Polyploid giant cancer cells: Unrecognized actuators of tumorigenesis, metastasis, and resistance.

Cancer led to the deaths of more than 9 million people worldwide in 2018, and most of these deaths were due to metastatic tumor burden. While in most cases, we still do not know why cancer is lethal, we know that a total tumor burden of 1 kg-equivalent to one trillion cells-is not compatible with life. While localized disease is curable through surgical removal or radiation, once cancer has spread, it is largely incurable. The inability to cure metastatic cancer lies, at least in part, to the fact that cancer is resistant to all known compounds and anticancer drugs. The source of this resistance remains undefined. In fact, the vast majority of metastatic cancers are resistant to all currently available anticancer therapies, including chemotherapy, hormone therapy, immunotherapy, and systemic radiation. Thus, despite decades-even centuries-of research, metastatic cancer remains lethal and incurable. We present historical and contemporary evidence that the key actuators of this process-of tumorigenesis, metastasis, and therapy resistance-are polyploid giant cancer cells.

High-throughput optofluidic screening for improved microbial cell factories via real-time micron-scale productivity monitoring.

The industrial synthetic biology sector has made huge investments to achieve relevant miniaturized screening systems for scalable fermentation. Here we present the first example of a high-throughput (>103 genotypes per week) perfusion-based screening system to improve small-molecule secretion from microbial strains. Using the Berkeley Lights Beacon® system, the productivity of each strain could be directly monitored in real time during continuous culture, yielding phenotypes that correlated strongly (r2 > 0.8, p < 0.0005) with behavior in industrially relevant bioreactor processes. This method allows a much closer approximation of a typical fed-batch fermentation than conventional batch-like droplet or microplate culture models, in addition to rich time-dependent data on growth and productivity. We demonstrate these advantages by application to the improvement of high-productivity strains using whole-genome random mutagenesis, yielding mutants with substantially improved (by up to 85%) peak specific productivities in bioreactors. Each screen of ∼5 × 103 mutants could be completed in under 8 days (including 5 days involving user intervention), saving ∼50-75% of the time required for conventional microplate-based screening methods.

An in vitro tumor swamp model of heterogeneous cellular and chemotherapeutic landscapes.

The heterogenous, highly metabolic stressed, poorly irrigated, solid tumor microenvironment - the tumor swamp - is widely recognized to play an important role in cancer progression as well as the development of therapeutic resistance. It is thus important to create realistic in vitro models within the therapeutic pipeline that can recapitulate the fundamental stress features of the tumor swamp. Here we describe a microfluidic system which generates a chemical gradient within connected microenvironments achieved through a static diffusion mechanism rather than active pumping. We show that the gradient can be stably maintained for over a week. Due to the accessibility and simplicity of the experimental platform, the system allows for not only well-controlled continuous studies of the interactions among various cell types at single-cell resolution, but also parallel experimentation for time-resolved downstream cellular assays on the time scale of weeks. This approach enables simple, compact implementation and is compatible with existing 6-well imaging technology for simultaneous experiments. As a proof-of-concept, we report the co-culture of a human bone marrow stromal cell line and a bone-metastatic prostate cancer cell line using the presented device, revealing on the same chip a transition in cancer cell survival as a function of drug concentration on the population level while exhibiting an enrichment of poly-aneuploid cancer cells (PACCs) as an evolutionary consequence of high stress. The device allows for the quantitative study of cancer cell dynamics on a stress landscape by real-time monitoring of various cell types with considerable experimental throughput.

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the "Evolution Accelerator," breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

The role of heterogeneous environment and docetaxel gradient in the emergence of polyploid, mesenchymal and resistant prostate cancer cells.

The ability of a population of PC3 prostate epithelial cancer cells to become resistant to docetaxel therapy and progress to a mesenchymal state remains a fundamental problem. The progression towards resistance is difficult to directly study in heterogeneous ecological environments such as tumors. In this work, we use a micro-fabricated "evolution accelerator" environment to create a complex heterogeneous yet controllable in-vitro environment with a spatially-varying drug concentration. With such a structure we observe the rapid emergence of a surprisingly large number of polyploid giant cancer cells (PGCCs) in regions of very high drug concentration, which does not occur in conventional cell culture of uniform concentration. This emergence of PGCCs in a high drug environment is due to migration of diploid epithelial cells from regions of low drug concentration, where they proliferate, to regions of high drug concentration, where they rapidly convert to PGCCs. Such a mechanism can only occur in spatially-varying rather than homogeneous environments. Further, PGCCs exhibit increased expression of the mesenchymal marker ZEB1 in the same high-drug regions where they are formed, suggesting the possible induction of an epithelial to mesenchymal transition (EMT) in these cells. This is consistent with prior work suggesting the PGCC cells are mediators of resistance in response to chemotherapeutic stress. Taken together, this work shows the key role of spatial heterogeneity and the migration of proliferative diploid cells to form PGCCs as a survival strategy for the cancer population, with implications for new therapies.

Cancer dormancy and criticality from a game theory perspective.

BACKGROUND: The physics of cancer dormancy, the time between initial cancer treatment and re-emergence after a protracted period, is a puzzle. Cancer cells interact with host cells via complex, non-linear population dynamics, which can lead to very non-intuitive but perhaps deterministic and understandable progression dynamics of cancer and dormancy. RESULTS: We explore here the dynamics of host-cancer cell populations in the presence of (1) payoffs gradients and (2) perturbations due to cell migration. CONCLUSIONS: We determine to what extent the time-dependence of the populations can be quantitively understood in spite of the underlying complexity of the individual agents and model the phenomena of dormancy.

Epithelial and mesenchymal prostate cancer cell population dynamics on a complex drug landscape.

We have improved our microfluidic cell culture device that generates an in vitro landscape of stress heterogeneity. We now can do continuous observations of different cancer cell lines and carry out downstream analysis of cell phenotype as a function of position on the stress landscape. We use this technology to probe adaption and evolution dynamics in prostate cancer cell metapopulations under a stress landscape of a chemotherapeutic drug (docetaxel). The utility of this approach is highlighted by analysis of heterogenous prostate cancer cell motility changes as a function of position in the stress landscape. Because the technology presented here is easily adapted to a standard epifluorescence microscope it has the potential for broad application in preclinical drug development and assays of likely drug efficacy.