Methods
Culture : Two cSCC cell lines (SCC-13 and A431) and two HDFs cell lines (AG13145 and CRL-2617) were cultured according to existing protocols.13,18,23 cSCCs and HDFs were studied to evaluate the cellular composition of a skin biopsy representative of AK/cSCC.
5-ALA Treatment : Twenty-four hours following plating, cSCC cell lines were treated with 0 or 0.5 mM 5-ALA (SUN pharmaceuticals) and incubated for thirty minutes on a custom-designed heating block at temperatures of 39 oC, according to existing protocols.13,18,23 Thermal incubation of 5-ALA enhances treatment efficacy in vitro.18
Blue Light Photoactivation : Cells were irradiated using a commercially available blue light device (BLU-U, SUN Pharmaceuticals) as per prior protocols.13,18,23 The blue light device produces a uniform field of blue light (420 ± 5 nm) on a black surface. The blue light has a power density of 10 W/cm2 at 5 cm. After 1000 seconds of irradiation with blue light, the total fluence was 10 J/cm2.
RNA Collection : At 0, 4, and 24 hours following blue light photoactivation, RNA was purified using miRNeasy Kit (Qiagen) with centrifuge and Qiashredder (Qiagen) extraction method, according to manufacturers’ protocols. The RNase-free DNA digestion set (Qiagen) included a DNA Lysis step (Qiagen). RNA concentrations and 260/280 scores were quantified using a Nanodrop One spectrophotometry (Thermofisher Scientific). Isolated RNA was stored at -80oC and submitted to the Icahn School of Medicine at Mount Sinai Genomics Core Facility for RNA-Seq.
RNA sequencing and bioinformatics : Libraries were prepared for RNA RNA-Seq using the Stranded Total RNA with RiboZero Plus library prep kit (Illumina). RNA-Seq was performed using NovaSeq 6000 SP Reagent Kit v1.5 (300 cycles) to achieve about 26 million reads per sample and paired-end 150bp (Illumina). Q30 and quality score are included in Supplemental Table 1.
Bioinformatics : Principal component analysis (PCA) was applied to the normalized samples to determine how samples cluster across cell lines, time points, and treatments. Differentially expressed genes between ALA-PDT and control samples were computed using the limma-voom.24 The limma-voom gene sets were generated by sorting the results using limma’s moderated t-statistic. From the sorted results, we analyzed the most significant up- and down-regulated gene sets with a p-value of < 0.05 without multiple hypotheses correction with the BH method. After BH correction, no statistically significant differentially expressed genes were identified. Enrichment analysis was performed using Enrichr against the GO Biological Processes 2021, KEGG 2021, ENCODE, and ChEA 2022 gene set libraries.25,26