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Supplementary Information Supplementary information Supplementary Discussion Radiation therapy is one of the key modalities in the management of HNSCC. As of today about 75% of patients with HNSCC are treated with radiotherapy alone or in adjuvant setting after surgery (1). Nevertheless, only few biological parameters have been identified so far as potent prognostic biomarkers for radiotherapy outcome and as potential targets for personalized treatment approaches of radiotherapy combined with targeted drugs. Markers for radiosensitivity include HPV positivity, which has been associated with improved survival and loco-regional control (2-4). On the other hand, tumor volume, the number and intrinsic radioresistance of CSC as well as tumor hypoxia and repopulation were shown to be associated with tumor radioresistance (2, 5- 12) . The tumor growth is maintained by a population of CSC which have unlimited self- renewal potential and cause tumor recurrence if not eradicated by treatment. The number of CSC is a promising biomarker for local tumor control especially for the primary RCTx setting when the number of CSC correlates with primary tumor volumes (8, 9). The tumor suppressor p53 is a key regulator of energy metabolism (13). Mutations in p53 are ubiquitous in HPV negative HNSCC (14). Cal33 HNSCC cells, which were used for the SLC3A2 gene knockout, harbour a p53R175H mutation, which has oncogenic functions and promotes tumor progression (15). A recent study showed that inducible expression of p53R175H in tumor cells increases mitochondrial oxygen consumption and cell proliferation as well as decreasing intracellular ROS levels (16). Interestingly, previous studies demonstrated that the p53R175H mutation prevents cell 1 cycle arrest and apoptosis by attenuation of the expression of stress related gene ATF3 (17, 18). Inhibition of the CD98hc-mediated amino acid transport in maKO cells resulted in suppression of the basal mitochondrial respiration, lowering cell proliferation rate and upregulation of the wild-type p53-dependent signalling including upregulation of stress related gene ATF3. Of note, expression level of ATF3 gene was found to be downregulated in HNSCC radioresistant cell lines suggesting that activation of cellular stress can be a strategy for HNSCC radiosensitization. Previous studies in HeLa and MCF7 cells have shown that CD98hc is required for amino acid exchange, activation of mTOR signalling pathway and cell growth (19). Amino acid transport mediated by CD98hc supplies the substrates to the Krebs cycle to sustain mitochondrial metabolism (20). Indeed, we found that CD98hc maKO cells exhibit a low level of mTOR activation, decreased levels of Krebs cycle intermediates as a source of energy and biosynthesis and, as a result, a downregulated proliferation rate. Supplementary methods Tissue sections and haematoxylin & eosin staining Before immunohistochemistry, all FFPE specimens were subjected to haematoxylin & eosin staining in order to confirm the presence of squamous cell carcinoma. For CD98hs staining, tumor tissues of the total cohort including 197 patients were available. The LAT1 staining was performed on tissue specimens of the patients of the monocentric Dresden cohort. Immunohistochemical staining of CD98hc 2 Following deparaffinization and antigen retrieval (antigen retrieval buffer pH6 (DAKO, Glostrup, DK)) for 28 min at 630 W, endogenous peroxidase activity was blocked (Peroxidase block, DAKO) for 10 min. Sections were then incubated with normal blocking serum (rabbit anti-goat serum) for 10 min to prevent unspecific binding of the secondary antibody followed by incubation with the polyclonal goat anti-human CD98 antibody (dilution 1:2500; Santa Cruz Biotechnology, Dallas, US; sc-7095). For negative controls, the corresponding IgG antibody was used. The staining was visualized by DAB immunostaining (Vectastain; Vector laboratories, Burlingame, US). Semiquantitative analyses of blinded samples were performed by two independent observers (D.D. and A.L.) with an interobserver variability <5%. Staining was evaluated both for percentage of positive stained tumor cells (≥10%) and for intensity (0, + vs ++, +++). For immunohistochemistry analysis, the respective isotype control was included to ensure specificity of the staining. During preparation of this manuscript, goat polyclonal anti-CD98 antibody from Santa Cruz Biotechnology (C-20, sc-7095) has been discontinued and replaced by mouse monoclonal antibody (E-5, sc-376815). We compared