Advances in the Characterization of Circulating Tumor Cells in Metastatic Breast Cancer: Single Cell Analyses and Interactions, and Patient-Derived Models for Drug Testing.

Metastasis is the major cause of breast cancer death worldwide. In metastatic breast cancer, circulating tumor cells (CTCs) can be captured from patient blood samples sequentially over time and thereby serve as surrogates to assess the biology of surviving cancer cells that may still persist in solitary or multiple metastatic sites following treatment. CTCs may thus function as potential real-time decision-making guides for selecting appropriate therapies during the course of disease or for the development and testing of new treatments. The heterogeneous nature of CTCs warrants the use of single cell platforms to better inform our understanding of these cancer cells. Current techniques for single cell analyses and techniques for investigating interactions between cancer and immune cells are discussed. In addition, methodologies for growing patient-derived CTCs in vitro or propagating them in vivo to facilitate CTC drug testing are reviewed. We advocate the use of CTCs in appropriate microenvironments to appraise the effectiveness of cancer chemotherapies, immunotherapies, and for the development of new cancer treatments, fundamental to personalizing and improving the clinical management of metastatic breast cancer.

Antitumor action of 3-bromopyruvate implicates reorganized tumor growth regulatory components of tumor milieu, cell cycle arrest and induction of mitochondria-dependent tumor cell death.

Evidences demonstrate that metabolic inhibitor 3-bromopyruvate (3-BP) exerts a potent antitumor action against a wide range of malignancies. However, the effect of 3-BP on progression of the tumors of thymic origin remains unexplored. Although, constituents of tumor microenvironment (TME) plays a pivotal role in regulation of tumor progression, it remains unclear if 3-BP can alter the composition of the crucial tumor growth regulatory components of the external surrounding of tumor cells. Thus, the present investigation attempts to understand the effect of 3-BP administration to a host bearing a progressively growing tumor of thymic origin on tumor growth regulatory soluble, cellular and biophysical components of tumor milieu vis-à-vis understanding its association with tumor progression, accompanying cell cycle events and mode of cell death. Further, the expression of cell survival regulatory molecules and hemodynamic characteristics of the tumor milieu were analysed to decipher mechanisms underlying the antitumor action of 3-BP. Administration of 3-BP to tumor-bearing hosts retarded tumor progression accompanied by induction of tumor cell death, cell cycle arrest, declined metabolism, inhibited mitochondrial membrane potential, elevated release of cytochrome c and altered hemodynamics. Moreover, 3-BP reconstituted the external milieu, in concurrence with deregulated glucose and pH homeostasis and increased tumor infiltration by NK cells, macrophages, and T lymphocytes. Further, 3-BP administration altered the expression of key regulatory molecules involved in glucose uptake, intracellular pH and tumor cell survival. The outcomes of this study will help in optimizing the therapeutic application of 3-BP by targeting crucial tumor growth regulatory components of tumor milieu.

Flavonoids inhibit chronically exposed arsenic-induced proliferation and malignant transformation of HaCaT cells.

BACKGROUND: Apart from exposure to UV-radiation, studies show relationship between skin cancer and chronic ingestion of arsenic through drinking water. Chemopreventive strategies could help in reducing the toxic effects of arsenic and arsenic-induced skin cancer. METHODS: Cytotoxicity of arsenic on human skin keratinocytes HaCaT cells was evaluated using MTT and trypan blue assays. Arsenic-induced malignant transformant HaCaT cells were selected through soft agar colony assay. Cell cycle progression was analyzed through FACS. The expressions of genes modulated by arsenic were studied through RT-PCR. RESULTS: The lower concentrations (0.1-0.5 µmol/L) of arsenic were non-toxic and transformed HaCaT cells on chronic exposure, and also enhanced the cell proliferation. Silibinin and fisetin reduced the arsenic-induced cell proliferation and malignant transformation. A slight increase in G2-M phase cell population was also observed. The anti-proliferation effects of flavonoids on HaCaT transformants were further enhanced when combined with gamma radiation. Chronic and acute exposure to arsenic modulated the expression of transformation-associated genes including Bcl-2A1, IGFL-1, Rab31, and TNC in HaCaT cells. CONCLUSIONS: Chronic exposure to lower arsenic concentrations caused malignant transformation of skin keratinocytes and that effect was attenuated by flavonoids silibinin and fisetin. Thus, chemoprevention could reduce arsenic-caused detrimental effects on skin cells.

Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy.

Y-box binding protein-1 (YB-1) is a proto-oncogenic protein associated with protein translation regulation. It plays a crucial role in the development and progression of triple-negative breast cancer (TNBC). In this study, we describe a promising approach to inhibit YB-1 using SU056, a small-molecule inhibitor. SU056 physically interacts with YB-1 and reduces its expression, which helps to restrain the progression of TNBC. Proteome profiling analysis indicates that the inhibition of YB-1 by SU056 can alter the proteins that regulate protein translation, an essential process for cancer cell growth. Preclinical studies on human cells, mice, and patient-derived xenograft tumor models show the effectiveness of SU056. Moreover, toxicological studies have shown that SU056 treatment and dosing are well tolerated without any adverse effects. Overall, our study provides a strong foundation for the further development of SU056 as a potential treatment option for patients with TNBC by targeting YB-1.

Microfluidic live tracking and transcriptomics of cancer-immune cell doublets link intercellular proximity and gene regulation.

