TGF-ß Type I Receptor Signaling in Melanoma Liver Metastases Increases Metastatic Outgrowth.

Despite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is needed. The multifunctional cytokine Transforming Growth Factor ß (TGF-ß) plays various roles in melanoma tumors and metastasis, affecting both tumor cells and cells from the surrounding tumor microenvironment. To study the role of TGF-ß in melanoma liver metastasis, we created a model to activate or repress the TGF-ß receptor pathway in vitro and in vivo in an inducible manner. For this, we engineered B16F10 melanoma cells to have inducible ectopic expression of a constitutively active (ca) or kinase-inactive (ki) TGF-ß receptor I, also termed activin receptor-like kinase (ALK5). In vitro, stimulation with TGF-ß signaling and ectopic caALK5 expression reduced B16F10 cell proliferation and migration. Contrasting results were found in vivo; sustained caALK5 expression in B16F10 cells in vivo increased the metastatic outgrowth in liver. Blocking microenvironmental TGF-ß did not affect metastatic liver outgrowth of both control and caALK5 expressing B16F10 cells. Upon characterizing the tumor microenvironment of control and caALk5 expressing B16F10 tumors, we observed reduced (cytotoxic) T cell presence and infiltration, as well as an increase in bone marrow-derived macrophages in caALK5 expressing B16F10 tumors. This suggests that caALK5 expression in B16F10 cells induces changes in the tumor microenvironment. A comparison of newly synthesized secreted proteins upon caALK5 expression by B16F10 cells revealed increased secretion of matrix remodeling proteins. Our results show that TGF-ß receptor activation in B16F10 melanoma cells can increase metastatic outgrowth in liver in vivo, possibly through remodeling of the tumor microenvironment leading to altered infiltration of immune cells. These results provide insights in the role of TGF-ß signaling in B16F10 liver metastasis and could have implications regarding the use of TGF-ß inhibitors for the treatment of melanoma patients with liver metastasis.

Low Transforming Growth Factor-ß Pathway Activity in Cervical Adenocarcinomas.

Cervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse prognosis. Over the last two decades, incidence rates of AC have increased, highlighting the importance of further understanding AC tumorigenesis, and the need to investigate new treatment options. The cytokine TGF-ß functions as a tumour suppressor in healthy tissue. However, in tumour cells this suppressive function can be overcome. Therefore there is an increasing interest in using TGF-ß inhibitors in the treatment of cancer. Here, we hypothesize that TGF-ß plays a different role in SCC and AC. Analysis of RNA-seq data from the TCGA, using a TGF-ß response signature, resulted in separate clustering of the two subtypes. We further investigated the expression of TGF-ß-signalling related proteins (TßR1/2, SMAD4, pSMAD2, PAI-1, αvß6 and MMP2/9) in a cohort of 62 AC patients. Low TßR2 and SMAD4 expression was associated with worse survival in AC patients and interestingly, high PAI-1 and αvß6 expression was also correlated with worse survival. Similar correlations of TßR2, PAI-1 and αvß6 with clinical parameters were found in previously reported SCC analyses. However, when comparing expression levels between SCC and AC patient samples, pSMAD2, SMAD4, PAI-1 and αvß6 showed lower expression in AC compared to SCC. Because of the low expression of core TßR1/2, (p-)SMAD2 and SMAD4 proteins and the correlation with worse prognosis, TGF-ß pathway most likely leads to tumour inhibitory effects in AC and therefore the use of TGF-ß inhibitors would not be recommended. However, given the correlation of PAI-1 and αvß6 with poor prognosis, the use of TGF- ß inhibitors might be of interest in SCC and in the subsets of AC patients with high expression of these TGF-ß associated proteins.

TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor-associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells. IMPLICATIONS: Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

Dynamic Visualization of TGF-ß/SMAD3 Transcriptional Responses in Single Living Cells.

Transforming growth factor-ß (TGF-ß) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-ß/SMAD3 signaling kinetics, we developed a dynamic TGF-ß/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-ß/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-ß challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-ß/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

Visualizing Dynamic Changes During TGF-ß-Induced Epithelial to Mesenchymal Transition.

Epithelial to mesenchymal transition (EMT) is crucial during embryonic development, tissue fibrosis, and cancer progression. Epithelial cells that display a cobblestone-like morphology can undergo a switch to mesenchymal-like phenotype, displaying an elongated spindle shape or a fibroblast-like morphology. EMT is characterized by timely and reversible alterations of molecular and cellular processes. The changes include loss of epithelial and gain of mesenchymal marker expression, loss of polarity, increased cell migratory and invasive properties. Epithelial cells can progress unevenly during this transition and attain hybrid E/M states or metastable EMT states, referred to as epithelial cell plasticity. To gain a deeper insight into the mechanism of EMT, understanding the dynamic aspects of this process is essential. One of the most prominent factors to induce EMT is the cytokine transforming growth factor-ß (TGF-ß). This chapter discusses molecular and cellular techniques to monitor TGF-ß-induced signaling and EMT changes in normal and cancer cell lines. These methods include measuring the TGF-ß-induced activation of its intracellular SMAD effectors proteins and changes in epithelial/mesenchymal marker expression and localization. Moreover, we describe assays of cell migration and dynamic reorganization of the actin cytoskeleton and stress filaments that are frequently part of the TGF-ß-induced EMT cellular response.

