A Scalable 3D High-Content Imaging Protocol for Measuring a Drug Induced DNA Damage Response Using Immunofluorescent Subnuclear γH2AX Spots in Patient Derived Ovarian Cancer Organoids.

The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to a lack of understanding of disease mechanisms and difficulties in developing new targeted therapies. Defective DNA damage detection and repair is one of the hallmarks of cancer cells and is a defining characteristic of ovarian cancer. Most in vitro studies to date involve viability measurements at scale using relevant cancer cell lines; however, the translation to the clinic is often lacking. The use of patient derived organoids is closing that translational gap, yet the 3D nature of organoid cultures presents challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale, providing a better understanding of the mechanism of action of drugs or genetic perturbagens. In this study we report a semiautomated and scalable immunofluorescence imaging assay utilizing the development of a 384-well plate based subnuclear staining and clearing protocol and optimization of 3D confocal image analysis for studying DNA damage dose response in human ovarian cancer organoids. The assay was validated in four organoid models and demonstrated a predictable response to etoposide drug treatment with the lowest efficacy observed in the clinically most resistant model. This imaging and analysis method can be applied to other 3D organoid and spheroid models for use in high content screening.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in in vitro and in vivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

P-, but not E- or L-, selectin-mediated rolling adhesion persistence in hemodynamic flow diverges between metastatic and leukocytic cells.

The ability of leukocytic cells to engage selectins via rolling adhesion is critical to inflammation, but selectins are also implicated in mediating metastatic dissemination. Using a microfluidic- and flow-based cell adhesion chromatography experimental and analytical technique, we interrogated the cell-subtype differences in engagement and sustainment of rolling adhesion on P-, E-, and L-selectin-functionalized surfaces in physiological flow. Our results indicate that, particularly at low concentrations of P-selectin, metastatic but not leukocytic cells exhibit reduced rolling adhesion persistence, whereas both cell subtypes exhibited reduced persistence on L-selectin and high persistence on E-selectin, differences not revealed by flow cytometry analysis or reflected in the extent or velocity of rolling adhesion. Conditions under which adhesion persistence was found to be significantly reduced corresponded to those exhibiting the greatest sensitivity to a selectin-antagonist. Our results suggest that potentially therapeutically exploitable differences in metastatic and leukocytic cell subtype interactions with selectins in physiological flow are identifiable through implementation of functional assays of adhesion persistence in hemodynamic flow utilizing this integrated, flow-based cell adhesion chromatography analytical technique.

P-Selectin and ICAM-1 synergy in mediating THP-1 monocyte adhesion in hemodynamic flow is length dependent.

The tightly orchestrated recruitment of monocytes, whose progeny are critical to the progression and resolution of various physiological and pathophysiological processes, is implicated in the time course, severity, and resolution of pathology. Using a microfluidic-based cell adhesion assay integrating spatiotemporal analyses and micropatterning of adhesive proteins, we interrogated the effects of adhesive molecule presentation length, which varies in vivo with disease and stage, on THP-1 monocyte cell rolling versus firm adhesion mediated by P-selectin and/or ICAM-1 in hemodynamic flow. Our results indicate that co-presentation of P-selectin and ICAM-1 substantially decreases the length of adhesive substrate required to sustain adhesion in flow and that P-selectin functions synergistically with ICAM-1 to substantially enhance THP-1 firm adhesion. This synergy was found to furthermore correlate with diminished cell rolling velocities and length-enhanced secondary cell capture. Our results suggest pathophysiological ramifications for local remodeling of the inflamed microvascular microenvironment in directing the efficiency of monocyte trafficking.