In vitro three-dimensional tumor microenvironment models for anticancer drug discovery

Anticancer drug discovery has long been hampered by the poor predictivity of the preclinical models. There is a growing realization that the tumor microenvironment is a critical determinant of the response of cancer cells to therapeutic agents. The past 5 years have seen a great deal of progress in our understanding of how the three-dimensional microenvironment modulates the signaling behavior of tumor cells. The present review discusses how three-dimensional in vitro cell culture models can benefit cancer drug discovery through an accurate modeling of the tumor microenvironment, leading to more physiologically relevant experimental outcomes

The Anti-Melanoma Activity of Dinaciclib, a Cyclin-Dependent Kinase Inhibitor, Is Dependent on p53 Signaling

<div><p>Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.</p> </div

Dinaciclib inhibits the growth and survival of melanoma cell lines grown under both 2D adherent culture and 3D organotypic cell culture conditions.

<p>A) Growth inhibition for melanoma cells treated with dinaciclib measured by the MTT assay. Cells were treated with increasing concentrations of dinaciclib (1 nM–10 µM) for 72 hours before treatment with the MTT reagent. Absorbances were read at 570 nm and expressed as a percentage of control. B) Dinaciclib reduces the viability and invasion of melanoma cells grown as three-dimensional collagen-implanted spheroids. Preformed melanoma spheroids that harbored either the BRAF V600E mutation (1205Lu) or NRAS mutation (WM1366) were embedded into collagen and overlaid with medium. Cells were then treated with either 10 nM or 30 nM dinaciclib for 72 hours before being stained with calcein-AM and ethidium bromide. Spheroids were visualized and photographed using an inverted fluoresce microscope. <i>Green</i>, viable cells; <i>red</i>, dead cells.</p

Dinaciclib treatment induces the up-regulation of p53 expression and caspase-3 cleavage and a downregulation of the pro-survival molecules Bcl-2, XIAP, and Mcl-1 in vitro.

<p>Western Blot data of dinaciclib treated 1205Lu cells treated with 30 nM of dinaciclib for increasing periods of time (0–48 hrs). Along with a decrease in pRBser807/811, dinaciclib induced a marked upregulation of p53, increase in cleaved caspase-3, and down regulation of the pro-survival molecules Bcl-2, XIAP, and Mcl-1. The membrane was probed for actin expression to ensure equal protein loading.</p

shRNA knockdown of p53 expression reverses dinaciclib-induced apoptosis.

<p>A) Knockdown of p53 levels using a lentiviral shRNA construct. WM35 melanoma cells were infected with a lentivirus encoding for shRNA against p53 and were selected by flow cytometric sorting for GFP (designated WM35 shp53). Western blot shows knockdown of p53 protein. Actin shows equal protein loading. B) WM35 cells expressing p53 shRNA show diminished cell accumulation in the sub-G1 phase of the cell cycle following dinaciclib treatment. Cells were treated with 30 nM of dinaciclib for either 12 hours or 24 hours, before being fixed and stained for DNA content with propidium iodide. C) Apoptosis in a wild-type p53 melanoma cell line is dependent upon p53. WM35 and WM35 shp53 cells were treated with 30 nM dinaciclib for 48 hrs and were assessed for apoptosis via Annexin-V staining. WM35 shp53 cells exhibited a dramatic reduction of apoptosis when compared to WM35. D) The knockdown of p53 led to decreased pRB (ser807/811) and a decrease in cleaved PARP in cells treated with dinaciclib. WM35 and WM35 shp53 cells were treated with 30 nM of dinaciclib for 8, 16, 24, and 48 hrs, after which protein were assessed for the levels of p53, pRBser807/811, PARP, and Mcl-1. Actin was used as loading control.</p

Dinaciclib causes a G2/M arrest and apoptosis in a panel of melanoma cell lines.

<p>A) Dinaciclib causes a G2/M arrest within 12 hours of treatment. Cell cycle analysis of a BRAF V600E (1205Lu) mutated or NRAS mutated (WM1366) cell line. Cells were treated with 30 nM of dinaciclib for either 12 hours or 24 hours; then cells were fixed for 24 hrs, and then stained for DNA content with propidium iodide. B) Dinaciclib causes apoptosis in a panel of melanoma cell lines independent of BRAF V600E <i>(blue),</i> NRAS <i>(red)</i> mutational status and CDK2 levels. Cells were treated with 30 nM dinaciclib for 48 hrs and apoptosis was assessed via Annexin-V staining. Data represent % of cells that were Annexin V +. Data shows mean of three independent experiments +/− S.E.</p