Breast cancer dormancy is associated with a 4NG1 state and not senescence.

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

An Experimental Liver Metastasis Mouse Model Suitable for Short and Long-Term Intravital Imaging.

The liver is a frequent site of cancer metastasis, but current treatment options for cancer patients with liver metastasis are limited, resulting in poor prognosis. Colonization of the liver by cancer cells is a multistep and temporally controlled process. Investigating this process in biological relevant settings in a dynamic manner may lead to new therapeutic avenues. Experimental mouse models of liver metastasis combined with high-resolution microscopy methods can facilitate study of the mechanisms that underlie the outgrowth of cancer cells in the liver. Intravital imaging can provide information on the behavior of tumor cells in their biological setting, in time frames of hours to days. In this unit, we describe the experimental induction of liver metastasis through administration of cancer cells into mice via mesenteric vein injection. The behavior of these injected cells can then be studied using intravital imaging by surgical exposure or through an abdominal imaging window. The approach is described for use with an upright multiphoton microscope, making it widely applicable. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Inducing liver metastasis through mesenteric vein injection Basic Protocol 2: Short-term imaging of tumor cells in mouse liver Basic Protocol 3: Long-term imaging of tumor cells in mouse liver using an abdominal imaging window.

Assessment of Microvessel Permeability in Murine Atherosclerotic Vein Grafts Using Two-Photon Intravital Microscopy.

Plaque angiogenesis and plaque hemorrhage are major players in the destabilization and rupture of atherosclerotic lesions. As these are dynamic processes, imaging of plaque angiogenesis, especially the integrity or leakiness of angiogenic vessels, can be an extremely useful tool in the studies on atherosclerosis pathophysiology. Visualizing plaque microvessels in 3D would enable us to study the architecture and permeability of adventitial and intimal plaque microvessels in advanced atherosclerotic lesions. We hypothesized that a comparison of the vascular permeability between healthy continuous and fenestrated as well as diseased leaky microvessels, would allow us to evaluate plaque microvessel leakiness. We developed and validated a two photon intravital microscopy (2P-IVM) method to assess the leakiness of plaque microvessels in murine atherosclerosis-prone ApoE3*Leiden vein grafts based on the quantification of fluorescent-dextrans extravasation in real-time. We describe a novel 2P-IVM set up to study vessels in the neck region of living mice. We show that microvessels in vein graft lesions are in their pathological state more permeable in comparison with healthy continuous and fenestrated microvessels. This 2P-IVM method is a promising approach to assess plaque angiogenesis and leakiness. Moreover, this method is an important advancement to validate therapeutic angiogenic interventions in preclinical atherosclerosis models.

TGF-ß signaling in liver metastasis.

The presence of liver metastases drastically worsens the prognosis of cancer patients. The liver is the second most prevalent metastatic site in cancer patients, but systemic therapeutic opportunities that target liver metastases are still limited. To aid the discovery of novel treatment options for metastatic liver disease, we provide insight into the cellular and molecular steps required for liver colonization. For successful colonization in the liver, adaptation of tumor cells and surrounding stroma is essential. This includes the formation of a pre-metastatic niche, the creation of a fibrotic and immune suppressive environment, angiogenesis, and adaptation of tumor cells. We illustrate that transforming growth factor ß (TGF-ß) is a central cytokine in all these processes. At last, we devise that future research should focus on TGF-ß inhibitory strategies, especially in combination with immunotherapy. This promising systemic treatment strategy has potential to eliminate distant metastases as the efficacy of immunotherapy will be enhanced.

In Vivo Assessment of Size-Selective Glomerular Sieving in Transplanted Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.

Photodynamic cancer therapy enhances accumulation of nanoparticles in tumor-associated myeloid cells.

In cancer treatment, nanomedicines may be employed in an attempt to improve the tumor localization of antineoplastic drugs e.g. immunotherapeutic agents either through passive or active targeting, thereby potentially enhancing therapeutic effect and reducing undesired off-target effects. However, a large number of administrated nanocarriers often fail to reach the tumor area. In the present study, we show that photodynamic therapy (PDT) enhances the tumor accumulation of systemically administered lipid-PEG layer coated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). Intravital microscopy and histological analysis of the tumor area reveal that the tumor vasculature was disrupted after PDT, disturbing blood flow and coinciding with entrapment of nanocarriers in the tumor area. We observed that the nanoparticles accumulating after treatment do not confine to specific locations within the tumor, but rather localize to various cells present throughout the tumor area. Finally, we show by flow cytometry that NP accumulation occurred mostly in immune cells of the myeloid lineage present in the tumor microenvironment (TME) as well as in tumor cells, albeit to a lower extent. These data expose opportunities for combination treatments of clinical PDT with NP-based immunotherapy to modulate the TME and improve antitumor immune responses.

