development and growth in both solid tumors, rat fibrosarcoma as well as murine melanoma, and significant inhibitory impact on the survival time of tumor-bearing animals. The progression of ascites hepatoma in rats was not influenced by chemical sympathectomy. Modulation of parasympathetic signalization by vagotomy or vagal nerve stimulation does not affect fibrosarcoma and ascites hepatoma growth and survival of the tumor-bearing rats. Based on the obtained data, it seems that the solid types of tumors are suitable substrate for the direct action of neurotransmitters released especially from the sympathetic nerves. In contrast, it appears that the malignant ascites are not under the direct autonomic nerves control; however, an indirect action via the immune functions modulation cannot be excluded.

Thrombomodulin promotes focal adhesion kinase activation and contributes to angiogenesis by binding to fibronectin.

Angiogenesis promotes tumor growth and metastasis. Cell adhesion molecules interact with the extracellular matrix (ECM) and increase cell adhesion and migration during angiogenesis. Thrombomodulin (TM) is a cell surface transmembrane glycoprotein expressed in endothelial cells. However, the function and significance of TM in cell-matrix interactions and angiogenesis remain unclear. Here, we first demonstrated that recombinant lectin-like domain of TM interacts with an ECM protein, fibronectin, and identified the N-terminal 70-kDa domain of fibronectin as the TM-binding site. Exogenous expression of TM in TM-deficient A2058 melanoma cells enhanced cell adhesion and migration on fibronectin and invasion on Matrigel. In addition, TM increased focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase-9 production. In mice bearing subcutaneous B16F10 melanoma tumors, immunofluorescence analysis indicated that TM was highly expressed and co-localized with fibronectin on the tumor vasculature. The interaction between TM and fibronectin in tumor blood vessels was also validated by the proximity ligation assay. In human umbilical vein endothelial cells, up-regulation of TM by vascular endothelial growth factor (VEGF), a tumor angiogenic factor, promoted cell adhesion and tube formation, whereas TM knockdown by RNA interference attenuated VEGF-induced cell adhesion and tube formation. In summary, TM promotes angiogenesis by enhancing cell adhesion, migration, and FAK activation through interaction with fibronectin. TM may represent a novel target for inhibiting tumor angiogenesis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Virotherapy: cancer gene therapy at last?

For decades, effective cancer gene therapy has been a tantalising prospect; for a therapeutic modality potentially able to elicit highly effective and selective responses, definitive efficacy outcomes have often seemed out of reach. However, steady progress in vector development and accumulated experience from previous clinical studies has finally led the field to its first licensed therapy. Following a pivotal phase III trial, Imlygic (talimogene laherparepvec/T-Vec) received US approval as a treatment for cutaneous and subcutaneous melanoma in October 2015, followed several weeks later by its European authorisation. These represent the first approvals for an oncolytic virotherapy. Imlygic is an advanced-generation herpesvirus-based vector optimised for oncolytic and immunomodulatory activities. Many other oncolytic agents currently remain in development, providing hope that current success will be followed by other diverse vectors that may ultimately come to constitute a new class of clinical anti-cancer agents. In this review, we discuss some of the key oncolytic viral agents developed in the adenovirus and herpesvirus classes, and the prospects for further enhancing their efficacy by combining them with novel immunotherapeutic approaches.

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

The immunomodulatory, antitumor and antimetastatic responses of melanoma-bearing normal and alcoholic mice to sunitinib and ALT-803: a combinatorial treatment approach.

ALT-803, a novel IL-15/IL-15 receptor alpha complex, and the tyrosine kinase inhibitor, sunitinib, were examined for their single and combined effects on the growth of subcutaneous B16BL6 melanoma and on lymph node and lung metastasis. The study was conducted in immunocompetent C57BL/6 mice drinking water (Water mice) and in mice that chronically consumed alcohol (Alcohol mice), which are deficient in CD8(+) T cells. Sunitinib inhibited melanoma growth and was more effective in Alcohol mice. ALT-803 did not alter tumor growth or survival in Water or Alcohol mice. Combined ALT-803 and sunitinib inhibited melanoma growth and increased survival, and these effects were greater than sunitinib alone in Water mice. ALT-803 and alcohol independently suppressed lymph node and lung metastasis, whereas sunitinib alone or in combination with ALT-803 increased lymph node and lung metastasis in Water and Alcohol mice. Initially, ALT-803 increased IFN-γ-producing CD8(+)CD44(hi) memory T cells and CD8(+)CD44(hi)CD62L(lo) effector memory T cells and sunitinib decreased immunosuppressive MDSC and T regulatory cells (Treg). However, the impact of these treatments diminished with time. Subcutaneous tumors from Water mice showed increased numbers of CD8(+) T cells, CD8(+)CD44(hi) T cells, NK cells, and MDSC cells and decreased Treg cells after ALT-803 treatment.

