Ismail M. Meraz

Activation of the Inflammasome and Enhanced Migration of Microparticle-Stimulated Dendritic Cells to the Draining Lymph Node

Porous silicon microparticles presenting pathogen-associated molecular patterns mimic pathogens, enhancing internalization of the microparticles and activation of antigen presenting dendritic cells. We demonstrate abundant uptake of microparticles bound by the TLR-4 ligands LPS and MPL by murine bone marrow-derived dendritic cells (BMDC). Labeled microparticles induce concentration-dependent production of IL-1β, with inhibition by the caspase inhibitor Z-VAD-FMK supporting activation of the NLRP3-dependent inflammasome. Inoculation of BALB/c mice with ligand-bound microparticles induces a significant increase in circulating levels of IL-1β, TNF-α, and IL-6. Stimulation of BMDC with ligand-bound microparticles increases surface expression of costimulatory and MHC molecules, and enhances migration of BMDC to the draining lymph node. LPS-microparticles stimulate in vivo C57BL/6 BMDC and OT-1 transgenic T cell interactions in the presence of OVA SIINFEKL peptide in lymph nodes, with intact nodes imaged using two-photon microscopy. Formation of in vivo and in vitro immunological synapses between BMDC, loaded with OVA peptide and LPS-microparticles, and OT-1 T cells are presented, as well as elevated intracellular interferon gamma levels in CD8(+) T cells stimulated by BMDC carrying peptide-loaded microparticles. In short, ligand-bound microparticles enhance (1) phagocytosis of microparticles; (2) BMDC inflammasome activation and upregulation of costimulatory and MHC molecules; (3) cellular migration of BMDC to lymphatic tissue; and (4) cellular interactions leading to T cell activation in the presence of antigen.

A New Imaging Platform for Visualizing Biological Effects of Non-Invasive Radiofrequency Electric-Field Cancer Hyperthermia.

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivo intravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors.

Adjuvant Cationic Liposomes Presenting MPL and IL-12 Induce Cell Death, Suppress Tumor Growth, and Alter the Cellular Phenotype of Tumors in a Murine Model of Breast Cancer

Dendritic cells (DC) process and present antigens to T lymphocytes, inducing potent immune responses when encountered in association with activating signals, such as pathogen-associated molecular patterns. Using the 4T1 murine model of breast cancer, cationic liposomes containing monophosphoryl lipid A (MPL) and interleukin (IL)-12 were administered by intratumoral injection. Combination multivalent presentation of the Toll-like receptor-4 ligand MPL and cytotoxic 1,2-dioleoyl-3-trmethylammonium-propane lipids induced cell death, decreased cellular proliferation, and increased serum levels of IL-1β and tumor necrosis factor (TNF)-α. The addition of recombinant IL-12 further suppressed tumor growth and increased expression of IL-1β, TNF-α, and interferon-γ. IL-12 also increased the percentage of cytolytic T cells, DC, and F4/80+ macrophages in the tumor. While single agent therapy elevated levels of nitric oxide synthase 3-fold above basal levels in the tumor, combination therapy with MPL cationic liposomes and IL-12 stimulated a 7-fold increase, supporting the observed cell cycle arrest (loss of Ki-67 expression) and apoptosis (TUNEL positive). In mice bearing dual tumors, the growth of distal, untreated tumors mirrored that of liposome-treated tumors, supporting the presence of a systemic immune response.

Multivalent Presentation of MPL by Porous Silicon Microparticles Favors T Helper 1 Polarization Enhancing the Anti-Tumor Efficacy of Doxorubicin Nanoliposomes

Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.

Characterization of Free and Porous Silicon-Encapsulated Superparamagnetic Iron Oxide Nanoparticles as Platforms for the Development of Theranostic Vaccines

Tracking vaccine components from the site of injection to their destination in lymphatic tissue, and simultaneously monitoring immune effects, sheds light on the influence of vaccine components on particle and immune cell trafficking and therapeutic efficacy. In this study, we create a hybrid particle vaccine platform comprised of porous silicon (pSi) and superparamagnetic iron oxide nanoparticles (SPIONs). The impact of nanoparticle size and mode of presentation on magnetic resonance contrast enhancement are examined. SPION-enhanced relaxivity increased as the core diameter of the nanoparticle increased, while encapsulation of SPIONs within a pSi matrix had only minor effects on T2 and no significant effect on T2* relaxation. Following intravenous injection of single and hybrid particles, there was an increase in negative contrast in the spleen, with changes in contrast being slightly greater for free compared to silicon encapsulated SPIONs. Incubation of bone marrow-derived dendritic cells (BMDC) with pSi microparticles loaded with SPIONs, SIINFEKL peptide, and lipopolysaccharide stimulated immune cell interactions and interferon gamma production in OT-1 TCR transgenic CD8+ T cells. Overall, the hybrid particle platform enabled presentation of a complex payload that was traceable, stimulated functional T cell and BMDC interactions, and resolved in cellular activation of T cells in response to a specific antigen.

