Iron-triggered signaling via ETS1 and the p38/JNK MAPK pathway regulates Bmp6 expression.

BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.

Immunological alterations in intracranial aneurysm: a prospective study on selected biomarker profiles in blood collected during endovascular neurointervention.

INTRODUCTION: Previous studies showed that the concentrations of selected chemokines are locally elevated in samples collected from the lumen of intracranial aneurysms (IA). Our objective was to determine whether the observed differences in analyte concentrations were influenced by the origin of the blood samples (i.e. cerebral versus peripheral), thus providing insight into the localised nature of these alterations and their significance in IA pathogenesis. MATERIAL AND METHODS: This prospective study included 24 patients with IA who underwent endovascular embolisation. Concentrations of selected analytes were analysed in blood samples from the IA lumen, feeding artery, and aorta. The analytes included MPO, Lipocalin-2/NGAL, sICAM-1, sVCAM-1, and serum amyloid A. RESULTS: Higher median plasma concentrations of MPO, lipocalin-2/NGAL, sVCAM-1, and SAA were found in samples obtained from the IA lumen and the feeding artery compared to the aorta. The concentration of sICAM-1 was significantly higher in the IA compared to the aorta, but did not differ between the proximal artery and the aorta. No significant differences in any analyte concentration were observed between the IA and the proximal artery. CONCLUSIONS: These findings suggest that the IA and the proximal vessel share similarities in the local immunological environment, which is different from that observed in the aorta. Further studies are needed to fully understand and elucidate these observations.

Tumor Microenvironment of Hepatocellular Carcinoma: Challenges and Opportunities for New Treatment Options.

The prevalence of liver cancer is constantly rising, with increasing incidence and mortality in Europe and the USA in recent decades. Among the different subtypes of liver cancers, hepatocellular carcinoma (HCC) is the most commonly diagnosed liver cancer. Besides advances in diagnosis and promising results of pre-clinical studies, HCC remains a highly lethal disease. In many cases, HCC is an effect of chronic liver inflammation, which leads to the formation of a complex tumor microenvironment (TME) composed of immune and stromal cells. The TME of HCC patients is a challenge for therapies, as it is involved in metastasis and the development of resistance. However, given that the TME is an intricate system of immune and stromal cells interacting with cancer cells, new immune-based therapies are being developed to target the TME of HCC. Therefore, understanding the complexity of the TME in HCC will provide new possibilities to design novel and more effective immunotherapeutics and combinatorial therapies to overcome resistance to treatment. In this review, we describe the role of inflammation during the development and progression of HCC by focusing on TME. We also describe the most recent therapeutic advances for HCC and possible combinatorial treatment options.

The CD200 Regulates Inflammation in Mice Independently of TNF-α Production.

Inflammatory bowel disease is characterized by the infiltration of immune cells and chronic inflammation. The immune inhibitory receptor, CD200R, is involved in the downregulation of the activation of immune cells to prevent excessive inflammation. We aimed to define the role of CD200R ligand-CD200 in the experimental model of intestinal inflammation in conventionally-reared mice. Mice were given a dextran sodium sulfate solution in drinking water. Bodyweight loss was monitored daily and the disease activity index was calculated, and a histological evaluation of the colon was performed. TNF-α production was measured in the culture of small fragments of the distal colon or bone marrow-derived macrophages (BMDMs) cocultured with CD200+ cells. We found that Cd200-/- mice displayed diminished severity of colitis when compared to WT mice. Inflammation significantly diminished CD200 expression in WT mice, particularly on vascular endothelial cells and immune cells. The co-culture of BMDMs with CD200+ cells inhibited TNF-α secretion. In vivo, acute colitis induced by DSS significantly increased TNF-α secretion in colon tissue in comparison to untreated controls. However, Cd200-/- mice secreted a similar level of TNF-α to WT mice in vivo. CD200 regulates the severity of DSS-induced colitis in conventionally-reared mice. The presence of CD200+ cells decreases TNF-α production by macrophages in vitro. However, during DDS-induced intestinal inflammation secretion of TNF-α is independent of CD200 expression.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine ß93 is the sole attachment moiety to the αß-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1).

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b-/- mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b-/- mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b-/- mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b-/- mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b-/- neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.

Effects of cannabinoids and their receptors on viral infections.