In vivo characteristics of human and mouse breast tumor cell lines.

Although two- and three-dimensional in vitro studies of breast tumor cell lines have increased our knowledge on tumor growth and metastasis formation, the complex in vivo microenvironment is not taken into consideration. The goal of our study was to illustrate the in vivo morphology and motility of widely used breast tumor cell lines. Intravital microscopy allows real-time visualization of individual cells inside tissues of living animals. We used this technique to study breast cancer migration in the complex orthotopic microenvironment. More specifically, we characterized cell morphology, cell-cell interactions, polarity and motility of mouse tumor cell lines 4T1 and mILC-1 and human tumor cell lines MDA-MB-231 and T47D. Almost all measured parameters were remarkably heterogeneous even between positions within the same tumor. Migrating tumor cells were circular in all tumor models, indicating predominantly amoeboid motility. This overview of the in vivo characteristics of mouse and human breast tumor cell lines illustrates their heterogeneity and complexity in real life, and additionally exemplifies caution should be taken to extrapolate in vitro assays of tumor invasiveness.

TGF-ß Family Signaling Pathways in Cellular Dormancy.

Individual cancer cells can switch, reversibly, to a non-proliferative dormant state, a process characterized by two principal stages: (i) establishment and maintenance, and (ii) the breaking of dormancy. This phenomenon is of clinical importance because dormant cells resist chemotherapy, and this can result in cancer relapse following years, if not decades, of clinical remission. Although the molecular mechanisms governing tumor cell dormancy have not been clearly delineated, accumulating evidence suggests that members of the transforming growth factor-ß (TGF-ß) family are integral. We summarize here recent findings which support the view that TGF-ß family signaling pathways play a pivotal role in cellular dormancy, and discuss how affected cells could be therapeutically targeted to prevent cancer relapse.

Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.

Procedures and applications of long-term intravital microscopy.

Intravital microscopy (IVM) is increasingly used in biomedical research to study dynamic processes at cellular and subcellular resolution in their natural environment. Long-term IVM especially can be applied to visualize migration and proliferation over days to months within the same animal without recurrent surgeries. Skin can be repetitively imaged without surgery. To intermittently visualize cells in other organs, such as liver, mammary gland and brain, different imaging windows including the abdominal imaging window (AIW), dermal imaging window (DIW) and cranial imaging window (CIW) have been developed. In this review, we describe the procedure of window implantation and pros and cons of each technique as well as methods to retrace a position of interest over time. In addition, different fluorescent biosensors to facilitate the tracking of cells for different purposes, such as monitoring cell migration and proliferation, are discussed. Finally, we consider new techniques and possibilities of how long-term IVM can be even further improved in the future.

Integrin ß1 activation induces an anti-melanoma host response.

TGF-ß is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-ß increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin ß1-mediated TGF-ß activation may also produce tumor suppression via an altered host response. In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin ß1-activation increases TGF-ß activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment. This is associated with attenuation of tumor growth and long-term survival benefit. Correspondingly, in human melanomas, TGF-ß1 correlates with integrin ß1/TGF-ß1 activation and the expression of markers for vasculature and stromal activation. Surprisingly, this integrin ß1/TGF-ß1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients. These correlations are unique to melanoma, in that we do not observe similar associations between ß1 integrin/TGF-ß1 activation and better long-term survival in other human tumor types. These results suggest that activation of TGF-ß1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study.

Two-Photon Intravital Microscopy Animal Preparation Protocol to Study Cellular Dynamics in Pathogenesis.

Two-photon intravital microscopy (2P-IVM) is an advanced imaging platform that allows the visualization of dynamic processes at subcellular resolution in vivo. Dynamic processes like cell migration, cell proliferation, cell-cell interactions, and cell signaling have an interactive character and occur in complex environments. Hence, it is of pivotal importance to study these processes in living animals, using for example 2P-IVM. 2P-IVM can be performed on a variety of tissues, from the skin of the animal to internal organs, and a variety of methods can be utilized to perform 2P-IVM on these tissues. Here, we discuss the protocols and considerations for four of those 2P-IVM methods, namely tissue explant imaging, skin imaging, surgical exposure imaging, and multi-day window imaging. We carefully compare and explain in depth how to set up each method. Lastly, in the notes section we mention some alternative solutions for the 2P-IVM methods described. In conclusion, this protocol can be used as a guide towards deciding which 2P-IVM method to use and to enable the setup of this method.

Vessel co-option mediates resistance to anti-angiogenic therapy in liver metastases.

The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of through the induction of angiogenesis, tumor vascularization can occur through the nonangiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option is also prevalent in human breast cancer liver metastases, a setting in which results with anti-angiogenic therapy have been disappointing. In preclinical mechanistic studies, we found that cancer cell motility mediated by the actin-related protein 2/3 complex (Arp2/3) is required for vessel co-option in liver metastases in vivo and that, in this setting, combined inhibition of angiogenesis and vessel co-option is more effective than the inhibition of angiogenesis alone. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option might be a warranted therapeutic strategy.

AKT Inhibition Promotes Nonautonomous Cancer Cell Survival.

Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.