Abstract CT021: Phase Ib study of a novel immunotherapy combination therapy of intralesional coxsackievirus A21 and systemic ipilimumab in patients with advanced melanoma

Abstract Background: CVA21 (CAVATAK) is a novel bio-selected oncolytic and immunotherapeutic strain of Coxsackievirus A21. Intratumoral (i.t.) CVA21 injection can induce preferential tumor cell infection, tumor immune-cell infiltration, up-regulation of γ-INF response genes, cell lysis and enhancement of a systemic anti-tumor immune response. Pre-clinical studies in an immune-competent mouse model of melanoma have revealed that combinations of intralesional CVA21 and anti-CTLA-4 or anti-PD-1 mAbs mediated significantly greater antitumor activity and compared to use of either agent alone. Presented are the interim data of the open-label, multicenter Phase Ib MITCI (Melanoma Intra-Tumoral Cavatak and Ipilimumab) study of novel immunotherapy combination Coxsackievirus A21 and ipilimumab in patients with advanced melanoma Methods: The Phase Ib MITCI study (NCT02307149) is investigating the efficacy and safety of i.t. CVA21 and i.v. ipilimumab in 26 patients with treated or untreated unresectable Stage IIIC-IVM1c melanoma. Pts received up to 3 × 10e8 TCID50 CVA21 i.t. on study days 1, 3, 5, 8 and 22, and then q3w for a further 6 series of injections. Ipilimumab (3 mg/kg) q3w was given as 4 i.v. infusions starting at Day 22. Treatment is continued until DLT, intolerance, all injectable tumors disappeared, or confirmed disease progression (PD) per immune-related (ir) WHO response criteria. The first response assessment occurs at study Day 106. Key eligibility criteria were ? 18 yrs old, ECOG PS 0-1, and at least 1 injectable cutaneous, subcutaneous, or nodal melanoma metastasis ? 1.0 cm. The primary endpoint was to assess safety of CAVATAK in combination with scheduled ipilimumab treatment. Secondary endpoints included irWHO objective response rate (ORR), responses in injected and non-injected lesions, durable response rate (continuous response ? 6 mos), median time to initial response, survival and QOL. Results: At present, of the 7 patients enrolled, no DLT's have been reported. Combination treatment has been generally well-tolerated with only 2 Gr 3 AEs reported, one with ipilimumab-related fatigue and the other with unrelated hypertension. Of 5 pts with investigator assessed response, ORR was 80%, with all responses occurring by 3.5 months and in both injected and non-injected lesions. Responses were observed in patients with Stage IIIC and Stage IV M1a/c disease. Response rates in individual injected and non-injected lesions were 80% (n = 10) and 87% (n = 15), respectively. Complete tumor responses were observed in some visceral and non-visceral lesions. Patients developed notable levels of anti-CVA21 serum neutralizing antibody by Day 22. Conclusions: Intralesional CVA21 + ipilimumab is a promising novel oncolytic immunotherapeutic combination for the treatment of unresectable Stage IIIC-IVM1c melanoma. At present no DLT has been achieved, with no Gr 3/4 CVA21-related AEs observed. The preliminary overall response rate (80%) is higher than published rates for either agent used alone (CVA21: 28.1% and ipilimumab:∼15-20%) in advanced melanoma patients. The combination immunotherapy treatment is generally well tolerated and has displayed antitumor activity in both local and systemic disease, also within environments of high levels of anti-CVA21 serum neutralizing antibody. Citation Format: Brendan Curti, Jon Richards, Mark Faries, Mark Grose, Roberta Karpathy, Darren R. Shafren. Phase Ib study of a novel immunotherapy combination therapy of intralesional coxsackievirus A21 and systemic ipilimumab in patients with advanced melanoma. [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 CT021.