Abstract 621: Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models

TUSC2, a pro-apoptotic tumor suppressor gene whose expression is lost or decreased in most lung cancers, activates the innate immune system through initiation of broad spectrum cytokine secretion and natural killer (NK) cell activation. TUSC2 delivered systemically by nanovesicles has mediated tumor regression in metastatic non-small cell lung cancer clinical trials. We studied the effect of TUSC2 on immune cell populations and the anti-tumor activity of TUSC2 in combination with anti-PD1 checkpoint blockade in two syngeneic mouse models: C57BL/6 mice subcutaneously injected with murine lung adenocarcinoma cell line CMT/167-luc cells (KrasG12V mutation) and 344SQ (KrasG12D allele and a knock-in Trp53R172HΔG allele) adenocarcinomas which metastasize to the lung in 129S2 mice. Tumor growth was monitored by scoring ex-vivo luminescence using the IVIS Imaging System 200. Multi-color flow cytometry was used for immune profiling of circulating immune cells after nanovesicle mediated TUSC2 intravenous injection. Cytokine gene expression in response to TUSC2 in sorted immune subpopulations was determined by real-time PCR. Tumor growth was significantly reduced with TUSC2 treatment compared with no treatment in both subcutaneous and metastatic mouse models. Synergistic anti-tumor activity was observed when TUSC2 was combined with anti-PD1 verified in five independent experiments. In the lung metastasis model, mice treated with TUSC2 + anti-PD1 lived significantly longer than with single agent treatment. Circulating NK cells increased three fold following TUSC2 nanovesicle intravenous injection both in tumor free and tumor bearing mice which correlated with tumor regression and survival. Cytotoxic T lymphocyte responses were increased whereas Tregs and MDSCs decreased with TUSC2 alone and TUSC2+anti-PD1 treatment. The levels of T cell checkpoint markers PD1, CTLA-4, LAG-3, and TIM-3 evaluated by flow cytometry were decreased after TUSC2 treatment. TUSC2 anti-tumor response was abolished when NK cells were depleted indicating NK cells are important mediators of the TUSC2 treatment effect. Single cell suspension analysis by flow cytometry showed high numbers of NK cells infiltrating lung tumor metastases after TUSC2 treatment. The number of tumor nodules in the lung was significantly less following treatment with TUSC2 nanovesicles compared with control. IL-15 gene expression which mediates NK cell proliferation, was increased by TUSC2. In conclusion, systemic TUSC2 nanovesicle immunogene therapy combined with checkpoint blockade showed synergistic anti-tumor efficacy and activated the immune system through upregulation of NK cells and CTL and downregulation of regulatory cells. Citation Format: Ismail M. Meraz, Mourad Majidi, RuPing Shao, Meng Feng, Xiaobo Cao, David Rice, Boris Sepesi, Lin Ji, Jack Roth. Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic 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 621. doi:10.1158/1538-7445.AM2017-621

Abstract 2594: Adjuvant cationic nanoliposomes induce anti-cancer immunity in a murine model of breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Nanoparticles, such as liposomes, provide opportunities to simultaneously present antigens and immune modulators. Using a 4T1 murine model of breast cancer, a cationic nanoliposomal formulation containing monophosphoryl lipid A and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane induced anti-tumor activity following intratumoral administration. Addition of recombinant interleukin-12 (IL-12) further suppressed tumor growth and augmented T helper-1 cell (Th-1) polarization, with enhanced tumor infiltration by cytotoxic T cells, dendritic cells, and M1 macrophages, and amplification of interferon gamma secretion. Mice bearing dual tumors displayed arrest of tumor growth in treated tumors as well as distal, untreated tumors following combination therapy with adjuvant nanoliposomes and IL-12. In summary, adjuvant MPL-liposomes combined with localized IL-12 therapy block tumor growth, stimulate a Th-1 bias of the tumor microenvironment, and induce cancer-specific immune responses. Citation Format: Ismail M. Meraz, David J. Savage, Jianhua Gu, Jessica Rhudy, Rita E. Serda. Adjuvant cationic nanoliposomes induce anti-cancer immunity in a murine model of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2014-2594

Biological Microniches Characterizing Pathological Lesions: Impact on Nanoparticle-Based Delivery of Therapeutics

Abstract Discrete pathologies have unique features that contribute to disease progression. Each lesion contains unique clusters of cells and other factors that make up specific micro-niches. Individual microniches present novel barriers that hamper the delivery of therapeutics, but it also contains characteristic elements that can favour accumulation of therapeutics within the lesion. This chapter discusses the unique biology associated with cancer, cardiovascular disease, and neurological disorders and describes how each unique presentation can be used to optimize nanoparticle design for targeting the lesion.

