Entinostat induces antitumor immune responses through immune editing of tumor neoantigens.

Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

Oxygen as a chemoattractant: confirming cellular hypoxia in paper-based invasion assays.

Low oxygen tension, or hypoxia, is a common occurrence in solid tumors. Hypoxia is a master regulator of cellular phenotype, and is associated with increased tumor invasion and aggressiveness as well as adverse patient prognosis. Oxygen has recently been linked with the selective movement of different cancer cell types in three-dimensional invasion assays utilizing paper-based scaffolds. It has remained unclear, however, if cells in these paper-based invasion assays are experiencing hypoxia. In this manuscript, we adapted cell-based methods to measure oxygen tension in our 3D invasion assays: the adduction of pimonidazole to free thiols in the cell, indicative of a reducing environment; the localization of hypoxia inducible factors to the nucleus; and the expression of hypoxia-regulated gene products. We utilized each method to compare the oxygen tension in different locations of the paper-based invasion stacks and found an oxygen gradient is indeed forming. Specifically, we found that the extent of pimonidazole binding, as well as the levels and activities of nucleus-localized HIF-α proteins, increase as the distance between the cells and the source of fresh medium increases. These complementary cell-based readouts not only confirm the selective invasion we observe is due to an oxygen gradient, they also show the gradient is temporal in nature and evolves with increasing culture period.

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

Real-time imaging of cancer cell chemotaxis in paper-based scaffolds.

Cellular migration is the movement of cells, cultured as a monolayer; cellular invasion is similar to migration, but requires the cells to move through a three-dimensional material such as basement membrane extract or a synthetic hydrogel. Migration assays, such as the transwell assay, are widely used to study cellular movement because they are amenable to high-throughput screens with minimal experimental setup. These assays offer limited information about cellular responses to gradients in vivo because they oversimplify the threedimensional (3D) environment of a tissue. There are a number of invasion assays that support 3D cultures, some of which provide experimental control over the spatial and temporal gradients imparted on the culture. These assays, in their current form, are difficult to setup and maintain, and often require specialized laboratory equipment or engineering expertise. Here we describe a paper-based invasion assay in which cellular movement can be monitored in real-time with fluorescence microscopy. These assays are easily prepared and utilize materials commonly found in any laboratory: a single sheet of paper. These sheets are wax patterned to contain channels in which cells suspended in a hydrogel are seeded and cultured. Cell-containing sheets of paper are placed in a custom-built holder that allows gradients to form along the length of the channels. In this work, we compare the invasion of cells cultured in the presence and absence of an oxygen gradient. Our result support previous findings that oxygen is a chemoattractant, and selectively directs cellular movement in a 3D culture environment.

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.