High-dimensional in situ proteomics imaging to assess γδ T cells in spatial biology.

This study presents a high-dimensional immunohistochemistry approach to assess human γδ T cell subsets in their native tissue microenvironments at spatial resolution, a hitherto unmet scientific goal due to the lack of established antibodies and required technology. We report an integrated approach based on multiplexed imaging and bioinformatic analysis to identify γδ T cells, characterize their phenotypes, and analyze the composition of their microenvironment. Twenty-eight γδ T cell microenvironments were identified in tissue samples from fresh frozen human colon and colorectal cancer where interaction partners of the immune system, but also cancer cells were discovered in close proximity to γδ T cells, visualizing their potential contributions to cancer immunosurveillance. While this proof-of-principle study demonstrates the potential of this cutting-edge technology to assess γδ T cell heterogeneity and to investigate their microenvironment, future comprehensive studies are warranted to associate phenotypes and microenvironment profiles with features such as relevant clinical characteristics.

Acidic ascites inhibits ovarian cancer cell proliferation and correlates with the metabolomic, lipidomic and inflammatory phenotype of human patients.

BACKGROUND: The poor prognosis of ovarian cancer patients is strongly related to peritoneal metastasis with the production of malignant ascites. However, it remains largely unclear how ascites in the peritoneal cavity influences tumor metabolism and recurrence. This study is an explorative approach aimed at for a deeper molecular and physical-chemical characterization of malignant ascites and to investigate their effect on in vitro ovarian cancer cell proliferation. METHODS: This study included 10 malignant ascites specimens from patients undergoing ovarian cancer resection. Ascites samples were deeply phenotyped by 1H-NMR based metabolomics, blood-gas analyzer based gas flow analysis and flow cytomertry based a 13-plex cytokine panel. Characteristics of tumor cells were investigated in a 3D spheroid model by SEM and metabolic activity, adhesion, anti-apoptosis, migratory ability evaluated by MTT assay, adhesion assay, flowcytometry and scratch assay. The effect of different pH values was assessed by adding 10% malignant ascites to the test samples. RESULTS:  The overall extracellular (peritoneal) environment was alkaline, with pH of ascites at stage II-III = 7.51 ± 0.16, and stage IV = 7.78 ± 0.16. Ovarian cancer spheroids grew rapidly in a slightly alkaline environment. Decreasing pH of the cell culture medium suppressed tumor features, metabolic activity, adhesion, anti-apoptosis, and migratory ability. However, 10% ascites could prevent tumor cells from being affected by acidic pH. Metabolomics analysis identified stage IV patients had significantly higher concentrations of alanine, isoleucine, phenylalanine, and glutamine than stage II-III patients, while stage II-III patients had significantly higher concentrations of 3-hydroxybutyrate. pH was positively correlated with acetate, and acetate positively correlated with lipid compounds. IL-8 was positively correlated with lipid metabolites and acetate. Glutathione and carnitine were negatively correlated with cytokines IL-6 and chemokines (IL-8 & MCP-1). CONCLUSION: Alkaline malignant ascites facilitated ovarian cancer progression. Additionally, deep ascites phenotyping by metabolomics and cytokine investigations allows for a refined stratification of ovarian cancer patients. These findings contribute to the understanding of ascites pathology in ovarian cancer.

Feasibility of pressurized intra peritoneal aerosol chemotherapy using an ultrasound aerosol generator (usPIPAC).

