Interleukin 6 plays a role in the migration of magnetically levitated mesenchymal stem cells spheroids

Mesenchymal stem cells (MSCs) reside quiescently within a specialised ‘niche’ environment in the bone marrow. However, following appropriate signalling cues, MSCs mobilise and migrate out from the niche, typically toward either sites of injury (a regenerative response) or toward primary tumours (an intrinsic homing response, which promotes MSCs as cellular vectors for therapeutic delivery). To date, very little is known about MSC mobilisation. By adopting a 3D MSC niche model, whereby MSC spheroids are cultured within a type I collagen gel, recent studies have highlighted interleukin-6 (IL-6) as a key cytokine involved in MSC migration. Herein, the ability of IL-6 to induce MSC migration was further investigated, and the key matrix metalloproteinases used to effect cell mobilisation were identified. Briefly, the impact of IL-6 on the MSC migration in a two-dimensional model systems was characterised—both visually using an Ibidi chemotaxis plate array (assessing for directional migration) and then via a standard 2D monolayer experiment, where cultured cells were challenged with IL-6 and extracted media tested using an Abcam Human MMP membrane antibody array. The 2D assay displayed a strong migratory response toward IL-6 and analysis of the membrane arrays data showed significant increases of several key MMPs. Both data sets indicated that IL-6 is important in MSC mobilisation and migration. We also investigated the impact of IL-6 induction on MSCs in 3D spheroid culture, serving as a simplistic model of the bone marrow niche, characterised by fluorescently tagged magnetic nanoparticles and identical membrane antibody arrays. An increase in MMP levels secreted by cells treated with 1 ng/mL IL-6 versus control conditions was noted in addition to migration of cells away from the central spheroid mass

Tumor cell migration in complex microenvironments

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable.National Science Foundation (U.S.) (Graduate Research Fellowship)Charles Stark Draper Laboratory (Research and Development Program (N.DL-H-550151))National Cancer Institute (U.S.) (R21CA140096

Silencing of Sinusoidal DDR1 Reduces Murine Liver Metastasis by Colon Carcinoma

Liver metastasis depends on the collagenous microenvironment generated by hepatic sinusoidal cells (SCs). DDR1 is an atypical collagen receptor linked to tumor progression, but whether SCs express DDR1 and its implication in liver metastasis remain unknown. Freshly isolated hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), that conform the SCs, expressed functional DDR1. HSCs expressed the largest amounts. C26 colon carcinoma secretomes increased DDR1 phosphorylation in HSCs and KCs by collagen I. Inhibition of kinase activity by DDR1-IN-1 or mRNA silencing of DDR1 reduced HSCs secretion of MMP2/9 and chemoattractant and proliferative factors for LSECs and C26 cells. DDR1-IN-1 did not modify MMP2/9 in KCs or LSECs secretomes, but decreased the enhancement of C26 migration and proliferation induced by their secretomes. Gene array showed that DDR1 silencing downregulated HSCs genes for collagens, MMPs, interleukins and chemokines. Silencing of DDR1 before tumor inoculation reduced hepatic C26 metastasis in mice. Silenced livers bore less tumor foci than controls. Metastatic foci in DDR1 silenced mice were smaller and contained an altered stroma with fewer SCs, proliferating cells, collagen and MMPs than foci in control mice. In conclusion, hepatic DDR1 promotes C26 liver metastasis and favors the pro-metastatic response of SCs to the tumor.We would like to acknowledge the following core facilities and individuals for their support: CIC bioGUNE Center for Cooperative Research in Biosciences, University of the Basque Country Animal Core Facility and SGIker Advanced Light Microscopy Core Facility. We thank Iratxe Basaldua for the in situ MMPs assay

Mutant p53 alters tumor microenvironment by reprogramming the cancer cell secretome via miR-30d

