Biology subjects

Toley, B. J.

Publications and source records attributed to Toley, B. J..

3 recordsLinked to original sources

bioRxiv2026

Enabling Spatially Resolved Gene Expression Analysis in a Multilayer Paper-Based In Vitro Tumor Model

Spatial heterogeneity is a defining feature of solid tumors, yet molecular analysis of three-dimensional (3D) in vitro tumor models is often performed as a bulk measurement, potentially obscuring position-dependent cellular responses. Here, we establish an analytical workflow for spatially resolved gene-expression analysis in a multilayer paper-based 3D tumor model by addressing the challenge of RNA recovery from cell-laden paper-Matrigel scaffolds. Using MDA-MB-231 breast cancer cells, multiple RNA extraction strategies were systematically evaluated across conventional culture, Matrigel, paper, and composite paper-Matrigel environments. Conventional silica-column extraction yielded only 3.39 {+/-} 0.93 ng/L RNA from the composite scaffold, whereas TRIzol-chloroform extraction increased recovery to 201.13 {+/-} 38.32 ng/L. The optimized workflow subsequently enabled independent RNA recovery from all eight layers of a multi-layer paper-based in vitro tumor, with concentrations ranging from 113.06 {+/-} 5.00 to 245.26 {+/-} 15.76 ng/L and purity suitable for downstream qRT-PCR analysis. Layer-resolved profiling of untreated cultures revealed distinct spatial patterns in HIF1A, HIF2A, VEGFA, and GLUT4 expression across the multilayer construct. To evaluate the workflow under pharmacological perturbation, cultures were exposed to 5 M doxorubicin (DOX) for 48 h. Relative cell viability exhibited a non-monotonic spatial profile, whereas DOX-induced HIF1A and VEGFA expression generally became more pronounced toward the deeper layers when each treated layer was compared with its corresponding untreated control. Collectively, these results establish a workflow for converting individually addressable regions of a multilayer 3D tumor model into spatially resolved transcriptional measurements, enabling analysis of both baseline spatial heterogeneity and position-dependent molecular responses to pharmacological perturbation.

bioengineering↗

bioRxiv2026

Wax-printing-free fabrication of paper-supported 3D cancer cell culture

Three-dimensional (3D) in vitro tumor models are critical for studying transport-limited drug efficacy in solid tumors; however, many existing platforms are technically complex and remain difficult to access. Stacked paper-based tumor models ("cells-in-gel-in-paper", CiGiP) address this challenge by enabling formation of diffusion-limited microenvironments while allowing direct access to cells from distinct tissue depths. Nevertheless, current CiGiP implementations rely on wax or hydrophobic barrier patterning of paper, which has become increasingly inaccessible. Here, we present a wax-printing-free approach for fabricating stacked paper-supported 3D tumor tissues using a simple 3D-printed press-fit enclosure that holds circular paper layers snugly together, thereby enforcing one-dimensional transport without lateral leakage. Using MDA-MB-231 breast cancer cells embedded in Matrigel, we demonstrate the formation of nutrient-limited microenvironments across the tissue depth, as evidenced by layer-dependent cell viability. The platform enables direct quantification of spatial and temporal drug responses, demonstrated using doxorubicin and paclitaxel, both individually and in combination. Layer-dependent cytotoxicity was measured, and combination treatment analysis revealed antagonistic interactions consistent with prior reports. By eliminating the need for hydrophobic patterning, this approach substantially lowers the technical barriers to constructing stacked paper tumor models and is expected to facilitate broader adoption of paper-supported 3D tissues for drug screening and mechanistic studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/705119v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@55f56aorg.highwire.dtl.DTLVardef@1633288org.highwire.dtl.DTLVardef@18aae9eorg.highwire.dtl.DTLVardef@1ce1532_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

A large-volume sputum dry storage and transportation device for molecular and culture-based diagnosis of tuberculosis

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in native Standard 17 glass fiber was stable for molecular testing after 10-day storage at 45{degrees}C, and for culture testing after 10- and 5-day storage at 37{degrees}C and 45{degrees}C, respectively. Compatibility with human sputum storage was demonstrated by dry storing Mycobacterium bovis-spiked pooled human sputum in SPECTRA-tube for 5 days at room temperature followed by successful qPCR detection. By significantly increasing the volume of samples that can be transported in the dry state and enabling recovery of the entire sample in liquid state, SPECTRA-tube presents a potential universal solution for the preservation and transportation of liquid specimens.

bioengineering↗