5 ala

All reagents were of the highest purity commercially available. SA, 5-ALA, 4-iodophenol, TMP, furosemide, saponin, IPP, doxycycline, luminol, and DHA were purchased from Sigma (St. Louis, MO, USA). 5-[¹³C₄]-ALA was purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA, USA).

PpIX photobleaches quickly.^{21 (link)} This makes it harder to see low levels of PpIX fluorescence with an operating microscope. Therefore, we stored ex vivo ultrasonic tissue fragments in a low-volume incubation chamber (Scientific Systems Design) and bathed the tissue fragments in 1 mM 5-ALA (Sigma: 5451-09-2) for 30 min prior to experiments, which was continued during experiments.^{21 (link)}The ultrasonic samples were imaged on an interface recording chamber^{22 (link)} (Scientific Systems Design), which was mounted on an Olympus BX51WI epifluorescence microscope. A custom filter set (excitation 402/15 nm, emission 654/75 nm; Chroma Technology Corp.) was fitted to the filter cube turret of the BX51WI microscope to detect PpIX fluorescence. The laboratory fluorescence images and brightfield images were acquired on a Spot RT sCMOS cooled 5MP camera (RT39M5, Spot Imaging) controlled by Spot Advanced imaging software (Spot Imaging).
The fluorescence signal was quantified in FIJI (https://imagej.net/Fiji).^{23 (link)} The mean pixel intensity of the background (no tissue) was subtracted from pixel intensity in the tissue fragment region of interest. The pixel intensities in the region of interest were summed and divided by the area of the region of interest to give a total fluorescence signal × 10⁷ per mm², which is expressed in relative fluorescence units (RFU).

We purchased 5-ALA from Sigma-Aldrich (Cat. No. A7793, USA) and dissolved it in phosphate buffered saline (PBS, pH 7.4) to prepare a 500 mM stock solution. It was sterilized by filtration using a 0.2 um pore filter, and wrapped in aluminum foil to avoid light exposure and stored at −20°C. Next, 5×10⁴ cells were seeded into 24-well plates and incubated in 500 μl media overnight. Cells were incubated with 5-ALA (final concentration 1 mM) for 1 hour, rinsed twice with PBS, then incubated in complete medium until analysis. Special care was taken to avoid exposure to light during the experiments.

Full description of methods is provided in Supplementary Methods but in brief, cell culture was undertaken with/without 1 mL 20 mM 5-ALA (Sigma-Aldrich) per 75 mL flask. Presto blue (Invitrogen) was performed according to the manufacturer’s protocol. Real-time PCR was performed with iQ SYBR Green Supermix 2x (Bio-Rad) and immunohistochemistry with DAKO reagents and secondary antibody. Transcriptomic libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB: E7490), the NEBNext Ultra Directional Library Kit for Illumina (NEB: E7420), and the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1; NEB: E7335L). Raw data have been deposited at ArrayExpress, accession number E-MTAB-8743. siRNA knockdown was performed using 25 nM of SERPINE1 targeting and nontargeting sequences (Dharmacon). Invasion assays were conducted using 24-well ThinCert cell culture inserts (Greiner) coated with 10 μg of collagen IV (Cultrex, Trevigen). In vivo patient-derived xenografts were performed in male Rag2/Il2rg (RAG) immunodeficient mice (10–12 weeks old) according to National Cancer Research Institute and Laboratory Animal Science Association guidelines.