Abstract B25: Porous silicon microparticles exhibit immunomodulatory effects leading to suppression of tumor growth.

Nanoparticles, such as polymeric delivery platforms, can exhibit intrinsic immunostimulant properties, dependent on size, charge, surface modification, and composition. To function as effective adjuvants, a balance between immunostimulatory properties and biocompatibility is essential. We have demonstrated that porous silicon (pSi) microparticles are effective delivery vehicles, with uptake by target cell populations. A peritonitis mouse model was used to access early innate immune responses 24 hours following administration of pSi microparticles. C57BL/6 mice were injected intraperitoneally with microparticles of various shapes and sizes and cytokine production and cell infiltration were assessed. pSi microparticles were found to have proinflammatory effects. The microparticles induced significant leukocyte infiltration into the site of injection and elevated IL-1β levels in lavage fluid. As cancer progresses, immune responses become more tolerant and cancer cells become more refractory to chemotherapies. Select chemotherapeutics, including cyclophosphamide, doxorubicin, and paclitaxel, have immunodulatory effects. Based on the immunopotentiating effects of pSi microparticles, and the ability of monophosphoryl lipid A (MPL) adsorbed pSi microparticles to activate antigen presenting cells, we sought to potentiate the doxorubicin-mediated immune response through co-delivery of MPL-pSi microparticles. When tumors in mice bearing intramammary 4T1-luc breast tumors reached a volume of 100 mm3, mice were injected with doxorubicin loaded liposomes (5 mg/kg) and MPL-pSi microparticles (5x108 microparticles; 10 µg MPL equivalent) by means of tail vein injection. Tumor growth was monitored by calipher measurements and luciferase expression using the IVIS Imaging System 200. While MPL-pSi microparticle-treated mice exhibited reduced tumor growth, mice receiving MPL-pSi microparticles and doxorubicin-loaded liposomes exhibited arrest of tumor growth. Thus pSi microparticles are an attractive immunopotentiating platform, with applications for both drug and antigen delivery. Citation Format: Ismail M. Meraz, Laura Williams, Marie Yang, Edward C. Lavelle, Rita E. Serda. Porous silicon microparticles exhibit immunomodulatory effects leading to suppression of tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B25.

Abstract 1565: Mesoporous silicon particle mediated dendritic cell based vaccine formulation showed stronger immunoresponse for the presentation of tumor antigens and adjuvant

Dendritic cell (DC) based immunotherapy is being used for cancer therapy in various clinical trials. During evaluation of recent clinical studies, it became apparent that efficient DC-based immunotherapy is dependent on a number of factors, such as the mode of antigen presentation, maturation of DC, injection site, and vaccination dosing. Major limiting factors for DC-based vaccines are insufficient loading of antigens by DC and poor migratory capacity of DC to lymph nodes. Nanotechnology provides tools to load large payloads of antigens and adjuvants in particles for uptake by DC. Toll-like receptor (TLR) ligands have been proposed as vaccine adjuvants for boosting adaptive immunity in cancer therapy. TLR signaling induces DC activation that is characterized by enhanced expression of costimulatory molecules and increased secretion of cytokines necessary for activation and differentiation of naive T cells. In this study, we have used porous silicon (pSi) microparticles to create a novel vaccine. We have demonstrated that porous silicon particles can be decorated with TLR-ligands using lipopolysaccharide (LPS) or monophosphoryl lipid (MPL). This decorated particles are substrates for bone marrow-derived DC internalization, leading to cellular uptake and activation and enhanced migration of DC to lymph nodes. Confocal and scanning electron microscopy, as well as flow cytometry studies supported higher uptake of LPS and MPL conjugated particles as compared to unlabeled particles. TLR ligands induced morphological changes in GM-CSF-stimulated bone marrow cells during particle uptake consistent with classical DC dentron formation. Stimulated antigen presenting cells also expressed elevated levels of costimulatory (e.g. CD80, CD86) and major histocompatibility molecules (MHC), and secreted pro-inflammatory cytokines both in vitro and in vivo. Ex-vivo processed DC loaded with TLR ligand coated pSi showed enhanced migration to lymph node as compared with empty DC when injected mice subcutaneously. As expected, LPS conjugation to particles showed toxicity towards DC whereas MPL conjugated pSi showed little or no toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1565. doi:1538-7445.AM2012-1565