BACKGROUND: We tested the feasibility of ultrasound technology for generating pressurized intraperitoneal aerosol chemotherapy (usPIPAC) and compared its performance vs. comparator (PIPAC). MATERIAL AND METHODS: A piezoelectric ultrasound aerosolizer (NextGen, Sinaptec) was compared with the available technology (Capnopen, Capnomed). Granulometry was measured for water, Glc 5%, and silicone oil using laser diffraction spectrometry. Two- and three-dimensional (2D and 3D) spraying patterns were determined with methylene blue. Tissue penetration of doxorubicin (DOX) was measured by fluorescence microscopy in the enhanced inverted Bovine Urinary Bladder model (eIBUB). Tissue DOX concentration was measured by high-performance liquid chromatography (HPLC). RESULTS: The droplets median aerodynamic diameter was (usPIPAC vs. PIPAC): H20: 40.4 (CI 10-90%: 19.0-102.3) vs. 34.8 (22.8-52.7) µm; Glc 5%: 52.8 (22.2-132.1) vs. 39.0 (23.7-65.2) µm; Silicone oil: 178.7 (55.7-501.8) vs. 43.0 (20.2-78.5) µm. 2D and 3D blue ink distribution pattern of usPIPAC was largely equivalent with PIPAC, as was DOX tissue concentration (usPIPAC: 0.65 (CI 5-95%: 0.44-0.86) vs. PIPAC: 0.88 (0.59-1.17) ng/ml, p = 0.29). DOX tissue penetration with usPIPAC was inferior to PIPAC: usPIPAC: 60.1 (CI 5.95%: 58.8-61.5) µm vs. PIPAC: 1172 (1157-1198) µm, p < 0.001). The homogeneity of spatial distribution (top, middle and bottom of the eIBUB) was comparable between modalities. DISCUSSION: usPIPAC is feasible, but its performance as a drug delivery system remains currently inferior to PIPAC, in particular for lipophilic solutions.

Influence of pre-analytical sample preparation on drug concentration measurements in peritoneal tissue: an ex-vivo study.

OBJECTIVES: Biopsy morphology (surface/depth ratio) and sample processing might affect pharmacological measurements in peritoneal tissue. METHODS: This is an ex-vivo study on inverted bovine urinary bladders (IBUB). We compared cisplatin (CIS) and doxorubicin (DOX) concentration in 81 standardized transmural punch biopsies of different diameters (6 and 12 mm). Then, we assessed the effect of dabbing the peritoneal surface before analysis. After automatized tissue homogenization with ceramic beads followed by lyophilisation, DOX concentration was quantified by high-performance liquid chromatography (HPLC), CIS concentration by atomic absorption spectroscopy. Experiments were performed in triplicate; the analysis was blinded to the sample origin. Comparisons were performed using non-parametric tests. RESULTS: Concentrations are given in mean (CI 5-95%). Results were reproducible between experiments (for CIS p=0.783, for DOX p=0.235) and between different localizations within the IBUB (for CIS p=0.032, for DOX p=0.663). Biopsy diameter had an influence on CIS tissue concentration (6 mm biopsies: 23.2 (20.3-26.1), vs. 12 mm biopsies: 8.1 (7.2-9.2) ng/mg, p<0.001) but not on DOX: (0.46, 0.29-0.62) vs. 0.43 (0.33-0.54) ng/mg respectively, p=0.248). Dabbing the peritoneal surface reduced DOX tissue concentration (dry biopsies: 0.28 (0.12-0.43) vs. wet biopsies: 0.64 (0.35-0.93) ng/mg, p=0.025) but not CIS (23.5 (19.0-28.0) vs. 22.9 (18.9-26.9) ng/mg, respectively, p=0.735). CONCLUSIONS: Measurements of drug concentration in peritoneal tissue can be influenced by the biopsy's surface/depth ratio and after drying the biopsy's surface. This influence can reach a factor three, depending on the drug tested. The biopsy technique and the pre-analytical sample preparation should be standardized to ensure reliable pharmacological measurements in peritoneal tissue.

Enhanced intraperitoneal delivery of charged, aerosolized curcumin nanoparticles by electrostatic precipitation.

Aims: To investigate the potential of curcumin-loaded polylactic-co-glycolic acid nanoparticles (CUR-PLGA-NPs), alone and with electrostatic precipitation, for improving tissue uptake during pressurized intraperitoneal aerosol chemotherapy (PIPAC). Methods: Positively and negatively charged CUR-PLGA-NPs were delivered as PIPAC into inverted bovine urinary bladders ex vivo. The experiment was repeated with the additional use of electrostatic precipitation pressurized intraperitoneal aerosol chemotherapy (electrostatic PIPAC). Results: Positively charged CUR-PLGA-NPs increased depth of tissue penetration by 81.5% and tissue concentration by 80%. Electrostatic precipitation further improved the uptake of positively charged CUR-PLGA-NPs by 41.8%. Conclusion: The combination of positive charge and electrostatic precipitation have significant potential to improve tissue uptake of nanoparticles during intraperitoneal chemotherapy.