Cannabinoids, the active ingredient in marijuana, and their derivatives have received remarkable attention in the last two decades because they can affect tumor growth and metastasis. There is a large body of evidence from in vivo and in vitro models showing that cannabinoids and their receptors influence the immune system, viral pathogenesis, and viral replication. The present study reviews current insights into the role of cannabinoids and their receptors on viral infections. The results reported here indicate that cannabinoids and their receptors have different sequels for viral infection. Although activation or inhibition of cannabinoid receptors in the majority of viral infections are proper targets for development of safe and effective treatments, caution is required before using pharmaceutical cannabinoids as a treatment agent for patients with viral infections.

Immune Cells in Cancer Therapy and Drug Delivery.

Recent studies indicate the critical role of tumour associated macrophages, tumour associated neutrophils, dendritic cells, T lymphocytes, and natural killer cells in tumourigenesis. These cells can have a significant impact on the tumour microenvironment via their production of cytokines and chemokines. Additionally, products secreted from all these cells have defined specific roles in regulating tumour cell proliferation, angiogenesis, and metastasis. They act in a protumour capacity in vivo as evidenced by the recent studies indicating that macrophages, T cells, and neutrophils may be manipulated to exhibit cytotoxic activity against tumours. Therefore therapy targeting these cells may be promising, or they may constitute drug or anticancer particles delivery systems to the tumours. Herein, we discussed all these possibilities that may be used in cancer treatment.

CD200 receptor controls sex-specific TLR7 responses to viral infection.

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200(-/-) mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.

Double-layer optical fiber interferometer with bio-layer-modified reflector for label-free biosensing of inflammatory proteins.

This work discusses label-free biosensing application of a double-layer optical fiber interferometer where the second layer tailors the reflection conditions at the external plain and supports changes in reflected optical spectrum when a bio-layer binds to it. The double-layer nanostructure consists of precisely tailored thin films, i.e., titanium (TiO2) and hafnium oxides (HfO2) deposited on single-mode fiber end-face by magnetron sputtering. It has been shown numerically and experimentally that the approach besides well spectrally defined interference pattern distinguishes refractive index (RI) changes taking place in a volume and on the sensor surface. These are of interest when label-free biosensing applications are considered. The case of myeloperoxidase (MPO) detection-a protein, which concentration rises during inflammation-is reported as an example of application. The response of the sensor to MPO in a concentration range of 1 × 10-11-5 × 10-6 g/mL was tested. An increase in the MPO concentration was followed by a redshift of the interference pattern and a decrease in reflected power. The negative control performed using ferritin proved specificity of the sensor. The results reported in this work indicate capability of the approach for diagnostic label-free biosensing, possibly also at in vivo conditions.

Opioid receptors control viral replication in the airways.

OBJECTIVE: Opioids are frequently used during mechanical ventilation for severe viral infection in infancy. Opioid receptors have immunomodulatory properties, but nothing is known about their antiviral effects. We therefore aimed to investigate the role of opioid receptors in virus-induced airway inflammation. PATIENTS AND INTERVENTIONS: Two single nucleotide polymorphisms in OPRM1 and OPRD1 were genotyped in 465 infants with severe respiratory syncytial virus infection and 930 control subjects. Subsequently, the mechanism by which opioid receptors affect clinical outcome in respiratory syncytial virus bronchiolitis was studied in BALB/c mice. Animals were injected daily with nalmefene, a nonselective opioid receptor antagonist, and infected by intranasal inoculation of respiratory syncytial virus 24 hrs after the first dose of nalmefene. The potential therapeutic effect of pharmaceutical opioids was studied using µ (DAMGO), κ (U50488), and Δ (DPDPE) opioid receptor agonists 48 hrs after infection. MEASUREMENTS AND MAIN RESULTS: In our human study, the A118G single nucleotide polymorphism rs1799971 was associated with respiratory syncytial virus disease severity (p = 0.015). In mice, nalmefene treatment increased viral titers and was associated with more pronounced weight loss. Increased viral replication was associated with increased levels of cytokines and chemokines in the bronchoalveolar lavage fluid, enhanced bronchoalveolar cellular influx, and exaggerated lung pathology. Pharmaceutical opioids, in particular DPDPE, did not affect viral replication. They did induce a decreased influx of neutrophils, but an increased influx of lymphocytes and monocytes into the bronchoalveolar lumen during respiratory syncytial virus infection. CONCLUSIONS: Using a human study and an experimental model, we show that opioid receptor signaling has a potential beneficial role in the outcome of respiratory viral disease. We show that opioid receptor signaling is required to control respiratory syncytial virus replication and thereby to control disease severity. However, we also show that caution is required before using pharmaceutical opioids as anti-inflammatory or antiviral treatment of patients with viral respiratory infection.

