Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer.

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8 T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy.

Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination.

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2/VEGF macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients.

SHP2 Inhibition Sensitizes Diverse Oncogene-Addicted Solid Tumors to Re-treatment with Targeted Therapy.

Unlabelled: Rationally targeted therapies have transformed cancer treatment, but many patients develop resistance through bypass signaling pathway activation. PF-07284892 (ARRY-558) is an allosteric SHP2 inhibitor designed to overcome bypass-signaling-mediated resistance when combined with inhibitors of various oncogenic drivers. Activity in this setting was confirmed in diverse tumor models.

Phase Ib/II Trial of Ribociclib in Combination with Binimetinib in Patients with NRAS-mutant Melanoma.

Purpose: Enhanced MAPK pathway signaling and cell-cycle checkpoint dysregulation are frequent in NRAS-mutant melanoma and, as such, the regimen of the MEK inhibitor binimetinib and the selective CDK4/6 inhibitor ribociclib is a rational combination.

Patients And Methods: This is a phase Ib/II, open-label study of ribociclib + binimetinib in patients with NRAS-mutant melanoma (NCT01781572). Primary objectives were to estimate the MTD/recommended phase II dose (RP2D) of the combination (phase Ib) and to characterize combination antitumor activity at the RP2D (phase II).

Homophilic CD44 Interactions Mediate Tumor Cell Aggregation and Polyclonal Metastasis in Patient-Derived Breast Cancer Models.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding.

A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation.

Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2 Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2 myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2/Vegf-A macrophages in the tumor microenvironment of metastasis (TMEM).

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.

Breast cancer cells disseminate through TIE2/MENA/MENA-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENA isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination.

The Multifaceted Role of Perivascular Macrophages in Tumors.

Evidence has emerged for macrophages in the perivascular niche of tumors regulating important processes like angiogenesis, various steps in the metastatic cascade, the recruitment and activity of other tumor-promoting leukocytes, and tumor responses to frontline therapies like irradiation and chemotherapy. Understanding the mechanisms controlling the recruitment, retention, and function of these cells could identify important targets for anti-cancer therapeutics.

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes.

Brightness-equalized quantum dots.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours.

Tuning cobalt(III) Schiff base complexes as activated protein inhibitors.

Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation.

Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA.

Unlabelled: Dissemination of tumor cells is an essential step in metastasis. Direct contact between a macrophage, mammalian-enabled (MENA)-overexpressing tumor cell, and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that transient vascular permeability and tumor cell intravasation occur simultaneously and exclusively at TMEM.

Synthesis, Characterization, and in vitro Testing of a Bacteria-Targeted MR Contrast Agent.

A bacteria-targeted MR contrast agent, Zn-, consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a Gd chelate has been synthesized and characterized. In vitro studies with and show that Zn- exhibits a significant improvement in bacteria labeling efficiency vs. control.

Axial ligand exchange of N-heterocyclic cobalt(III) Schiff base complexes: molecular structure and NMR solution dynamics.

The kinetic and thermodynamic ligand exchange dynamics are important considerations in the rational design of metal-based therapeutics and therefore, require detailed investigation. Co(III) Schiff base complex derivatives of bis(acetylacetone)ethylenediimine [acacen] have been found to be potent enzyme and transcription factor inhibitors. These complexes undergo solution exchange of labile axial ligands.

Kinetics and thermodynamics of irreversible inhibition of matrix metalloproteinase 2 by a Co(III) Schiff base complex.

Cobalt(III) Schiff base complexes have been used as potent inhibitors of protein function through the coordination to histidine residues essential for activity. The kinetics and thermodynamics of the binding mechanism of Co(acacen)(NH(3))(2)Cl [Co(acacen); where H(2)acacen is bis(acetylacetone)ethylenediimine] enzyme inhibition has been examined through the inactivation of matrix metalloproteinase 2 (MMP-2) protease activity. Co(acacen) is an irreversible inhibitor that exhibits time- and concentration-dependent inactivation of MMP-2.

Targeted inactivation of Snail family EMT regulatory factors by a Co(III)-Ebox conjugate.

Snail family proteins are core EMT (epithelial-mesenchymal transition) regulatory factors that play essential roles in both development and disease processes and have been associated with metastasis in carcinomas. Snail factors are required for the formation of neural crest stem cells in most vertebrate embryos, as well as for the migratory invasive behavior of these cells. Snail factors have recently been linked to the formation of cancer stem cells, and expression of Snail proteins may be associated with tumor recurrence and resistance to chemotherapy and radiotherapy.

Specific inhibition of the transcription factor Ci by a cobalt(III) Schiff base-DNA conjugate.

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas.

Molecular imaging of in vivo gene expression.

Molecular imaging provides spatial and temporal information on cellular changes that occur during development and in disease. MRI and optical imaging of reporter genes allows for the visualization of promoter activity, protein-protein interactions, protein stability and the tracking of individual proteins and cells. Reporter genes can be genetically encoded in transgenic animals or detected through the administration of an exogenous contrast agent.

Chiral porphyrazine near-IR optical imaging agent exhibiting preferential tumor accumulation.

A chiral porphyrazine (pz), H(2)[pz(trans-A(2)B(2))] (247), has been prepared that exhibits preferential in vivo accumulation in the cells of tumors. Pz 247 exhibits near-infrared (NIR) emission with lambda > 700 nm in the required wavelength range for maximum tissue penetration. When MDA-MB-231 breast tumor cells are treated with 247, the agent shows strong intracellular fluorescence with an emission maximum, 704 nm, which indicates that it localizes within a hydrophobic microenvironment.

Targeted inhibition of Snail family zinc finger transcription factors by oligonucleotide-Co(III) Schiff base conjugate.

A transition metal complex targeted for the inhibition of a subset of zinc finger transcription factors has been synthesized and tested in Xenopus laevis. A Co(III) Schiff base complex modified with a 17-bp DNA sequence is designed to selectively inhibit Snail family transcription factors. The oligonucleotide-conjugated Co(III) complex prevents Slug, Snail, and Sip1 from binding their DNA targets whereas other transcription factors are still able to interact with their target DNA.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces suppressor of cytokine signaling 2 in murine B cells.

The B cell, a major component of humoral immunity, is a sensitive target for the immunotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), possibly by rendering cells less responsive to antigenic or mitogenic stimulation. Potential mechanisms of TCDD action on B cells were examined in murine B cell lymphoma cells (CH12.LX) treated with 3 nM TCDD or dimethyl sulfoxide vehicle using sequence-verified cDNA microarrays.