Fluorescent molecular rotors as versatile in situ sensors for protein quantitation.

Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.

Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation.

Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.

Chiral resolution of a caged xanthone and evaluation across a broad spectrum of breast cancer subtypes.

Racemic resolution of (+/-)-MAD28, a representative caged xanthone, was accomplished using (1S, 4R)-(-)-camphanic chloride as the chiral agent. Selective crystallization of the resulting diastereomers in acetonitrile produced, after hydrolysis, the pure enantiomers. Screening of racemic MAD28 and both enantiomers across a broad spectrum of breast cancer cell lines revealed that they: (a) are equipotent in each of the breast cancer subtypes examined; and (b) exhibit a higher degree of cytotoxicity against breast cancer cell lines of basal-like subtype and triple negative receptor status. The results support the notion that MAD28 and related caged xanthones are promising drug leads against chemoresistant and metastatic cancers.

Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-ring modified caged xanthones in a preclinical model of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and related caged xanthones in spheroidsMARY-X, an in vitro preclinical IBC model, we constructed a library of synthetic analogs and performed structure-activity relationship studies. The studies revealed that functionalizing the A-ring of the caged xanthone framework can significantly affect potency. Specifically, introduction of hydroxyl or fluorine groups at discrete positions of the A-ring leads to enhanced cytotoxicity at submicromolar concentrations. These compounds induce complete dissolution of spheroidsMARY-X with subsequent apoptosis of both the peripherally- and centrally-located cells, proliferative and quiescent-prone (e.g. hypoxic), respectively. These results highlight the structural flexibility and pharmacological potential of the caged xanthone motif for the design of IBC-targeting therapeutics.

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

HDAC6 activity is a non-oncogene addiction hub for inflammatory breast cancers.

INTRODUCTION: Inflammatory breast cancer (IBC) is the most lethal form of breast cancers with a 5-year survival rate of only 40 %. Despite its lethality, IBC remains poorly understood which has greatly limited its therapeutic management. We thus decided to utilize an integrative functional genomic strategy to identify the Achilles' heel of IBC cells. METHODS: We have pioneered the development of genetic tools as well as experimental and analytical strategies to perform RNAi-based loss-of-function studies at a genome-wide level. Importantly, we and others have demonstrated that these functional screens are able to identify essential functions linked to certain cancer phenotypes. Thus, we decided to use this approach to identify IBC specific sensitivities. RESULTS: We identified and validated HDAC6 as a functionally necessary gene to maintain IBC cell viability, while being non-essential for other breast cancer subtypes. Importantly, small molecule inhibitors for HDAC6 already exist and are in clinical trials for other tumor types. We thus demonstrated that Ricolinostat (ACY1215), a leading HDAC6 inhibitor, efficiently controls IBC cell proliferation both in vitro and in vivo. Critically, functional HDAC6 dependency is not associated with genomic alterations at its locus and thus represents a non-oncogene addiction. Despite HDAC6 not being overexpressed, we found that its activity is significantly higher in IBC compared to non-IBC cells, suggesting a possible rationale supporting the observed dependency. CONCLUSION: Our finding that IBC cells are sensitive to HDAC6 inhibition provides a foundation to rapidly develop novel, efficient, and well-tolerated targeted therapy strategies for IBC patients.

Breast Cancer-Derived Extracellular Vesicles: Characterization and Contribution to the Metastatic Phenotype.

The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers.

Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening.

The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 µM for gambogic acid and 0.66 +/-0.02 µM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 µM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore.

Ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

Synthesis of purine-scaffold fluorescent probes for heat shock protein 90 with use in flow cytometry and fluorescence microscopy.

Fluorescent ligands for the heat shock protein 90 (Hsp90) were synthesized containing either fluorescein isothiocyanate (FITC), 4-nitrobenzo[1,2,5]oxadiazole (NBD) or the red shifted dye sulforhodamine 101 (Texas Red) conjugated to PU-H71. Two of the compounds, PU-H71-FITC2 (9) and PU-H71-NBD1 (8), were shown to be suitable for fluorescence-activated flow cytometry and fluorescence microscopy. Thus these molecules serve as useful probes for studying Hsp90 in heterogeneous live cell populations.

Breast carcinomatous tumoral emboli can result from encircling lymphovasculogenesis rather than lymphovascular invasion.

The canonical view of the origin of tumor lymphovascular emboli is that they usually originate from lymphovascular invasion as part of a multistep metastatic process. Recent experimental evidence has suggested that metastasis can occur earlier than previously thought and we found evidence that tumor emboli formation can result from the short-circuiting step of encircling lymphovasculogenesis. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids in vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of in situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the in situ clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The in situclusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion.

Gain in cellular organization of inflammatory breast cancer: A 3D in vitro model that mimics the in vivo metastasis.

BACKGROUND: The initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT), occurs via the loss of adherens junctions (e.g. cadherins) by the tumor embolus. This leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC) over-expresses E-cadherin. The human xenograft model of IBC (MARY-X), like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI) in situ by tumor emboli. An intact E-cadherin/alpha, beta-catenin axis mediates the tight, compact clump of cells found both in vitro and in vivo as spheroids and tumor emboli, respectively. METHODS: Using electron microscopy and focused ion beam milling to acquire in situ sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization. RESULTS: Here we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis) structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features. CONCLUSIONS: This study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture.These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an in vitro model with tractable in vivo applications.

Molecular plasticity of E-cadherin and sialyl lewis x expression, in two comparative models of mammary tumorigenesis.