Abstract 3048: Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−

Abstract Melanoma is a cancer that has become increasingly frequent over the last decades in the western world. Initial treatment for primary and locoregional melanoma is surgery. In metastatic disease, systematic treatment and recently immunotherapy has been the mainstay. At this stage however, the prognostic outlook is still bleak. Thus, new treatments are urgently needed. Melanoma is considered to be a radioresistant cancer. The mechanisms underlying radioresistance are multiple and incompletely characterized. In some tumors, radioresistance is mediated by increased synthesis of anti-oxidants that scavenge reactive oxygen species (ROS), induced by radiotherapy. Glutathione (GSH) is an anti-oxidant synthesized from cystine and constitutes a major defense system against oxidative stress in mammalian cells. Cystine is taken up through system Xc− (SXC), an anti-port transmembrane protein with catalytic subunit xCT. Sulfasalazine, a drug approved in the 1950s for treatment of rheumatoid arthritis and inflammatory bowel disease, has been shown to block SXC. Based on previous reports by others, and our own recent findings, we hypothesize that: I) xCT represents a mechanism for radioresistance and is expressed in radioresistant melanoma cancer and II) xCT inhibitors can act as radiosensitizers to potentiate the efficacy of radiotherapy. Expression of the catalytic subunit of SXC, xCT, was found in tissue micro array of primary and secondary melanoma biopsies. In addition, SAS treatment dramatically reduced cystine-uptake and GSH levels in melanoma cells in vitro, and markedly increased the levels of reactive oxygen species (ROS). Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the anti-oxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and irradiation synergistically reduced subcutaneous melanoma tumor growth in vivo, compared to controls or either treatment alone. Thus, future experimental studies are warranted to validate SAS as a radiosensitizer in the treatment of metastatic melanoma. Future studies will also be aimed at assessing the effect on pulmonary melanoma metastasis. Citation Format: Hilde Elise Førde, Linda Sleire, Heidi Espedal, Jan Ingemann Heggdal, Frode Selheim, Paal-Henning Pedersen, Per Øyvind Enger. Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−. [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 3048.

Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

Patient-derived tumor xenograft strategies for informed management of patients with metastatic melanoma.

Metastatic melanoma has benefited from immunotherapy and targeted therapy advances. Faced with the inescapable onset of treatment resistance, the choice of a second-line treatment can be guided by a patient-derived tumor xenograft (PDTX). This new approach requires an excellent multidisciplinary collaboration where the surgeon has a key role to play. Each patient included (stage IIIC or IV) presented with subcutaneous melanoma metastasis that could be surgically resected. The surgeon performed orthotopic PDTX on CB17-SCID mice. To validate the model, tumor material was amplified over three successive generations of animals to obtain cohorts compatible with carrying out a study to compare treatment response by targeted therapy (vemurafenib versus controls). Tumors were characterized (histologically and genetically) at all stages of the generations' amplification. Functional imaging by fluorine-18 fluorodeoxyglucose PET scan was performed for the third generation PDTX. Seventeen patients with a mutated BRAF V600E subcutaneous metastasis were included, yielding 257 PDTX. Clinical, histological, and genetic characteristics of the grafted tumors were stable over the three mice generations. The treatment response to vemurafenib was observed for all PDTX. The fluorine-18 fluorodeoxyglucose PET scan evidenced a decreased in glucose uptake in the treated tumors. PDTX models are being widely used in fundamental research and are more compatible with clinical issues. If PDTX are simple and easily reproducible in metastatic melanoma, an organized multidisciplinary platform is essential to implement them. In our experience, surgeons have a key role to play in the cohesion of this new therapeutic approach.

Induced Pluripotent Stem Cell-conditioned Medium Suppressed Melanoma Tumorigenicity Through the Enhancement of Natural-Killer Cellular Immunity.

Induced pluripotent stem cells (iPSCs) can secrete cytokines that are involved in T-cell development and affect cytotoxic activity. To assess the effect of iPSC-conditioned medium on tumorigenicity, we retrieved splenocytes from B6 mice and cocultured them with or without irradiated B16 melanoma cells, mouse interleukin-2 (mIL-2), or iPSC-conditioned medium. Splenocyte cytotoxicity assays against B16 melanoma cells [as cytotoxic T lymphocyte (CTL) activity] and P815 cells [as natural killer (NK) activity] were performed. IL-10 and interferon-γ concentrations were measured. An in vivo subcutaneous B16 melanoma growth model was performed in B6 mice and treated with iPSC-conditioned medium. The lymphocyte subpopulation depletion test was performed to determine effectors against B16 melanoma cells. We found that unstimulated splenocytes had little cytotoxic activity. Without tumor cells, mIL-2 could augment iPSC-conditioned medium-treated CTL and NK activities (P<0.01). With irradiated tumor cells, mIL-2 treatment of splenocytes could not enhance CTL or NK activity, but iPSC-conditioned medium could enhance CTL and NK activity (P<0.001). Irradiated tumor cells induced mice splenocytes to secrete more IL-10, similar to mIL-2 treatment, but not iPSC-conditioned medium treatment. mIL-2 had better efficacy than conditioned medium in inducing splenocyte interferon-γ production. The CTL and NK cell depletion test showed that the immunostimulating effect of iPSC-conditioned medium on splenocytes was through the enhancement of NK cellular activity (P<0.05). The subcutaneous melanoma growth model showed that B16-bearing mice treated with an iPSC-conditioned medium intraperitoneal injection had a decreased tumor growth rate (P<0.01). Our study suggests that iPSC-conditioned medium had a protective effect against tumor-induced immunosuppression through the enhancement of host NK cellular activity.

