Engineering bacteria for cancer immunotherapy.

Bacterial therapeutics have emerged as promising delivery systems to target tumors. These engineered live therapeutics can be harnessed to modulate the tumor microenvironment or to deliver and selectively release therapeutic payloads to tumors. A major challenge is to deliver bacteria systemically without causing widespread inflammation, which is critical for the many tumors that are not accessible to direct intratumoral injection. We describe potential strategies to address this challenge, along with approaches for specific payload delivery and biocontainment to ensure safety. These strategies will pave the way for the development of cost-effective, widely applicable next-generation cancer therapeutics.

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows.

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers.

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification.

We have developed a method for spatially resolved genetic analysis of formalin-fixed paraffin-embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 µm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF-7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Nip the bubble in the bud: a guide to avoid gas nucleation in microfluidics.

Gas bubbles are almost a routine occurrence encountered by researchers working in the field of microfluidics. The spontaneous and unexpected nature of gas bubbles represents a major challenge for experimentalists and a stumbling block for the translation of microfluidic concepts to commercial products. This is a startling example of successful scientific results in the field overshadowing the practical hurdles of day-to-day usage. We however believe such hurdles can be overcome with a sound understanding of the underlying conditions that lead to bubble formation. In this tutorial, we focus on the two main conditions that result in bubble nucleation: surface nuclei and gas supersaturation in liquids. Key theoretical concepts such as Henry's law, Laplace pressure, the role of surface properties, nanobubbles and surfactants are presented along with a view of practical implementations that serve as preventive and curative measures. These considerations include not only microfluidic chip design and bubble traps but also often-overlooked conditions that regulate bubble formation, such as gas saturation under pressure or temperature gradients. Scenarios involving electrolysis, laser and acoustic cavitation or T-junction/co-flow geometries are also explored to provide the reader with a broader understanding on the topic. Interestingly, despite their often-disruptive nature, gas bubbles have also been cleverly utilized for certain practical applications, which we briefly review. We hope this tutorial will provide a reference guide in helping to deal with a familiar foe, the "bubble".