ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

The bone-marrow (BM) niche is the spatial environment composed by a network of multiple stromal components regulating adult hematopoiesis. We use multi-omics and computational tools to analyze multiple BM environmental compartments and decipher their mutual interactions in the context of acute myeloid leukemia (AML) xenografts. Under homeostatic conditions, we find a considerable overlap between niche populations identified using current markers. Our analysis defines eight functional clusters of genes informing on the cellular identity and function of the different subpopulations and pointing at specific stromal interrelationships. We describe how these transcriptomic profiles change during human AML development and, by using a proximity-based molecular approach, we identify early disease onset deregulated genes in the mesenchymal compartment. Finally, we analyze the BM proteomic secretome in the presence of AML and integrate it with the transcriptome to predict signaling nodes involved in niche alteration in AML.

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

Despite substantial clinical benefit of targeted and immune checkpoint blockade-based therapies in melanoma, resistance inevitably develops. We show cytoskeletal remodeling and changes in expression and activity of ROCK-myosin II pathway during acquisition of resistance to MAPK inhibitors. MAPK regulates myosin II activity, but after initial therapy response, drug-resistant clones restore myosin II activity to increase survival. High ROCK-myosin II activity correlates with aggressiveness, identifying targeted therapy- and immunotherapy-resistant melanomas. Survival of resistant cells is myosin II dependent, regardless of the therapy. ROCK-myosin II ablation specifically kills resistant cells via intrinsic lethal reactive oxygen species and unresolved DNA damage and limits extrinsic myeloid and lymphoid immunosuppression. Efficacy of targeted therapies and immunotherapies can be improved by combination with ROCK inhibitors.

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation.

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy.

Radiotherapy is one of the most effective approaches to achieve tumor control in cancer patients, although healthy tissue injury due to off-target radiation exposure can occur. In this study, we used a model of acute radiation injury to the lung, in the context of cancer metastasis, to understand the biological link between tissue damage and cancer progression. We exposed healthy mouse lung tissue to radiation before the induction of metastasis and observed a strong enhancement of cancer cell growth. We found that locally activated neutrophils were key drivers of the tumor-supportive preconditioning of the lung microenvironment, governed by enhanced regenerative Notch signaling. Importantly, these tissue perturbations endowed arriving cancer cells with an augmented stemness phenotype. By preventing neutrophil-dependent Notch activation, via blocking degranulation, we were able to significantly offset the radiation-enhanced metastases. This work highlights a pro-tumorigenic activity of neutrophils, which is likely linked to their tissue regenerative functions.

An elevated number of circulating neutrophils is a poor prognostic factor for breast cancer, where evidence of bone marrow cancer-dependent priming is found. However, how early this priming is detectable remains unclear.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 μm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1β. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Basement membrane matrix proteins, such as matrigel, are able to improve the efficiency of tumour transplantation. This assay represents the gold standard to measure tumour initiation potential in vivo of a limited number of cancer cells. However, in culture conditions, matrigel directly signals to cancer cells altering their phenotype. We here investigate how matrigel influences the tumour reconstitution dynamics of breast cancer cells in vivo. This is particularly relevant in the setting of limiting dilution assay where cells are transplanted in a relatively high amount of Matrigel. We show that matrigel initially induces a normalized growth of transplanted MMTV-PyMT breast tumours cells. This occurs in the context of a matrigel-segregation effect where cancer cells are transiently isolated from host tissue. We identify macrophages as gatekeepers of the cancer-host cell interaction: depriving transplants from macrophages locked cancer cells in this isolated environment where they fail to form tumours despite retaining their intrinsic tumorigenic potential. This is a decisive proof of concept that cancer cells' malignant behaviour can be dominated by their microenvironment. Moreover, considering that diverse breast cancer cells are differently subjected to a segregation effect, this needs to be considered when comparing tumour initiation potential of different cancer cells.

Cancer cells thrive when embedded in a fine-tuned cellular and extracellular environment or tumour microenvironment (TME). There is a general understanding of a co-evolution between cancer cells and their surrounding TME, pointing at a functional connection between cancer cells characteristics and the perturbations induced in their surrounding tissue. However, it has never been formally proven whether this functional connection needs to be set from the start or if aggressive cancer cells always dominate their microenvironmental any point in time. This would require a dedicated experimental setting where malignant cells are challenged to grow in a different TME from the one they would naturally create. Here we generated an experimental setting where we transiently perturb the secretory profile of aggressive breast cancer cells without affecting their intrinsic growth ability, which led to the initial establishment of an atypical TME. Interestingly, even if initially tumours are formed, this atypical TME evolves to impair long term in vivo cancer growth. Using a combination of in vivo transcriptomics, protein arrays and in vitro co-cultures, we found that the atypical TME culminates in the infiltration of macrophages with STAT1high activity. These macrophages show strong anti-tumoural functions which reduce long-term tumour growth, despite lacking canonical M1 markers. Importantly, gene signatures of the mesenchymal compartment of the TME, as well as the anti-tumoural macrophages, show striking prognostic power that correlates with less aggressive human breast cancers.

Centrosomal abnormalities, in particular centrosome amplification, are recurrent features of human tumors. Enforced centrosome amplification in vivo plays a role in tumor initiation and progression. However, centrosome amplification occurs only in a subset of cancer cells, and thus, partly due to this heterogeneity, the contribution of centrosome amplification to tumors is unknown. Here, we show that supernumerary centrosomes induce a paracrine-signaling axis via the secretion of proteins, including interleukin-8 (IL-8), which leads to non-cell-autonomous invasion in 3D mammary organoids and zebrafish models. This extra centrosomes-associated secretory phenotype (ECASP) promotes invasion of human mammary cells via HER2 signaling activation. Further, we demonstrate that centrosome amplification induces an early oxidative stress response via increased NOX-generated reactive oxygen species (ROS), which in turn mediates secretion of pro-invasive factors. The discovery that cells with extra centrosomes can manipulate the surrounding cells highlights unexpected and far-reaching consequences of these abnormalities in cancer.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Understanding cell-cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell-cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2-3 weeks to generate the labeling cells and 1-2 weeks to test their labeling ability.