Biology subjects

Ball, A. G.

Publications and source records attributed to Ball, A. G..

5 recordsLinked to original sources

bioRxiv2024

Ex vivo model of breast cancer cell invasion in live lymph node tissue

Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naive LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naive LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

bioengineering↗

bioRxiv2024

Multi-organ immunity on a 3D-printed multi-tissue chip with tubing-free impeller pump

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement, due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump with the capacity to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D-printed mesh supports for at least 24 hr. In a two-compartment model of a LN and an upstream injection site in mock tissue, vaccination of the multi-compartment chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

bioengineering↗

bioRxiv2024

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naive lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naive lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/573955v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@22ae83org.highwire.dtl.DTLVardef@ef1c94org.highwire.dtl.DTLVardef@3d3906org.highwire.dtl.DTLVardef@16ae318_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Detergent wash improves vaccinated lymph node handling ex vivo

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 - 75 % to 0 - 25 %, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

immunology↗

bioRxiv2019

Acute lymph node slices are a functional model system to study immunity ex vivo

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-hr viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

immunology↗