Development and validation of an advanced ex vivo brain slice invasion assay to model glioblastoma cell invasion into the complex brain microenvironment.

Organotypic cultures of murine brain slices are well-established tools in neuroscience research, including electrophysiology studies, modeling neurodegeneration, and cancer research. Here, we present an optimized ex vivo brain slice invasion assay that models glioblastoma multiforme (GBM) cell invasion into organotypic brain slices. Using this model, human GBM spheroids can be implanted with precision onto murine brain slices and cultured ex vivo to allow tumour cell invasion into the brain tissue. Traditional top-down confocal microscopy allows for imaging of GBM cell migration along the top of the brain slice, but there is limited resolution of tumour cell invasion into the slice. Our novel imaging and quantification technique involves embedding stained brain slices into an agar block, re-sectioning the slice in the Z-direction onto slides, and then using confocal microscopy to image cellular invasion into the brain tissue. This imaging technique allows for the visualization of invasive structures beneath the spheroid that would otherwise go undetected using traditional microscopy approaches. Our ImageJ macro (BraInZ) allows for the quantification of GBM brain slice invasion in the Z-direction. Importantly, we note striking differences in the modes of motility observed when GBM cells invade into Matrigel in vitro versus into brain tissue ex vivo highlighting the importance of incorporating the brain microenvironment when studying GBM invasion. In summary, our version of the ex vivo brain slice invasion assay improves upon previously published models by more clearly differentiating between migration along the top of the brain slice versus invasion into the slice.

Germinal center hypoxia in tumor-draining lymph nodes negatively regulates tumor-induced humoral immune responses in mouse models of breast cancer.

Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.