Reversal of Anchorage-Independent Multicellular Spheroid into a Monolayer Mimics a Metastatic Model

Kunjithapatham, Rani; Karthikeyan, Swathi; Geschwind, Jean François H.; Kieserman, Esther K.; Lin, Ming De; Fu, Dax; Ganapathy-Kanniappan, Shanmugasundaram

doi:10.1038/srep06816

Cited by 30 publications

(46 citation statements)

References 21 publications

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“…The lysates were centrifuged at 12,000 × g for 15 min at 4°C to remove any cell debris. The clear supernatants were collected and the protein concentrations were determined using a 2-D Quant protein assay Kit (GE Healthcare) as described [17]. Then, protein samples were resolved on a 4–12% Bis-Tris gel by electrophoresis with MOPS buffer or 12% Bis-Tris gel electrophoresis with MES running buffer (for low molecular weight targets) and blotted onto PVDF membranes (BioRad, Hercules, CA, USA) followed by immunoblotting with specific antibodies.…”

Section: Methodsmentioning

confidence: 99%

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model

Karthikeyan

Potter

Geschwind

et al. 2016

Biochemical and Biophysical Research Communications

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Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

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Section: Methodsmentioning

confidence: 99%

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model

Karthikeyan

Potter

Geschwind

et al. 2016

Biochemical and Biophysical Research Communications

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“…However, the molecular mechanisms governing the formation and progression of PCa are still not well understood 19,20. Of the many techniques to study PCa formation and development, prostaspheres produced from PCa cells grown suspended in a purified ECM gel/matrix in vitro2,16,21 or within a hanging drop2 represent the most commonly used systems in the field. Other commonly used techniques for spheroid formation may include spontaneous aggregation, spinner flasks, rotatory cell culture systems, liquid overlay, low binding plates, microencapsulation, and cell cultivation in ultra-low attachment round-bottom plates where cell aggregation occurs as a result of mechanical forces or surface properties, preventing cell attachment to the bottom of the well 7,11,15,22–26.…”

Section: Introductionmentioning

confidence: 99%

Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK(TPP) peptide

Haq¹,

Samuel²,

Haxho³

et al. 2017

OTT

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BackgroundProstaspheres-based three dimensional (3D) culture models have provided insight into prostate cancer (PCa) biology, highlighting the importance of cell–cell interactions and the extracellular matrix (EMC) in the tumor microenvironment. Although these 3D classical spheroid platforms provide a significant advance over 2D models mimicking in vivo tumors, the limitations involve no control of assembly and structure with only limited spatial or glandular organization. Here, matrix-free prostaspheres from human metastatic prostate carcinoma PC3 and DU145 cell lines and their respective gemcitabine resistant (GemR) variants were generated by using cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)).Materials and methodsMicroscopic imaging, immunocytochemistry (ICC), flow cytometry, sialidase, and WST-1 cell viability assays were used to evaluate the formation of multicellular tumor spheroid (MCTS), cell survival, morphologic changes, and expression levels of α2,6 and α2,3 sialic acid (SA) and E- and N-cadherin in DU145, PC3, and their GemR variants.ResultsBy using the cyclo-RGDfK(TPP) peptide platform in a dose- and time-dependent manner, both DU145 and DU145GemR cells formed small MCTS. In contrast, PC3 and PC3GemR cells formed irregular multicellular aggregates at all concentrations of cyclo-RGDfK(TPP) peptide, even after 6 days of incubation. ICC and flow cytometry results revealed that DU145 cells expressed higher amounts of E-cadherin but lower N-cadherin compared with PC3 cells. By using Maackia amurensis (α2,3-SA-specific MAL-II) and Sambucus nigra (α2,6-SA specific SNA) lectin-based cytochemistry staining and flow cytometry, it was found that DU145 and DU145GemR cells expressed 5 times more α2,6-SA than α2,3-SA on the cell surface. PC3 cells expressed 4 times more α2,3-SA than α2,6-SA, and the PC3GemR cells showed 1.4 times higher α2,6-SA than α2,3-SA. MCTS volume was dose-dependently reduced following pretreatment with α2,6-SA-specific neuraminidase (Vibrio cholerae). Oseltamivir phosphate enhanced cell aggregation and compaction of 3D MCTS formed with PC3 cells.ConclusionThe relative levels of specific sialoglycan structures on the cell surface correlate with the ability of PCa cells to form avascular multicellular prostaspheres.

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“…MCTS would thus serve as an ideal model for the screening of novel anticancer drugs, especially for tumors that have demonstrated chemoresistance to past treatments 29,30. Furthermore, MCTS in matrigel or in ECM-based matrixes is a valuable tool for the study of cancer cell processes and chemotherapeutic responses in vitro 28,31. Recently, matrix-free spheroids have been developed to investigate cell growth, mutations, invasion, cell motility, and metastasis that contribute to the pathophysiology of tumors 32–36.…”

Section: Biological Models For Breast Cancer Researchmentioning

confidence: 99%

Alternative therapies for metastatic breast cancer: multimodal approach targeting tumor cell heterogeneity

Sambi¹,

Haq²,

Samuel³

et al. 2017

BCTT

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One of the primary challenges in developing effective therapies for malignant tumors is the specific targeting of a heterogeneous cancer cell population within the tumor. The cancerous tumor is made up of a variety of distinct cells with specialized receptors and proteins that could potentially be viable targets for drugs. In addition, the diverse signals from the local microenvironment may also contribute to the induction of tumor growth and metastasis. Collectively, these factors must be strategically studied and targeted in order to develop an effective treatment protocol. Targeted multimodal approaches need to be strategically studied in order to develop a treatment protocol that is successful in controlling tumor growth and preventing metastatic burden. Breast cancer, in particular, presents a unique problem because of the variety of subtypes of cancer that can arise and the multiple drug targets that could be exploited. For example, the tumor stage and subtypes often dictate the appropriate treatment regimen. Alternate multimodal therapies should consider the importance of time-dependent drug administration, as well as targeting the local and systemic tumor environment. Many reviews and papers have briefly touched on the clinical implications of this cellular heterogeneity; however, there has been very little discussion on the development of study models that reflect this diversity and on multimodal therapies that could target these subpopulations. Here, we summarize the current understanding of the origins of intratumoral heterogeneity in breast cancer subtypes, and its implications for tumor progression, metastatic potential, and treatment regimens. We also discuss the advantages and disadvantages of utilizing specific breast cancer models for research, including in vitro monolayer systems and three-dimensional mammospheres, as well as in vivo murine models that may have the capacity to encompass this heterogeneity. Lastly, we summarize some of the current advancements in the development of multitarget therapeutics that have shown promising results in clinical and preclinical studies when used alone or in combination with traditional regimens of surgery, chemotherapy, and/or radiation.

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Reversal of Anchorage-Independent Multicellular Spheroid into a Monolayer Mimics a Metastatic Model

Cited by 30 publications

References 21 publications

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model

Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model

Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK(TPP) peptide

Alternative therapies for metastatic breast cancer: multimodal approach targeting tumor cell heterogeneity

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