197. Balancing Anti-Tumor Efficacy with Local Inflammatory Toxicity for the Treatment of Diffuse Intrinsic Pontine Glioma and Other Brain Tumors

We have shown previously that multiple rounds of a systemic treatment of GM-CSF, followed by intravenous reovirus, leads to effective treatment of subcutaneous melanomas (Ilett et al., Mol. Ther. 2015). We show here that a similar regimen is also effective at treating both melanoma (B16) and glioma (GL261) tumors growing intra-cranially. As a result of these pre-clinical studies, we initiated a Phase I clinical trial in paediatric patients with gliomas of GM-CSF and reovirus therapy. To date, three patients have been treated. In two of three of these patients there were possible indications of pseudoprogression and intra-cranial inflammation, both of which were clinically resolved upon treatment with dexamethasone. Whilst this trial continues to recruit, we have investigated additional treatments for paediatric brain tumors, especially those in which direct virus injection may allow greater local access to tumor. In this respect, there is a constant tension between the pro-inflammatory, anti-tumor nature of oncolytic viroimmunotherapy and the need to reduce potentially toxic local inflammatory reactions within the brain. Our overall hypothesis is that it will be possible to balance the anti tumor effects of viral oncolysis, in part caused by inflammatory reactions to the virus, with the associated toxicity. Nowhere is this more relevant than for the treatment of Diffuse Pontine Gliomas (DIPG), which grow in the brain stem. Our experiments show that it is indeed possible to treat tumors growing in the pons/and or medulla, with inflammatory, immune based therapies (both T cell mediated and locally cytotoxic) without increasing toxicity over that caused by tumor growth alone. Therefore, we have screened multiple oncolytic virus types for their ability to kill DIPG cell lines both in vitro and in vivo. In particular, we have compared viruses with different speeds of oncolysis and different inflammatory properties to investigate how the efficacy can be balanced with local toxicity, using standard of care anti inflammatory treatments (with dexamethasone) as well as with novel agents and viruses designed to suppress local inflammatory reactions.

IL-1 Contributes to the Anti-Cancer Efficacy of Ingenol Mebutate.

Ingenol mebutate is approved for the topical treatment of actinic keratoses and may ultimately also find utility in treating skin cancers. Here we show that relapse rates of subcutaneous B16 melanoma tumours treated topically with ingenol mebutate were not significantly different in C57BL/6 and Rag1-/- mice, suggesting B and T cells do not play a major role in the anti-cancer efficacy of ingenol mebutate. Relapse rates were, however, significantly increased in MyD88-/- mice and in C57BL/6 mice treated with the anti-IL-1 agent, anakinra. Ingenol mebutate treatment induces a pronounced infiltration of neutrophils, which have been shown to have anti-cancer activity in mice. Herein we provide evidence that IL-1 promotes neutrophil recruitment to the tumour, decreases apoptosis of infiltrating neutrophils and increases neutrophil tumour killing activity. These studies suggest IL-1, via its action on neutrophils, promotes the anti-cancer efficacy of ingenol mebutate, with ingenol mebutate treatment causing both IL-1β induction and IL-1α released from keratinocytes.

Electrochemotherapy by pulsed electromagnetic field treatment (PEMF) in mouse melanoma B16F10 in vivo.

IntroductionPulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably increases membrane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, contactless PEMF could represent a promising approach for drug delivery.Materials and methodsNoninvasive electroporation was performed by magnetic field pulse generator connected to an applicator consisting of round coil. Subcutaneous mouse B16F10 melanoma tumors were treated with intravenously injection of cisplatin (CDDP) (4 mg/kg), PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 μs) or with the combination of both therapies (electrochemotherapy − PEMF + CDDP). Antitumor effectiveness of treatments was evaluated by tumor growth delay assay. In addition, the platinum (Pt) uptake in tumors and serum, as well as Pt bound to the DNA in the cells and Pt in the extracellular fraction were measured by inductively coupled plasma mass spectrometry.ResultsThe antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF was comparable to the conventional electrochemotherapy with CDDP, with the induction of 2.3 days and 3.0 days tumor growth delay, respectively. The exposure of tumors to PEMF only, had no effect on tumor growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured.ConclusionsThe obtained results in mouse melanoma model in vivo demonstrate the possible use of PEMF induced electroporation for biomedical applications, such as electrochemotherapy. The main advantages of electroporation mediated by PEMF are contactless and painless application, as well as effective electroporation compared to conventional electroporation.

