Apoptotic Bodies in the Pancreatic Tumor Cell Culture Media Enable Label‐Free Drug Sensitivity Assessment by Impedance Cytometry

Abstract

The ability to rapidly and sensitively predict drug response and toxicity using in vitro models of patient‐derived tumors is essential for assessing chemotherapy efficacy. Currently, drug sensitivity assessment for solid tumors relies on imaging adherent cells or by flow cytometry of cells lifted from drug‐treated cultures after fluorescent staining for apoptotic markers. Subcellular apoptotic bodies (ABs), including microvesicles that are secreted into the culture media under drug treatment can potentially serve as markers for drug sensitivity, without the need to lift cells under culture. However, their stratification to quantify cell disassembly is challenging due to their compositional diversity, with tailored labeling strategies currently needed for the recognition and cytometry of each AB type. It is shown that the high frequency impedance phase versus size distribution of ABs determined by high‐throughput single‐particle impedance cytometry of supernatants in the media of gemcitabine‐treated pancreatic tumor cultures exhibits phenotypic resemblance to lifted apoptotic cells and enables shape‐based stratification within distinct size ranges, which is not possible by flow cytometry. It is envisioned that this tool can be applied in conjunction with the appropriate pancreatic tumor microenvironment model to assess drug sensitivity and toxicity of patient‐derived tumors, without the need to lift cells from cultures.

Automated biophysical classification of apoptotic pancreatic cancer cell subpopulations by using machine learning approaches with impedance cytometry

“…Furthermore, with the onset of the "secondary necrosis" onward from late apoptosis, the plasma membrane becomes progressively permeabilized to lead to alterations similar to those discussed previously for necrosis, i.e., a significant decrease in both ϕZ 0.5 MHz and ϕZ 2 MHz (≤ **p < 0.01 & ≤ **p < 0.01, respectively), and a significant increase in ϕZ 18 MHz and ϕZ 30 MHz (≤ **p < 0.01 & ≤ *p < 0.05, respectively) due to ion exchange between the intracellular and external media. The transition from early to late apoptosis also leads to the formation and shedding of apoptotic bodies that vary in size, shape and composition during drug-induced cellular disassembly for removing fragmented internal components, 35 which would lead to a sharp decrease in cell size, as detected by electrical diameter from our simulations (Fig. S6D †) and measured results (Fig.…”

“…33,34 Furthermore, the frequency spectra of single-cell impedance phenotypes can be fit to dielectric shell models to obtain a biophysical picture to identify each cell type in a heterogeneous sample and gauge the myriad subcellular alterations over a spectrum of drug-induced transformations. 35 Prior dielectric approaches have recognized the cell death mechanism, 36 trapped specific cells for molecular analysis, 37 and classified their drug-induced transformations within the viable, necrotic, and apoptotic categories, 38 but subpopulations over the progression of apoptotic alterations were not quantified. Furthermore, quantification of the subpopulations was by manual gating strategies, which is limited by overlap of the respective data clusters, motivating the consideration of automated strategies for impedance data classification.…”

On‐chip microfluidic buffer swap of biological samples in‐line with downstream dielectrophoresis

“…Hence, label‐free cellular separations based on their inherent biophysical properties are gaining interest, with microfluidic systems offering a dynamic platform with sufficient force fields for controlled deflection of a particular cell type from heterogeneous samples. Platforms for cell separations based on biophysical differences [8] in size [9], shape [10], deformability [11], and electrical properties [12] are particularly of great interest to quantify heterogeneity of cellular systems.…”

Rapid assessment of susceptibility of bacteria and erythrocytes to antimicrobial peptides by single-cell impedance cytometry

“…The technique measures the electrical phenotype of individual biological cells and has been applied to mammalian cells, human pathogens, yeast cells, and plant cells (Daguerre et al, 2020;de Bruijn et al, 2021;Gökçe et al, 2021;Honrado et al, 2021b;Kruit et al, 2022;Petchakup et al, 2018;Tang et al, 2021;Wang et al, 2022). The sensitivity of the technique to alterations of cell size, membrane, and interior composition makes it particularly suitable for cell viability applications (De Ninno et al, 2020;Honrado et al, 2022Honrado et al, , 2021aZhong et al, 2021). David et al performed impedance-based viability analysis of Bacillus megaterium (B. megaterium) with cells at different growth stages and heat-inactivated cells (David et al, 2012).…”