Non-radioactive imaging strategies for in vivo immune cell tracking.

In vivo tracking of administered cells chosen for specific disease treatment may be conducted by diagnostic imaging techniques preceded by cell labeling with special contrast agents. The most commonly used agents are those with radioactive properties, however their use in research is often impossible. This review paper focuses on the essential aspect of cell tracking with the exclusion of radioisotope tracers, therefore we compare application of different types of non-radioactive contrast agents (cell tracers), methods of cell labeling and application of various techniques for cell tracking, which are commonly used in preclinical or clinical studies. We discuss diagnostic imaging methods belonging to three groups: (1) Contrast-enhanced X-ray imaging, (2) Magnetic resonance imaging, and (3) Optical imaging. In addition, we present some interesting data from our own research on tracking immune cell with the use of discussed methods. Finally, we introduce an algorithm which may be useful for researchers planning leukocyte targeting studies, which may help to choose the appropriate cell type, contrast agent and diagnostic technique for particular disease study.

Lack of CD200 enhances pathological T cell responses during influenza infection.

Influenza virus infection can be accompanied by life-threatening immune pathology most likely due to excessive antiviral responses. Inhibitory immune receptors may restrain such overactive immune responses. To study the role of the inhibitory immune receptor CD200R and its ligand CD200 during influenza infection, we challenged wild-type and CD200(-/-) mice with influenza virus. We found that CD200(-/-) mice in comparison to wild-type controls when inoculated with influenza virus developed more severe disease, associated with increased lung infiltration and lung endothelium damage. CD200(-/-) mice did develop adequate adaptive immune responses and were able to control viral load, suggesting that the severe disease was caused by a lack of control of the immune response. Interestingly, development of disease was completely prevented by depletion of T cells before infection, despite dramatically increased viral load, indicating that T cells are essential for the development of disease symptoms. Our data show that lack of CD200-CD200R signaling increases immune pathology during influenza infection, which can be reduced by T cell depletion.

The Role of Macrophages in Cancer Development and Therapy.

Macrophages are critical mediators of tissue homeostasis and influence various aspects of immunity. Tumor-associated macrophages are one of the main cellular components of the tumor microenvironment. Depending on their activation status, macrophages can exert a dual influence on tumorigenesis by either antagonizing the cytotoxic activity of immune cells or, less frequently, by enhancing antitumor responses. In most situations, TAMs suppress T cell recruitment and function or regulate other aspects of tumor immunity. The importance of TAMs targeting in cancer therapy is derived from the strong association between the high infiltration of TAMs in the tumor tissue with poor patient prognosis. Several macrophage-targeting approaches in anticancer therapy are developed, including TAM depletion, inhibition of new TAM differentiation, or re-education of TAM activation for cancer cell phagocytosis. In this review, we will describe the role of TAMs in tumor development, including such aspects as protumorigenic inflammation, immune suppression, neoangiogenesis, and enhancement of tissue invasion and distant metastasis. Furthermore, we will discuss therapeutic approaches that aim to deplete TAMs or, on the contrary, re-educate TAMs for cancer cell phagocytosis and antitumor immunity.

Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma.

Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex.

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin-based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1-Rac signaling. Tiam1 interacts with Par3 and aPKCzeta, which are two components of the conserved Par3-Par6-aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCzeta, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3-Par6-aPKC polarity complex.

The pro-tumor effect of CD200 expression is not mimicked by agonistic CD200R antibodies.

Tumor-infiltrating immune cells can impact tumor growth and progression. The inhibitory CD200 receptor (CD200R) suppresses the activation of myeloid cells and lack of this pathway results in a reduction of tumor growth, conversely a tumorigenic effect of CD200R triggering was also described. Here we investigated the role of CD200R activation in syngeneic mouse tumor models. We showed that agonistic CD200R antibody reached tumors, but had no significant impact on tumor growth and minor effect on infiltration of immune myeloid cells. These effects were reproduced using two different anti-CD200R clones. In contrast, we showed that CD200-deficiency did decrease melanoma tumor burden. The presence of either endogenous or tumor-expressed CD200 restored the growth of metastatic melanoma foci. On the basis of these findings, we conclude that blockade of the endogenous ligand CD200 prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, whereas agonistic anti-CD200R has no effect on tumor development.