Oral Delivery of a High Quercetin Payload Nanosized Emulsion: In Vitro and In Vivo Activity Against B16-F10 Melanoma.

Quercetin is a natural compound that has several biological activities including anticancer activity. However, the use of this drug has been limited mostly because of its poor water solubility and low bioavailability. Therefore, the development of quercetin-loaded nanocarrier systems may be considered a promising advance to exploit its therapeutic properties in clinical setting including cancer treatment. This study evaluates the effect of orally administered nanosized emulsion containing quercetin (QU-NE) on the cytotoxicity activity against B16-F10 cells in vitro, and on subcutaneous melanoma in mice inoculated with B16-F1O cells. In vivo experiments, also evaluate the co-administration of quercetin with cisplatin in order to predict synergic effects and the renal and hepatic toxicity. The nanocarriers were prepared through the hot solvent diffusion associated with the phase inversion temperature methods. In vitro study showed reduction of cell viability in a concentration-depend manner for free quercetin and QU-NE. In vivo study, quercetin either as a free drug or colloidal dispersion was administrated at a dose of 5 mg kg(-1) twice a week for 17 days via oral route. Cisplatin was administrated at dose of 1 mg kg(-1) once a week intraperitoneally. Free quercetin and QU-NE reduced tumor growth, however, the reduction observed for QU-NE (P < 0.001 vs. control) was significantly higher than free quercetin (P < 0.05 vs. control). The association of both drugs did not show synergic effect. Besides, no renal or hepatic toxicities were observed after administration of free quercetin and QU-NE. These results suggest that an improvement in the oral bioavailability of quercetin occurred when this compound was dissolved in the oily phase of a nanosized emulsion, indicating that it might have a potential application in the treatment of melanoma.

Combination Therapy With Reovirus and Anti-PD-1 Blockade Controls Tumor Growth Through Innate and Adaptive Immune Responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both preclinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and antitumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive antitumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (P < 0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced T(reg) activity, and increased the adaptive CD8(+) T-cell-dependent antitumor T-cell response. PD-1 blockade also enhanced the antiviral immune response but through effector mechanisms which overlapped with but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus viroimmunotherapy.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.

First oncolytic virus vaccine for advanced melanoma

First oncolytic virus vaccine for advanced melanoma

Abstract A18: Modified ZD6474 (Vandetanib) derivatives with VEGFR2 and non-peptide integrin antagonist (IA) slows tumor growth

Abstract Tumor angiogenesis is an important component of cancer biology driven in part by the hypothesis that tumor vessel growth is a necessary requirement for tumor growth. Angiogenesis does not only depend on endothelial cell invasion and proliferation, it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF). Vandetanib, also known as ZD6474, is an orally bioavailable small molecule tyrosine kinase inhibitor of multiple growth factors that is an antagonist of the vascular endothelial growth factor receptor-2 (VEGFR-2). ZD6474 was purchased and modified to add a fluorescent dye (6-FAM). A linker was used to couple the ZD6474 monomer to create dimers, and similarly linked to FAM fluorescent dye. The two compounds were compared using human endothelial cell (HUVECs) and the cancer cell lines U-87-MG and MDA-MB-231. We compared cellular uptake and binding specificity, as well as effects on cellular viability and angiogenesis in a series of in vitro and in vivo studies. ZD6474 dimer demonstrated improved uptake in HUVECs and other VEGFR expressing cells over ZD6474 monomer in vitro. Therapeutic effects were mixed, with in vitro studies showing the dimer having the same or weaker effects compared to the monomer, while an in vivo study using pseudotumors (matrigel plug assay) seemed to indicate stronger effects could be obtained from the dimer. Finally, in biodistribution study in a xenograft tumor model, the dimer accumulated 20× greater concentration in the tumor than in the liver, spleen, or kidneys, and also 20× greater than the accumulation of the monomer or the dye alone, all at 24 h following compound injection. This study provides a rationale for the evaluation of dimeric ZD6474 as a potent imaging agent of angiogenic activity in vivo. Multiagency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvβ3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p &amp;lt; 10-7) by 36 ± 9%, 49 ± 8%, and 71 ± 7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p = 0.00011). These results suggest that the major target of these anti-αvβ3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvβ3 multivalent ligand showing clear enhancement in antitumor effectiveness. Citation Format: Yoo-shin Kim, Brian E. O'Neill. Modified ZD6474 (Vandetanib) derivatives with VEGFR2 and non-peptide integrin antagonist (IA) slows tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A18.