Cervical lymph nodes were harvested from C57Bl/6 mice held at the Moores Cancer Center, UCSD. These mice are housed in individually ventilated cages in a facility at temperatures between 18-23°C and humidity 40-60%. All personnel handling animals are required to wear full scrubs, face mask, hair net, vivarium-approved footwear, and disposable gloves. Once harvested, cervical lymph nodes were washed for 15 minutes and 2 wash cycles in ice-cold PBS before being placed in 0.075% SDS in PBS for 16 hours at slight agitation. After decellularization, the nodes were washed for one hour and 4 wash cycles in ice-cold PBS and stored for analysis.

Hind limbs from C57Bl/6 mice were harvested and washed in 100% ethanol before moving into sterile PBS. Skin and muscle tissue were removed and femur and tibia were isolated. Bone caps were removed with scissors and marrow was flushed into a conical tube with sterile PBS in a 25 gauge needle and 10cc syringe. 3 million cells were then resuspended into 3 mLs of BMDC media containing granulocyte macrophage colony-stimulating factor (GM-CSF), and cultured in a 6-well plate for 10 days, with an additional spike of GM-CSF on day 8.

Bone-marrow derived dendritic cells were activated with CpG for 12 hours, before the cells were spiked with the model antigen ovalbumin (OVA). After 8 hours, the activated DCs were seeded into the dLN scaffold via the centrifugation method, and cultured for 24 hours. 500uL of blood from an OT-1 transgenic mouse was collected and lymphocytes isolated using Lympholyte (Tebu-bio; Offenbach, Germany). Lymphocytes were cultured in a 96 well U-bottom plate with seeded dLN at 40,000 cells/well for 24 hours. B16-OVA cells were plated in a transparent 96 well flat-bottom plate at 4,000 cells/well, and the activated lymphocytes were added at 40,000 cells/well, alongside control conditions of B16-OVA alone, B16-OVA + 20% DMSO, lymphocytes alone, and B16-OVA + lymphocytes activated by plate-bound DCs. After 24 hours, the wells were evacuated and washed to remove splenocytes, and the AquaBluer redox indicator was added (MultiTarget Pharm; Colorado Springs, CO) and incubated for 2 hours. The %RFU was calculated as the percent difference of the data point from the average fluorescence value of the B16-OVA control samples.

For samples in Figure 1, native and decellularized lymph nodes were fixed in zinc formalin fixative for 4 hours, then stored in 70% ethanol and sent to HistoServ for staining and sectioning (Germantown, MD). Slides were then scanned at the Moores Cancer Center Biorepository and Tissue Technology Core (La Jolla, CA) and analyzed using Aperio ImageScope. For samples in Figures 2 and 3, native, decellularized, and seeded lymph node samples were fixed in 4% paraformaldehyde for 4 hours, then submerged in 15% sucrose for 12 hours and 30% sucrose for 24 hours to prevent freezing damage. Samples were then frozen in OCT and sectioned into 10 um sections at -20^oC using a Leica Biosystems cryostat at the Sanford Consortium for Regenerative Medicine. The resulting slides were then stained with hematoxylin and eosin (H&E) and Alcian Blue (Vector Laboratories; Burlingame, CA) to visualize tissue morphology and quantify residual SDS. Slides processed in the Christman Lab were also scanned using an Aperio ScanScope slide scanner and analyzed with Aperio ImageScope (Leica Biosystems; Wetzlar, Germany).

ImageJ (NIH; Bethesda, MD) was used to measure intensity of Alcian Blue staining in each of the sections. Briefly, a histogram of pixel intensity from 0-255 was taken from the region of interest within the native or decellularized lymph node. The average pixel intensity of all non-background colored pixels was calculated for each sample and subtracted from 255. Intensity values for each decellularized lymph node were normalized against the average intensity of their respective native lymph nodes to account for changes in the staining protocol.

ImageJ (NIH; Bethesda, MD) was used to obtain size measurements on images of 10 um thick H&E stained histological sections of the decellularized lymph nodes. Briefly, the scale bar was used to set the scale of each image. Then the perimeter of the lymph nodes were traced with the freehand selection tool and measured to find the area, feret diameter, circularity and aspect ratio.

Double-stranded DNA present within each sample was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen; Carlsbad, CA). In brief, samples were diluted to a final volume of 100uL in TE buffer, and 100uL of the Quant-iT PicoGreen reagent was added to each sample. The samples were plated onto a 96 well plate, alongside standards of stock dsDNA concentrations. These samples were then read on a plate reader (Ex=480nm, Em=520nm) to measure fluorescence, and a standard curve was generated to quantify the amount of double-stranded DNA in each sample.

Evans Blue was injected into subsites of the oral cavity, the tongue and buccal mucosa, and visibly spread into the left and right superficial nodal basins (via tongue) and into the ipsilateral superficial nodal basin (via the buccal mucosa) (data not shown). To confirm that the vessels stained were indeed lymphatic, a siinfekl peptide based antigen and an immunostimulant were injected into the same subsites of the Evans Blue injection. The nodal basins were then extracted and run with flow cytometry to check that the siinfekl peptide was presented by antigen presenting cells located in the stained vessels (data not shown).

To properly capture the complex 3D architecture of the lymphatic system in the head and neck cavity of our mice models, the clearing-enhanced 3D (C_e3D) tissue clearing method developed by Weizhe Li, Ronald N. Germain, and Michael Y. Gerner was used. Key characteristics defining the quality of tissue preparation for microscopy imaging include tissue transparency, preserving brightness of reporter protein fluorescence, and strong immunolabeling, all of which the C_e3D tissue clearing method achieved. Briefly, the lymphatic vasculature in the head and neck cavity were harvested from each of the mice models, fixed overnight, and incubated with a blocking buffer for at least 8 hours. The lymphatic tissues were then incubated with the C_e3D medium placed on a rotor at room temperature for 12-72 hours. Sequentially, the lymphatic tissues were stained with LYVE (Lymphatic Vessel Endothelial) to highlight lymphatic vasculature and provide a magenta fluorescence when observed with IMARIS. The tissues were then placed in a glass slide for microscopy imaging. These tissue samples were prepared and imaged for us by Dr. Robert Saddawi-Konefka, and these procedures were used to prepare tissue samples from wild type, HNSCC tumor-bearing, and neck dissection mice models, individually. For more information on the C_e3D tissue clearing protocol, please access the cited article³².

The microscopy images were first filtered to emphasize the LYVE staining of the lymphatic vasculature by eliminating the display of autofluorescence. The Surface Segmentation feature was then used to translate the microscopy images, based on the brightness and presence of the LYVE staining, into solid 3D models. The entire image was processed and background artifacts removed through thresholding. The Slicing feature of IMARIS allowed for measuring of the size of lymphatic vessels, and these measurements were used to filter out undesired components of the model IMARIS created. Creating the solid model allowed us to extract statistics that IMARIS automatically produces based on the segmented surfaces. IMARIS provided the volumes of individual components of the model, for which the numbers were totaled for each whole mouse model to compare the physiological states of their lymphatic vasculature. This procedure was then repeated for a region of interest behind the tongue in each model to achieve normalization in measuring vessel count, average volume per vessel, and total volume of stained vessels.

Figure cartoons were created using BioRender software (Toronto, Canada). Data graphs and statistical analyses were formed with the GraphPad Prism version 7 program (San Diego, CA). All data is reported as mean ± standard error of the mean. Asterisks denote statistical significance, with ns = not significant (p>0.05), * = p<0.05, ** = p<0.01, *** = p<0.001, and **** = p<0.0001)