Antitumor Activity of TLR7 Is Potentiated by CD200R Antibody Leading to Changes in the Tumor Microenvironment.

Stimulation of Toll-like receptor 7 (TLR7) activates myeloid cells and boosts the immune response. Previously, we have shown that stimulation of the inhibitory CD200 receptor (CD200R) suppresses TLR7 signaling and that the absence of CD200R signaling leads to a decreased number of papillomas in mice. Here, we investigated the effects of agonistic anti-CD200R on the antitumor activity of a TLR7 agonist (R848) in a syngeneic mouse tumor model. Intratumoral administration of R848 inhibited the growth of the CT26 colon carcinoma and simultaneously decreased CD200R expression in tumor-infiltrating immune cells. The antitumor effects of R848 were potentiated by anti-CD200R. Successfully treated mice were resistant to rechallenge with the same tumor cells. However, the immediate antitumor effects were independent of lymphocytes, because treatment efficacy was similar in wild-type and Rag1tm1Mom mice. Administration of R848, particularly in combination with anti-CD200R, changed the phenotype of intratumoral myeloid cells. The infiltration with immature MHC-II+ macrophages decreased and in parallel monocytes and immature MHC-II- macrophages increased. Combined treatment decreased the expression of the macrophage markers F4/80, CD206, CD86, CD115, and the ability to produce IL1ß, suggesting a shift in the composition of intratumor myeloid cells. Adoptively transferred CD11b+ myeloid cells, isolated from the tumors of mice treated with R848 and anti-CD200R, inhibited tumor outgrowth in recipient mice. We conclude that administration of agonistic anti-CD200R improves the antitumor effects of TLR7 signaling and changes the local tumor microenvironment, which becomes less supportive of tumor progression. Cancer Immunol Res; 6(8); 930-40. ©2018 AACR.

Changes in hypoxia level of CT26 tumors during various stages of development and comparing different methods of hypoxia determination.

The aim of this study was to evaluate hypoxia level at various tumor developmental stages and to compare various methods of hypoxia evaluation in pre-clinical CT26 tumor model. Using three methods of hypoxia determination, we evaluated hypoxia levels during CT26 tumor development in BALB/c mice from day 4 till day 19, in 2-3 days intervals. Molecular method was based on the analysis of selected genes expression related to hypoxia (HIF1A, ANGPTL4, TGFB1, VEGFA, ERBB3, CA9) or specific for inflammation in hypoxic sites (CCL2, CCL5) at various time points after CT26 cancer cells inoculation. Imaging methods of hypoxia evaluation included: positron-emission tomography (PET) imaging using [18F]fluoromisonidazole ([18F]FMISO) and a fluorescence microscope imaging of pimonidazole (PIMO)-positive tumor areas at various time points. Our results showed that tumor hypoxia at molecular level was relatively high at early stage of tumor development as reflected by initially high HIF1A and VEGFA expression levels and their subsequent decrease. However, imaging methods (both PET and fluorescence microscopy) showed that hypoxia increased till day 14 of tumor development. Additionally, necrotic regions dominated the tumor tissue at later stages of development, decreasing the number of hypoxic areas and completely eliminating normoxic regions (observed by PET). These results showed that molecular methods of hypoxia determination are more sensitive to show changes undergoing at cellular level, however in order to measure and visualize hypoxia in the whole organ, especially at later stages of tumor development, PET is the preferred tool. Furthermore we concluded, that during development of tumor, two peaks of hypoxia occur.

The Rac activator Tiam1 and Ras-induced oncogenesis.

The Tiam1 gene encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho-like GTPase Rac. In vitro studies indicate that Tiam1 localizes to adherens junctions and plays a role in the formation and maintenance of cadherin-based cell adhesions, thereby regulating migration of epithelial cells. In vivo studies implicate Tiam1 in various aspects of tumorigenesis. In this chapter, we discuss the use of the DMBA/TPA chemical carcinogenesis protocol in Tiam1-deficient mice to study the role of Tiam1 in Ras-induced skin tumors. This two-stage carcinogenesis protocol allows us to study initiation, promotion, and progression of tumors in a Tiam1-positive and Tiam1-negative background. Moreover, we describe methods to study the role of Tiam1 in susceptibility to apoptosis, cell growth, and Ras transformation by in vivo and in vitro experiments. The latter makes use of tumor cells and primary embryonic fibroblasts and keratinocytes isolated from mice.