Herein we present methods for synthesizing monodisperse mesoporous silica particles and silica particles with bimodal porosity by templating with surfactant micelle and microemulsion phases. The fabrication of monodisperse mesoporous silica particles is based on the formation of well-defined equally sized emulsion droplets using a microfluidic approach. The droplets contain the silica precursor/surfactant solution and are suspended in hexadecane as the continuous oil phase. The solvent is then expelled from the droplets, leading to concentration and micellization of the surfactant. At the same time, the silica solidifies around the surfactant structures, forming equally sized mesoporous particles. We show that hierarchically bimodal porous structures can be obtained by templating silica microparticles with a specially designed surfactant micelle/microemulsion mixture. Oil, water, and surfactant liquid mixtures exhibit very complex phase behavior. Depending on the conditions, such mixtures give rise to highly organized structures. A proper selection of the type and concentration of surfactants determines the structuring at the nanoscale level. Tuning the phase state by adjusting the surfactant composition and concentration allows for the controlled design of a system where microemulsion droplets coexist with smaller surfactant micellar structures. The microemulsion droplet and micellar dimensions determine the two types of pore sizes.

Immunotherapies have shown promise in treatment of cancer, but more potent and targeted therapies are needed. Natural killer (NK) cells are lymphocytes with innate ability to recognize and lyse tumor cells. When activated, they also produce type II interferon (IFNγ) to orchestrate the activity of other immune cells. Strategies to elicit NK cell activation in vivo have potential usefulness in anti-tumor immunotherapies. Here, we report on a strategy to stimulate NK cell activation and anti-tumor activity in mice with established B16.F10 murine melanomas. We and others previously observed that NK cells are rapidly activated during infection by pathogens such as the bacterium Listeria monocytogenes (Lm). A secreted Lm virulence protein, p60, and a fragment of p60 termed L1S were previously shown to stimulate innate immune responses and promote NK cell activation. We purified recombinant L1S and characterized its activity in cell culture studies. Recombinant L1S protein was also observed to promote accumulation and robust NK cell activation in the lungs when given via intratracheal instillation to control and tumor-bearing mice. Importantly, therapeutic administration of a single L1S dose was found to significantly reduce the number and area of “metastatic” tumor nodules on the lungs of mice with established B16.F10 murine melanomas. Depletion studies showed that these antitumor effects were dependent on NK cells and IFNγ. These data provide proof of concept that administration of a single immune-modulating microbial polypeptide can be used to therapeutically boost NK cell in vivo activation and promote anti-tumor responses.

Introduction: It is strikingly difficult to develop successful treatments for PDAC; even with curative resection, most patients die from early occult metastases. Prior studies identified the presence of tumor-infiltrating lymphocytes (TILs) in primary PDAC tumors as having prognostic significance in the PDAC adjuvant setting, sharpening the questions of what fraction of patients have immune-infiltrated tumors and what therapeutic strategies should be pursued in these patients vs. the non-infiltrated group. The phase 3 APACT trial evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with PDAC who had undergone primary tumor resection, with the primary endpoint of disease-free survival evaluated by independent review. We extended studies of the tumor microenvironment of PDAC to a large set of resected APACT primary tumors in an effort to further refine features of tumor or immune infiltrate that influence disease progression and to determine if chemotherapy regimen–specific predictive signatures are identifiable. Tissue analyses for a large subset of APACT samples included RNA-seq, DNA-seq, multiplexed immunohistochemistry (IHC), and proteomics. Methods: We imaged and quantified markers for tumor cells, 7 different immune cells, and 2 immune checkpoint markers using bright-field chromogenic multiplexed IHC from pretreatment samples for more than 500 APACT primary tumor samples. We computationally defined the tumor, tumor margin, and distal stromal (> 150 μm from tumor boundary) regions, and quantified densities and distributions of immune cells in these regions. As part of an initial analysis of more than 400 samples, we applied both unsupervised clustering and supervised classification to these IHC measurements to identify patient subgroups with similar spatial arrangements of immune cells relative to tumor regions. Results: The preliminary analysis of normalized cell densities across all 3 tissue regions revealed 3 patient subgroups: one in which immune cells are mixed within the tumor regions; a second where immune cells approach the tumor boundary but are depleted within the tumor; and a third in which immune cells are depleted in both tumor and its margin, remaining at high densities only in the distal stromal regions. Within these latter subgroups, CD20+, CD4+, and CD8+ cells were more prevalently depleted from tumor and/or margin, whereas CD163+ and CD163+CMAF+ cells showed less of this arrangement. Nearly 85% of patients fell in the second or third patient group. Conclusions: We are pursuing analyses of these data in conjunction with upcoming molecular and genetic profiling data to further elucidate the association of the immune cell populations and these subgroups with clinical outcomes. These data will provide an unprecedented opportunity for exploratory analysis and discovery of immune, molecular, and genetic biomarkers for PDAC patient stratification. Citation Format: David J. Reiss, Thomas Lila, Suzana Couto, Sitharthan Kamalakaran, Yan Ren, Doug Bowman, Amber Ortiz, Maria Wang, Clifton Drew, Kao-Tai Tsai, Mathieu Marella, Brian Fox, Garth McGrath, Matthew Trotter, Fadi Towfic, Ian Cushman, Alexander Ratushny, Brian Lu, Daniel Pierce, Jim Cassidy. Spatial organization of pancreatic ductal adenocarcinoma (PDAC)–associated immune cells from the Adjuvant Pancreatic Adenocarcinoma Clinical Trial (APACT) study [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A43.