In vivo microscopy reveals macrophage polarization locally promotes coherent microtubule dynamics in migrating cancer cells

Gaurav Luthria,Stephanie Wang,Miles A. Miller,Mark Prytyskach,Douglas A. Lauffenburger,Ralph Weissleder,Rainer H. Kohler,Ran Li,Timothy J. Mitchison

doi:10.1038/s41467-020-17147-y

Abstract

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Yet in vivo analysis of cancer cell MT behavior within the tumor microenvironment remains challenging. Here we developed an imaging pipeline using plus-end tip tracking and intravital microscopy to quantify MT dynamics in live xenograft tumor models. Among analyzed features, cancer cells in vivo displayed higher coherent orientation of MT dynamics along their cell major axes compared with 2D in vitro cultures, and distinct from 3D collagen gel cultures. This in vivo MT phenotype was reproduced in vitro when cells were co-cultured with IL4-polarized MΦ. MΦ depletion, MT disruption, targeted kinase inhibition, and altered MΦ polarization via IL10R blockade all reduced MT coherence and/or tumor cell elongation. We show that MT coherence is a defining feature for in vivo tumor cell dynamics and migration, modulated by local signaling from pro-tumor macrophages.

Highlights

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer
We developed an integrated pipeline combining in vivo confocal microscopy[17], automated plus-end tip tracking of individual MTs18, and multivariate statistics to study MT dynamics in live xenograft models of cancer
We examined a spectrum of MT dynamic features across thousands of individual MTs and applied multivariate statistical methods to interpret patterns in MT behavior across experimental conditions

Summary

Introduction

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Multiple signaling pathways influence MT dynamics, including via receptor tyrosine kinases (RTKs) and G-protein coupled receptors[15,16] It is unclear how these act in the TME where multiple such pathways operate simultaneously, begging the question: if MT dynamics and cell migration occur so distinctly in various tissue culture models, how do MTs behave in vivo?. We developed an integrated pipeline combining in vivo confocal (intravital) microscopy[17], automated plus-end tip tracking of individual MTs18, and multivariate statistics to study MT dynamics in live xenograft models of cancer In multiple models, this approach revealed that in vivo, MT growth in cancer cells was aligned along the cell major axis and coherently with each other in the same cell, which correlated with elongated cell morphology, formation of MT-rich pseudopod-like structures, and cell migration. We present a platform for examining the in situ dynamics of MTs in live xenograft models of cancer, revealing MT coherence as a defining feature of in vivo tumor cell motility, and that pro-tumor MΦ signaling can produce such MT coherence in neighboring tumor cells

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Conclusion