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