Solid tumors can be considered as complex organs, in which neoplastic cells are surrounded by a tumor microenvironment (TME) that consists of an extracellular matrix (ECM) and many non-transformed cell populations (e.g. fibroblasts, endothelial cells). Cancer cells shape their TME through secretion of soluble and insoluble factors, including proteins, metabolites, extracellular vesicles (EVs), and circulating nucleic acids, among which miRNAs. The cancer secretome has both local and systemic effects on tumor growth, dissemination and metastatic colonization. Several cancer-related pathways concur to reprogram the secretome, and thereby alter the TME to sustain cancer development. In human cancers, one of the most frequently mutated gene encodes for the transcription factor and tumor suppressor TP53. The majority of these mutations occur within the DNA binding domain of TP53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the oncosuppressive features of the wild-type form, but may also acquire novel oncogenic properties, generally referred to as gain of function (GOF). Indeed, mut-p53 proteins are capable of promoting cancer progression, metastasis and chemoresistance. mut-p53 can exert its oncogenic functions by different mechanisms, a major one being the alteration of coding and non-coding (including miRNAs) gene expression profiles. In the laboratory where this PhD project has been developed, miR-30d was recently identified as a novel target of mut-p53 and an effector of its oncogenic functions in breast cancer cells. In an attempt to dissect the molecular mechanisms underlying the effects exerted by the mut-p53/miR-30d axis, we have demonstrated that miR-30d impacts the cancer secretome, suggesting an effect on TME remodeling. Starting from these observations, here I provided evidence that the mut-p53/miR-30d-dependent secretome is able to promote in vitro paracrine effects towards different components of TME, such as ECM, endothelial cells, fibroblasts, as well as other cancer cells. Moreover, the mut-p53/miR-30d axis also contributes to the loading of proteins within EVs. In addition, I showed that miR-30d secreted by cancer cells can exert a pro-oncogenic effect in non-transformed receiving cells and may represent a promising secreted biomarker associated with p53 status in vivo. This study provides new insights into the mechanisms by which mut-p53, through miR-30d induction, can exert pro-tumorigenic functions in a paracrine fashion, and highlights potential noninvasive biomarkers and therapeutic targets to treat tumors harboring mut-p53

Human plasmacytoid dendritic cells and cutaneous melanoma

The prognosis of metastatic melanoma (MM) patients has remained poor for a long time. However, the recent introduction of effective target therapies (BRAF and MEK inhibitors for BRAFV600-mutated MM) and immunotherapies (anti-CTLA-4 and anti-PD-1) has significantly improved the survival of MM patients. Notably, all these responses are highly dependent on the fitness of the host immune system, including the innate compartment. Among immune cells involved in cancer immunity, properly activated plasmacytoid dendritic cells (pDCs) exert an important role, bridging the innate and adaptive immune responses and directly eliminating cancer cells. A distinctive feature of pDCs is the production of high amount of type I Interferon (I-IFN), through the Toll-like receptor (TLR) 7 and 9 signaling pathway activation. However, published data indicate that melanoma-associated escape mechanisms are in place to hijack pDC functions. We have recently reported that pDC recruitment is recurrent in the early phases of melanoma, but the entire pDC compartment collapses over melanoma progression. Here, we summarize recent advances on pDC biology and function within the context of melanoma immunity

