Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors

Abstract

Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

Intratumoral Heterogeneity Promotes Collective Cancer Invasion Through NOTCH1 Variation

“…Most microtumors stayed at the medium-Matrigel interface or in the Matrigel, and no cells were able to reach into the collagen layer (i.e., 0%). This observation is consistent with the view that DLL4 supports collective invasion of cancer (Dean et al, 2016;Riahi et al, 2015;Torab et al, 2020). On the other hand, NOTCH1 transient knockdown was performed on individual cell line or both cell lines in the co-culture invasion assay (Fig.…”

Nrf2 modulates the hybrid epithelial/mesenchymal phenotype and Notch signaling during collective cancer migration

“…Biosensors were added prior to the wound healing assay to ensure uniform probe internalization. 23,35 Images of live migrating cells were acquired 24 hours post wound scratch to characterize the gene expression. Left panel shows fluorescence images for WT and sulforaphane cases measuring miR-200c, Notch1, and Dll4 with the single cell biosensor ( Figure 5A-C, left panel ) and in the FISH assay ( Figure 5D-F, left panel ).…”

“…Notch signaling has been shown to control collective cell migration. 19,22,23,[30][31][32][33] We therefore measured the distributions of Notch markers (i.e., Notch1, Dll4, and Jagged1) in the WT and under Nrf2 perturbations (Figure 4A-C). In agreement with previous studies, 19,23 Notch signaling was upregulated near the leading edge.…”

“…19,22,23,[30][31][32][33] We therefore measured the distributions of Notch markers (i.e., Notch1, Dll4, and Jagged1) in the WT and under Nrf2 perturbations (Figure 4A-C). In agreement with previous studies, 19,23 Notch signaling was upregulated near the leading edge. Spatial gradients of Notch1, Dll4, and Jagged1 near the leading edge were observed, and the expression levels were dependent on Nrf2.…”

A gapmer aptamer nanobiosensor for real-time monitoring of transcription and translation in single cells

“…5). Unlike transfection of molecular beacons and double-stranded probes 28–30 , the nanobiosensor was delivered into the cytoplasm without nuclear accumulation (Supplementary Fig. 1c).…”