BACKGROUND: The process of metastasis involves a series of steps and interactions between the tumor embolus and the microenvironment. Key alterations in adhesion molecules are known to dictate progression from the invasive to malignant phenotype followed by colonization at a distant site. The invasive phenotype results from the loss of expression of the E-cadherin adhesion molecule, whereas the malignant phenotype is associated with an increased expression of the carbohydrate ligand-binding epitopes, (e.g. Sialyl Lewis (x/a)) that bind endothelial E-selectin of the lymphatics and vasculature. METHODOLOGY: Our study analyzed the expression of two adhesion molecules, E-cadherin and Sialyl Lewis x (sLe(x)), in both a canine mammary carcinoma and human inflammatory breast cancer (IBC) model, using double labelled immunofluorescence staining. RESULTS: Our results demonstrate that canine mammary carcinoma and human IBC exhibit an inversely correlated cellular expression of E-cadherin and sLe(x) within the same tumor embolus. CONCLUSIONS: Our results in these two comparative models (canine and human) suggest the existence of a biologically coordinated mechanism of E-cadherin and sLe(x) expression (i.e. molecular plasticity) essential for tumor establishment and metastatic progression.

Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast carcinoma.

Inflammatory breast carcinoma (IBC) is characterized by florid tumor emboli within lymphovascular spaces called lymphovascular invasion. These emboli have a unique microscopic appearance of compact clumps of tumor cells retracted away from the surrounding endothelial cell layer. Using a human SCID model of IBC (MARY-X), we, in previous studies, demonstrated that the tumor cell embolus (IBC spheroid) forms on the basis of an intact and overexpressed E-cadherin/alpha,beta-catenin axis that mediates tumor cell-tumor cell adhesion. In the present study we examine the mechanism behind the apparent lack of binding of the tumor embolus to the surrounding endothelium. We find that this lack of tumor cell binding is because of markedly decreased sialyl-Lewis(x/a) (sLe(x/a)) carbohydrate ligand-binding epitopes on its overexpressed MUC1 and other surface molecules that bind endothelial E-selectin. Decreased sLe(x/a) is because of decreased alpha3/4-fucosyltransferase activity in MARY-X. The decreased sLe(x/a) fail to confer electrostatic repulsions between tumor cells, which further contributes to the compactness of the MARY-X spheroid by allowing the E-cadherin homodimeric interactions to go unopposed. MARY-X spheroids were retrovirally transfected with FucT-III cDNA, significantly raising their levels of fucosyltransferase activity and surface sLe(x/a). In parallel experiments, enzymatic transfers with a milk alpha1,3-fucosyltransferase and an alpha2,3-sialyltransferase (ST3GalIV) were performed on the MARY-X spheroids and increased surface sLe(x/a). The addition of sLe(x/a) by either manipulation caused disadherence of the MARY-X spheroids and the disruption of the E-cadherin homodimers mediating cell adhesion. Our findings support the cooperative relationship of sLe(x/a) underexpression and E-cadherin overexpression in the genesis of the lymphovascular embolus of IBC.

Reversible model of spheroid formation allows for high efficiency of gene delivery ex vivo and accurate gene assessment in vivo.

The native three-dimensional architecture of carcinomas, which governs numerous autocrine-paracrine interactions related to tumor progression, cannot be faithfully recreated in most in vitro models. Even when the three-dimensional architecture is recreated in artificial scaffolds such as soft agar, this approach does not truly recreate the natural microenvironment of the tumor. Multicellular spheroids can reasonably recreate in vitro the natural three-dimensional architecture of carcinomas, but even the most efficient gene delivery vectors will penetrate only the outer layers of these structures and hence only a small fraction of cells will receive the gene of interest. If the multicellular spheroids are disrupted into a single-cell suspension in order to achieve high transfection efficiency, the single-cell production may have so altered the gene expression profile of the spheroid as to bring into question its present relevancy to in vivo tumor progression. Our laboratory has developed a human-SCID (severe combined immunodeficient) mouse model of inflammatory breast cancer, MARY-X, which grows as tight multicellular spheroids in vitro and as lymphovascular emboli in vivo. The spheroids, which express only low levels of surface sialyl-Lewis(x/a) (sLe(x/a)), are able to form compact homotypic cell clumps mediated by an intact, overexpressed E-cadherin/alpha,beta-catenin axis. The spheroids can be fully disrupted by trypsin proteolysis, anti-E-cadherin antibodies, or Ca(2+) depletion. Of these approaches the disruption with depleted Ca(2+), complete after 30 min, is fully reversible by the readdition of Ca(2+) within 6 hr. This time interval allows for a transfection "window" in which successful gene delivery can be achieved before spheroid reformation. Retroviruses (10(6)-10(7) CFU/ml) carrying the gene encoding either green fluorescent protein (GFP), a dominant-negative E-cadherin mutant (H-2K(d)-E-cad), its control (H-2K(d)-E-cad Delta C25), or alpha-1,3-fucosyltransferase III (FucT-III), an enzyme that increases surface sLe(x/a), were used to transfect either intact (wild-type) or disadhered/readhered (reformed) spheroids. There were marked differences in transfection efficiency in the reformed versus wild-type spheroids. Retroviral transfection of GFP resulted in successful delivery of this reporter gene to only the outer layer of cells of the wild-type spheroids, but to all layers of the reformed spheroids. A single retroviral transfection of H-2K(d)-E-cad, H-2K(d)-E-cad Delta C25, or FucT-III produced evidence of their respective gene expression at 72 hr throughout all layers of the reformed spheroids, but only H-2K(d)-E-cad and FucT-III produced progressive disadherence. Both H-2K(d)-E-cad-MARY-X and FucT-III-MARY-X lost their ability to form lymphovascular emboli in SCID mice. This reversible model of spheroid formation has provided us with insight into the pathogenesis of inflammatory breast carcinoma. If more broadly applied, this model could be used to examine the effects of any gene, using any gene delivery system in the three-dimensional context of native tumoral architecture.