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB

The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis

Background The tumor microenvironment has complex effects in cancer pathophysiology that are not fully understood. Most cancer therapies are directed against malignant cells specifically, leaving pro-malignant signals from the microenvironment unaddressed. Defining specific mechanisms by which the tumor microenvironment contributes to breast cancer metastasis may lead to new therapeutic approaches against advanced breast cancer. Methods We use a novel method for manipulating three-dimensional mixed cell co-cultures, along with studies in mouse xenograft models of human breast cancer and a histologic study of human breast cancer samples, to investigate how breast cancer-associated fibroblasts affect the malignant behaviors of breast cancer cells. Results Altering fibroblast Tiam1 expression induces changes in invasion, migration, epithelial-mesenchymal transition, and cancer stem cell characteristics in associated breast cancer cells. These changes are both dependent on fibroblast secretion of osteopontin and also long-lasting even after cancer cell dissociation from the fibroblasts, indicating a novel Tiam1-osteopontin pathway in breast cancer-associated fibroblasts. Notably, inhibition of fibroblast osteopontin with low doses of a novel small molecule prevents lung metastasis in a mouse model of human breast cancer metastasis. Moreover, fibroblast expression patterns of Tiam1 and osteopontin in human breast cancers show converse changes correlating with invasion, supporting the hypothesis that this pathway in tumor-associated fibroblasts regulates breast cancer invasiveness in human disease and is thus clinically relevant. Conclusions These findings suggest a new therapeutic paradigm for preventing breast cancer metastasis. Pro-malignant signals from the tumor microenvironment with long-lasting effects on associated cancer cells may perpetuate the metastatic potential of developing cancers. Inhibition of these microenvironment signals represents a new therapeutic strategy against cancer metastasis that enables targeting of stromal cells with less genetic plasticity than associated cancer cells and opens new avenues for investigation of novel therapeutic targets and agents.National Institute of General Medical Sciences (U.S.) (GM074825

Inhibition of Mutant EGFR in NSCLC Promotes Endothelin-1-Mediated NSCLC Disease Progression

Angiogenesis in NSCLC has been identified as important therapeutic target in combination with EGFR TKIs. However, only small incremental advancements have been made for the use of angiogenesis inhibitors in NSCLC and it remains elusive why the inhibition of VEGF-mediated neovascularization is not therapeutically efficacious. I present experimental evidence that a subpopulation of NSCLC cells with EGFR TKI-induced EMT contributes toward the attenuation of the response to EGFR TKI therapy. One of the hallmarks of cancer is heterogeneity and I have previously demonstrated that tumor heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations gives rise to divergent resistance mechanisms in response to treatment. In vivo admix models are instructive in studying intratumoral heterogeneity and in elucidating therapeutic responses and tumor-host interactions. While NSCLC cells with acquired EGFR TKI resistance and EMT phenotype did not exhibit growth advantage in vitro, a 50% epithelial EGFR TKI sensitive and 50% mesenchymal EGFR TKI resistant admix provided significant growth advantage in vivo assessed by caliper measurement. This preliminary result led us to hypothesize that changes in angiogenic growth factor expression during the EMT process might lead to the in vivo growth advantage I observed. To test the hypothesis, I utilized the Luminex multiplex assay system to quantify secreted growth factors, cytokines, and chemokines important in angiogenesis. I have discovered that epithelial EGFR TKI sensitive cells secrete a significant amount of VEGF-A and cells with acquired/transient EGFR TKI resistance with an EMT phenotype secrete substantial amount of EDN1. Using an in vitro tube formation assay, I showed that secreted VEGF-A and EDN1 in admix conditions work synergistically to promote endothelial cell differentiation. Furthermore, this synergistic effect can be attenuated by VEGFR2/EDNRA dual inhibition. Surprisingly, ectopic overexpression of EDN1 in EGFR- mutated HCC827 cells resulted in significant growth retardation in vivo. Informed by a literature search, I hypothesized that the presence of EDN1 in the tumor microenvironment contributes positively to EGFR TKI resistance, possibly through the vasoconstrictive property of EDN1. I observed that epithelial/mesenchymal admix tumors and ectopic overexpression of EDN1 in EGFR-mutated HCC827 cells conferred significantly more resistance to gefitinib in vivo. This result led us to hypothesize that EDN1 may reduce MVD in EGFR-mutated NSCLC tumors leading to poor EGFR TKI penetrance in vivo. I tested this through CD31 IHC staining and MVD calculation. I indirectly tested poor EGFR TKI penetrance by examining phosphorylated EGFR and found maintenance of the signal in admix and mesenchymal tumors. Taken together, I suggest that inhibition of the EDN1 signaling system may be an important component to a blood vascular-based approach to treatment of EGFR-mutation positive NSCLC

Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039