Journal: Nature Communications

Article Title: Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

doi: 10.1038/s41467-021-22985-5

Figure Lengend Snippet: a Super-resolution STED imaging used to generate 3D views of T cells on ICAM1-PAAG nanotopographies ( G ’ = 50 kPa in this example) to characterize T-cell interactions with nanotopography. Views: 1, stereometric; 2, cross-section; 3, interface from atop (arrowheads, “in-groove” invasions); and 4, F-actin (phalloidin). Colors: white, ICAM1; fire, F-actin; blue, nuclei. b Projection schematics of nanotexture–T cell interactions, showing simultaneous “on-ridge” and “in-groove” dynamics. The nano-“ridge/groove” configuration supports both (1) unconfined, multidirectional “on-ridge” lamellipodium (LP), and (2) “in-groove” sterically interactive pseudopodium. “In-groove” T-cell dentations are sterically confined and are guided along the nanogrooves, driving T-cell contact guidance. c T-cell motility in 3D extracellular matrix where extension of invasive pseudopodium and rear retraction propel T cells through the 3D environment. Although 3D pseudopodia may not be confined to singular motility direction, they remain analogous to directed “in-groove” dentations. d Our model for the mechanomodulated “in-groove” (i.e., more amoeboid-like phenotype with actin-rich pseudopodia) and “on-ridge” (i.e., more mesenchymal-like phenotype, which for T cells is defined as lamellipodia driven protrusions during migration, particular on flat environments) dynamics can be competitive and regulate the amoeboid-mesenchymal plasticity balance. Our model predicts “on-ridge” mesenchymal spreading enhancement on rigid nanotopography outbalances and antagonizes “in-groove” amoeboid invasiveness. Alternatively, soft nanotextures shift the balance towards amoeboid “in-groove” steric cell invasiveness. e Metrics (left) and measurement (right) of T-cell “in-groove” relative invasiveness as a ratio between area of invasive regions and entire T-cell area at the topography interface (see Eq. in the “Methods” section). Invasiveness is a function of nanotopography rigidity G ’ and microtubule stability. G ’ range: 16–50 kPa (PAAG) to ≫1000 kPa (pU: polyurethane plastic). Remarkably, at ICAM1-PAAG nanotextures of G ’ < 16 kPa hCD4+ T cells fail to attach to the nanotopographies, indicating G ’ ≈ 16 kPa may be the lower mechanosensing limit for hCD4+ T cells on ICAM1. Individual dots correspond to the individual cells. Box plots depict the 25th percentile, median, and 75th percentile, and whiskers depict the 95% confidence intervals. Statistical tests are one-way ANOVA, Tukey’s multiple comparisons tests. All n - and p -values are shown on the plots. Number of replicates per condition: 3. Source data are provided as a Source Data file.

Article Snippet: The resultant fluorescent PAA nanopatterns of Fab anti-Fc antibody fragment protein were incubated overnight with 1 mg/mL ICAM1-Fc chimeric protein (Sino Biological, China) in cold PBS (Gibco, USA) at 4 °C, rinsed, and used for experiments.

Techniques: Imaging, Migration

Journal: Nature Communications

Article Title: Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

doi: 10.1038/s41467-021-22985-5

Figure Lengend Snippet: a Left to right: schematic, 3D micrograph, and “in-groove” cross-section and side views of human T cells on ICAM1 nanotextures (grooves/ridges widths = 800 nm). Top to bottom: control (+DMSO), destabilized MTs (+Nocodazole), and stabilized MTs (+Taxol) showing increased “in-groove” actin-rich pseudopodia in cells with destabilized MTs, in contrast to more mesenchymal-like behavior with Taxol. Nocodazole-induced MT disassembly and Taxol-induced MT stabilization are shown on immunofluorescence panels (green). Arrowheads indicate “in-groove” protrusions. Colors: light blue, polyurethane nanotexture; green, microtubules; red, F-actin; dark blue, nuclei. b T cells migration tracks on compliant ( G ’ = 16 kPa), intermediate ( G ’ = 50 kPa), or rigid ( G ’ ≫ 1000 kPa) ICAM1 nanotextures, where compliant nanotopographies enhance contact guidance. Top to bottom: T-cell migration under control (+DMSO), Nocodazole, or Taxol treatment conditions, where MT disassembly results in the enhanced directed migration across all rigidities. All n -values are shown on the plots. Number of replicates for per case: 3. Source data are provided as a Source Data file. c T-cell averaged per cell speeds on ICAM1 nanotextures of various rigidities (16, 50, and ≫1000 kPa) for control (+DMSO, top), Nocodazole (middle), and Taxol (bottom) conditions. Individual dots correspond to the individual cells. Statistical tests are one-way ANOVA, Tukey’s multiple comparisons test. All n - and p -values are depicted on the plots. Number of replicates for each condition: 3. Source data are provided as a Source Data file. d T-cell phenotype transitions from the mixed “in-groove” and “on-ridge” phenotype to the more amoeboid “in-groove” dominant phenotype after MT disassembly from Nocodazole treatment. Micrographs are generated with human CD4+ T cells on rigid polyacrylamide gels ( G ’ = 50 kPa), functionalized with human ICAM1. Sequence 1 capture (see Supplementary Movies and ) displays a well-developed lamellipodium and non-aligned motility during vehicle treatment as a control condition, whereas Nocodazole treatment induces amoeboid dynamics, pseudopodia, and migration along the nanogrooves (Sequence 2 capture; Supplementary Movies and ). Corresponding STED super-resolution images show detailed T cell–nanogrooves interactions. Arrowheads indicate lamellipodia in DMSO (left) or pseudopodia in Nocodazole (right). e Left, schematics of our model for T cell–ICAM1 nanotexture interactions. Right, mechanosensitive “in-groove” steric invasion and “on-ridge” mesenchymal-like (lamellipodial-based) interactions as a function of the mechanical rigidity of the microenvironment and cell contractility. f Quantification of directionality of human T cells migration during contact guidance as a function of substrate mechanical rigidity and the state of microtubules. Corresponding T-cell migration tracks are shown in the same matrix order in b . Measurements represent frequency distributions of cell-to-nanogroove angles every 10 s step. All n -values are indicated on the plot. Number of replicates for each condition: 3. Source data are provided as a Source Data file.

Article Snippet: The resultant fluorescent PAA nanopatterns of Fab anti-Fc antibody fragment protein were incubated overnight with 1 mg/mL ICAM1-Fc chimeric protein (Sino Biological, China) in cold PBS (Gibco, USA) at 4 °C, rinsed, and used for experiments.

Techniques: Immunofluorescence, Migration, Generated, Sequencing