A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation

Abstract

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

Dynamic Magneto-Softening of 3D Hydrogel Reverses Malignant Transformation of Cancer Cells and Enhances Drug Efficacy

“…In contrast, no obvious lumen formation was found in “all-stiffened” hydrogel, and the spheroid had denser cell organization (Figure S16b). In addition, previous studies have shown that lumen formation of MCF-7 is related to cell apoptosis due to hypoxia or autophagy. , However, it is worth noting that their spheroid size is much larger than ours (they used anchorage-independent growth protocol instead of our 3D scaffolds), and a dark area in the central region which represented “cell death region” was not observed here. To better understand the structure of the spheroid, we used DAPI to stain the cell nucleus on day 17.…”

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

“…Three-dimensional tumor models, such as spheroids, offers an improved model to assess molecular and physiological aspects that are essential for drug development including drug penetration, hypoxic/necrotic environment, stemness and cell interaction, among many others 19,20 . Traditionally, spheroid models have been technically challenging, especially from a proteomics perspective, due to the low protein amount obtained from single spheroids, which typically requires pooling of several spheroids per condition to achieve a reasonable proteome coverage [21][22][23] . These limitations are even more evident when studying the phosphoproteome layer, due to the need for phosphoenrichment prior to LC-MS/MS measurements.…”

Claudin-18.2 mediated interaction of gastric Cancer cells and Cancer-associated fibroblasts drives tumor progression

“…We used proximity labeling technology [ 37 ] to elucidate CLDN18.2’s mechanism in promoting the juxtacrine interaction between gastric cancer cells and CAFs. We overexpressed CLDN18.2 in gastric cancer cells with an overexpressed biotin tag, and after subcutaneous injection into a mouse tumor model, we used mass spectrometry to confirmed that CLDN18.2 may produce a series of tumor-promoting effects by interacting with S100A4 in the tumor microenvironment.…”

“…We used proximity labeling technology [37] to elucidate CLDN18.2's mechanism in promoting the juxtacrine interaction between gastric cancer cells and CAFs. We The N-terminus of CLDN18.2 was coupled to HA-turboID with a linker, HA-turboID was coupled to the N-terminus of PDFGRB with a linker.…”