The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types1,2. Intratumoral host–microbiota studies have so far largely relied on bulk tissue analysis1–3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies4 and single-cell RNA sequencing5 to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host–microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling6, we show that bacterial communities populate microniches that are less vascularized, highly immuno‑suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion–adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.

Immunotherapies based on the autologous adoptive transfer of ex vivo-manipulated T cells are rapidly evolving for the treatment of both metastatic and primary malignancies. However, extended ex vivo culturing reduces the functionality of isolated T cells. Cryopreservation of rapidly expanded T cells for subsequent use throughout an immunotherapeutic regimen is a highly desirable recourse, thus far encumbered by a lack of studies investigating its effects on effector T-cell functionality. Here we directly compare murine tumour-reactive CD8+ T cells cryopreserved during ex vivo expansion to freshly isolated populations. We show that cryopreservation fully conserves the differentiation potential of effector T cells, secretion of pro-inflammatory cytokines, cytotoxic function and does not impair the three-dimensional scanning motility of T cells or their capacity to infiltrate and reject tumours.

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8 + T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activationinduced gains in fluorescence were also observed in naïve and CD4 + T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Scientific Reports 6: Article number: 37909; published online: 02 December 2016; updated: 07 March 2017. The original version of this Article contained an error in the spelling of the author Jorge Luis Galeano Niño, which was incorrectly given as Jorge Luis Galenano-Niño. Additionally, there were typographical errors in Affiliation 9 which was incorrectly listed as ‘Sydney Medical School, The University of Sydney, NSW, 2006, Australia.

The potential to migrate is one of the most fundamental functions for various epithelial, mesenchymal and immune cells. Image analysis of motile cell populations, both primary and cultured, typically reveals an intercellular variability in migration speeds. However, cell migration chromatography, the sorting of large populations of cells based on their migratory characteristics, cannot be easily performed. The lack of such methods has hindered our understanding of the direct correlation between the capacity to migrate and other cellular properties. Here, we report two novel, easily implementable and readily scalable methods to sort millions of live migratory cancer and immune cells based on their spontaneous migration in 2D and 3D microenvironments respectively. Correlative downstream transcriptomic, molecular and functional tests reveal marked differences between the fast and slow subpopulations in patient-derived cancer cells. We further employ our method to reveal that sorting the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This phenotypic assay opens new avenues for the precise characterization of the mechanisms underlying hitherto unexplained heterogeneities in migratory phenotypes within a cell population, and for the targeted enrichment of the most potent migratory leukocytes in immunotherapies. [Media: see text] [Media: see text] [Media: see text]