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Two related papers show that cells disseminated from malignant lesions at early time points during tumorigenesis can contribute to metastases at distant organs and provide insights into the molecular basis of dissemination. A potential mechanism for metastases The origin of metastases in cancer remains an open question. In a pair of linked papers, Christoph Klein, Julio Aguirre-Ghiso and colleagues now show in mouse models that cells disseminated from tumours early in tumorigenesis can contribute to metastases at distant organs at such early time points. Both papers also provide insights into the molecular basis of dissemination, which may be useful as targets to prevent metastasis. Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant 1 . Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours 2 . However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases 3 , 4 , 5 . These findings, and those in pancreatic cancer 6 and melanoma 7 models, indicate that dissemination might occur during the early stages of tumour evolution 3 , 8 , 9 . However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown 8 . Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2 + p-p38 lo p-Atf2 lo Twist1 hi E-cad lo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2 + eDCC precursors invaded locally, intravasated and lodged in target organs. Her2 + eDCCs activated a Wnt-dependent epithelial–mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1 hi E-cad lo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.

The window for high-resolution imaging of the lung (WHRIL) enables longitudinal imaging of the same region of murine lung tissue over a period of weeks, and this enables the visualization of spontaneous cancer metastasis from the earliest stages.Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy. The understanding of the mechanisms underlying the ability of disseminated tumor cells (DTCs) to form metastasis is incomplete. Here, by using high-resolution intravital imaging of the murine lung to track the fate of breast-derived DTCs, the authors show that macrophages within the primary tumor induce a pro-dissemination and pro-dormancy phenotype in tumor cells, favouring their extravasation in the lung.

Tumor-associated macrophages (TAMs) are a phenotypically diverse, highly plastic population of cells in the tumor microenvironment (TME) that have long been known to promote cancer progression. In this review, we summarize TAM ontogeny and polarization, and then explore how TAMs enhance tumor cell migration through the TME, thus facilitating metastasis. We also discuss how chemotherapy and host factors including diet, obesity, and race, impact TAM phenotype and cancer progression. In brief, TAMs induce epithelial-mesenchymal transition (EMT) in tumor cells, giving them a migratory phenotype. They promote extracellular matrix (ECM) remodeling, allowing tumor cells to migrate more easily. TAMs also provide chemotactic signals that promote tumor cell directional migration towards blood vessels, and then participate in the signaling cascade at the blood vessel that allows tumor cells to intravasate and disseminate throughout the body. Furthermore, while chemotherapy can repolarize TAMs to induce an anti-tumor response, these cytotoxic drugs can also lead to macrophage-mediated tumor relapse and metastasis. Patient response to chemotherapy may be dependent on patient-specific factors such as diet, obesity, and race, as these factors have been shown to alter macrophage phenotype and affect cancer-related outcomes. More research on how chemotherapy and patient-specific factors impact TAMs and cancer progression is needed to refine treatment strategies for cancer patients.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours. The key feature is independent tunability of emission wavelength, extinction coefficient and quantum yield through distinct structural domains in the nanocrystal. Precise tuning eliminates a 100-fold red-to-green brightness mismatch of size-tuned quantum dots at the ensemble and single-particle levels, which substantially improves quantitative imaging accuracy in biological tissue. We anticipate that these materials engineering principles will vastly expand the optical engineering landscape of fluorescent probes, facilitate quantitative multicolour imaging in living tissue and improve colour tuning in light-emitting devices. Quantum dots with different size emit light at different wavelengths but also different brightness, which complicates analysis of fluorescence images. Here, the authors synthesize multicolour brightness-equalized quantum dots by controlling the composition and structure of core-shell HgCdSeS-CdZnS nanocrystals.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites. Intravital imaging reveals macrophage-driven de novo induction of cancer stem cells in vivo, and their dramatic enrichment on dissemination through TMEM doorways. These findings provide a mechanism for the validated ability of TMEM doorway density to be prognostic for distant recurrence of metastatic tumors in breast cancer patients.

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin regulatory protein Mena, encoded by the ENAH gene, that endows tumor cells with transendothelial migration activity, allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer, we further determined that for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling activation and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Although cancer immunotherapy has resulted in unpreceded survival benefits to subsets of oncology patients, accumulating evidence from preclinical animal models suggests that the immunosuppressive tumor microenvironment remains a detrimental factor limiting benefit for many patient subgroups. Recent efforts on lymphocyte-mediated immunotherapies are primarily focused on eliminating cancer foci at primary and metastatic sites, but few studies have investigated the impact of these therapies on the highly complex process of cancer cell dissemination. The metastatic cascade involves the directional streaming of invasive/migratory tumor cells toward specialized blood vessel intravasation gateways, called TMEM doorways, to the peripheral circulation. Importantly, this process occurs under the auspices of a specialized tumor microenvironment, herewith referred to as “Dissemination Trajectory”, which is supported by an ample array of tumor-associated macrophages (TAMs), skewed towards an M2-like polarization spectrum, and which is also vital for providing microenvironmental cues for cancer cell invasion, migration and stemness. Based on pre-existing evidence from preclinical animal models, this article outlines the hypothesis that dissemination trajectories do not only support the metastatic cascade, but also embody immunosuppressive niches, capable of providing transient and localized immunosubversion cues to the migratory/invasive cancer cell subpopulation while in the act of departing from a primary tumor. So long as these dissemination trajectories function as “immune deserts”, the migratory tumor cell subpopulation remains efficient in evading immunological destruction and seeding metastatic sites, despite administration of cancer immunotherapy and/or other cytotoxic treatments. A deeper understanding of the molecular and cellular composition, as well as the signaling circuitries governing the function of these dissemination trajectories will further our overall understanding on TAM-mediated immunosuppression and will be paramount for the development of new therapeutic strategies for the advancement of optimal cancer chemotherapies, immunotherapies, and targeted therapies.

Tissues are heterogeneous with respect to cellular and non-cellular components and in the dynamic interactions between these elements. To study the behaviour and fate of individual cells in these complex tissues, intravital microscopy (IVM) techniques such as multiphoton microscopy have been developed to visualize intact and live tissues at cellular and subcellular resolution. IVM experiments have revealed unique insights into the dynamic interplay between different cell types and their local environment, and how this drives morphogenesis and homeostasis of tissues, inflammation and immune responses, and the development of various diseases. This Primer introduces researchers to IVM technologies, with a focus on multiphoton microscopy of rodents, and discusses challenges, solutions and practical tips on how to perform IVM. To illustrate the unique potential of IVM, several examples of results are highlighted. Finally, we discuss data reproducibility and how to handle big imaging data sets.