Cell-cell communication and physical interactions play a vital role in cancer initiation, homeostasis, progression, and immune response. Here, we report a system that combines live capture of different cell types, co-incubation, time-lapse imaging, and gene expression profiling of doublets using a microfluidic integrated fluidic circuit that enables measurement of physical distances between cells and the associated transcriptional profiles due to cell-cell interactions. We track the temporal variations in natural killer-triple-negative breast cancer cell distances and compare them with terminal cellular transcriptome profiles. The results show the time-bound activities of regulatory modules and allude to the existence of transcriptional memory. Our experimental and bioinformatic approaches serve as a proof of concept for interrogating live-cell interactions at doublet resolution. Together, our findings highlight the use of our approach across different cancers and cell types.

Combination of Resveratrol and Quercetin Causes Cell Growth Inhibition, DNA Damage, Cell Cycle Arrest, and Apoptosis in Oral Cancer Cells.

Resveratrol and quercetin alone are well reported to have anticancer potential, but their combination studies are very inadequate. We have examined their combination in Cal-33 and SCC-15 oral cancer cells (OCCs) and noncancerous HEK-293 cells. Combination of 10 µM concentration of each resveratrol and quercetin brought additive effect on cellular growth, DNA damage, S-phase cell cycle arrest, and cell death in Cal-33 cells but not in the HEK-293 cells. Augmentation of the cell cycle regulatory protein, Cyclin E, and downregulation of Cyclin A possibly caused S-phase arrest in Cal-33 cancer cells. Comet formation and presence of gamma-H2AX foci confirmed DNA damage, and cleavage of PARP1 and upregulation in Bax level specified apoptosis after combined treatment. Ratio of transcription activation and repression histone marks was found increased after alone as well as combined treatment. Histone deacetylase (HDAC)1, HDAC3, and HDAC8 were downregulated by resveratrol alone and combined treatment. Conclusively, combination of resveratrol and quercetin brings cell growth inhibition, DNA damage, and cell cycle arrest in OCCs but not in normal cells. Additionally, combined treatment causes downregulation of HDACs and apoptosis in cancer cells and it could be an incisive strategy against oral cancer.

Protective and recuperative effects of 3-bromopyruvate on immunological, hepatic and renal homeostasis in a murine host bearing ascitic lymphoma: Implication of niche dependent differential roles of macrophages.

3-bromopyruvate (3-BP) possesses promising antineoplastic potential, however, its effects on immunological homeostasis vis a vis hepatic and renal functions in a tumor bearing host remain unclear. Therefore, the effect of 3-BP administration to a murine host bearing a progressively growing tumor of thymoma origin, designated as Dalton's lymphoma (DL), on immunological, renal and hepatic homeostasis was investigated. Administration of 3-BP (4 mg/kg) to the tumor bearing host reversed tumor growth associated thymic atrophy and splenomegaly, accompanied by altered cell survival and repertoire of splenic, bone marrow and tumor associated macrophages (TAM). TAM displayed augmented phagocytic, tumoricidal activities and production of IL-1 and TNF-α. 3-BP-induced activation of TAM was of indirect nature, mediated by IFN-γ. Blood count of T lymphocytes (CD4+ & CD8+) and NK cells showed a rise in 3-BP administered tumor bearing mice. Moreover, 3-BP administration triggered modulation of immunomodulatory cytokines in serum along with refurbished hepatic and renal functions. The study indicates the role of altered cytokines balance, site specific differential macrophage functions and myelopoiesis in restoration of lymphoid organ homeostasis in 3-BP administered tumor bearing host. These observations will have long lasting impact in understanding of alternate mechanisms underlying the antitumor action of 3-BP accompanying appraisal of safety issues for optimizing its antineoplastic actions.

Antitumor and chemosensitizing action of 3-bromopyruvate: Implication of deregulated metabolism.

3-Bromopyruvate (3-BP), brominated derivative of pyruvate, possesses strong antitumor potential, owing to its ability to inhibit multiple target molecules crucial for survival of neoplastic cells. Although, 3-BP displays cytotoxicity against a wide variety of tumors, there is no report with respect to malignancies of thymic origin. Therefore, we investigated its antineoplastic action in vitro against tumor cells of a murine transplantable lymphoma of thymoma origin, designated as Dalton's lymphoma (DL). 3-BP treatment of tumor cells inhibited metabolism and survival with augmented induction of apoptosis and necrosis. 3-BP treatment suppressed lactate release, glucose uptake, deregulated pH homeostasis and augmented chemosensitization. It also altered expression of metabolism, chemosensitivity and cell survival regulatory molecules including HK 2, GAPDH, LDH, SDH, HIF-1α, MDR-1 & GLUT-1 and cytokine repertoire of IFN-γ, IL-6, IL-10, & VEGF. Pretreatment with MCT-1 inhibitor α-cyano-4-hydroxycinnamate and siRNA gene silencing of HK 2 implicated the role of MCT-1 and HK 2 in 3-BP cytotoxicity. 3-BP also altered expression of cell death regulatory Bcl-2, Mcl-1, caspase-3 accompanied by increased cytochrome c release, indicating mitochondrial mode of cell death. The study collates possible molecular mechanisms of cytotoxic action of 3-BP, which will help to optimize the therapeutic efficacy of 3-BP against tumors of thymic origin.

Angiogenesis Assays.

Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.