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.

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 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.

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.