both antibodies by the immunochistochemical staining of HNSCC specimens and achieved comparable staining (Figure S 8 B). Immunohistochemical staining of LAT1 Following deparaffinization and antigen retrieval (antigen retrieval buffer pH9 (DAKO, Glostrup, DK) for 28 min at 630 W, endogenous peroxidase activity was blocked (Peroxidase block, DAKO) for 10 min. This was followed by incubation with the monoclonal rabbit anti-human LAT1 antibody (dilution 1:5000; Abcam, Cambridge, UK; 3 EPR17573). For negative controls, the corresponding IgG antibody was used. The staining was visualized by DAB immunostaining (Dako REAL EnVision Detection System, Peroxidase/DAB, rabbit/mouse). Semiquantitative analyses of blinded samples were performed by two independent observers (D.D. and A.L.) with an interobserver variability <5%. Staining was evaluated both, for percentage of positive stained tumor cells (≥10%) and for intensity (0,+ vs ++,+++). For immunohistochemistry analysis, the respective isotype control was included to ensure specificity of the staining (Figure S 8 C). Clonogenic cell survival assay Clonogenic cell survival assay was performed as described previously (11, 12). Cells were counted via Casy® Cell Counter TCC and plated in triplicates at a density of 500-2000 cells/well depending on the cell line. Assays were performed either in the regular two-dimensional (2-D) culture conditions in 6-well plates or in 3-D conditions when cells were embedded in Matrigel (BD Biosciences, GE) and plated in triplicates in ultra-low attachment plates (Corning, BE). Eighteen hours after cell plating cells were irradiated with doses of 2, 4, 6 and 8 Gy of 200 kV X-rays (Yxlon Y.TU 320; 200 kV X- rays, dose rate 1.3Gy/min at 20 mA) filtered with 0.5 mm Cu. The absorbed dose was measured using a Duplex dosimeter (PTW). After 10-14 days of culturing, the colonies were fixed with 10% formaldehyde (VWR International, GE) and stained with 0.05% crystal violet (Sigma-Aldrich, GE). Colonies containing more than 50 cells were counted using a stereo microscope (Zeiss, GE). Plating efficacies (PE = counted colonies/seeded cells x 100) and surviving fractions (SF = counted colonies/(seeded cells xPE) x 100) were calculated. 4 Microarray analysis of the HNSCC cancer cell lines Gene expression profiling of the Cal33 WT1, Cal33 WT2, Cal33 maKO1, Cal33 maKO2 cells was performed using SurePrint G3 Human Gene Expression 8x60K v3 Microarray Kit (Design ID 039494, Agilent Technologies) according to manufacturer's recommendations as described previously (11). Briefly, cells were irradiated with 4Gy of X-rays or sham irradiated and collected 24 h after irradiation. Total RNA was isolated from cell pellets using the RNeasy kit (Qiagen, Valencia, CA, USA). Sample preparation for analysis was carried out according to the protocol detailed by Agilent Technologies (Santa Clara, CA, USA). Arrays were processed using standard Agilent protocols. Probe values from image files were obtained using Agilent Feature Extraction Software. The dataset was analysed using SUMO software package: http://angiogenesis.dkfz.de/oncoexpress/software/sumo/. Data deposition: all data is MIAME compliant. The raw data has been deposited in the Gene Expression Omnibus (GEO) database, accession no GSE116162. CRISPR/Cas9 mediated gene editing Due to alternative splicing the SLC3A2 gene encodes multiple isoforms of CD98hc whose translation is initiated from different exons. Therefore it was not possible to completely knockout SLC3A2 via single indel mutation upon CRISPR/Cas9 gene editing and we decided to delete the whole gene by targeting simultaneously two sequences, upstream and downstream of the gene. In order to delete the whole SLC3A2 gene two sgRNAs were designed using Cas-Designer online tool http://www.rgenome.net/cas-designer/. First, sgRNA with targeting position upstream of SLC3A2 had sequence GT TGT GGG TAG AGA GTT CCA and was cloned into 5 pCasHF-EGFP vector. Second sgRNA with targeting position downstream of SLC3A2 had sequence GG ACC CTA CAT AAA TAA TGA and was cloned into pCasHF- mCherry vector. Prior to that pCasHF-EGFP and pCasHF-mCherry were generated from pSpCas9(BB)-2A-GFP ( gift from Feng Zhang, Addgene plasmid # 48138) and pU6-(BbsI)_CBh-Cas9-T2A-mCherry (gift from Ralf Kuehn, Addgene plasmid # 64324) correspondingly via replacing original Cas9 by its high fidelity version Cas9-HF1 (gift from Aliona Bogdanova, Max Planck Institute of Molecular Cell Biology and Genetics). Cal33 RR cells were co-transfected with both sgRNA constructs using DOTAP:DOPE (1:1) cationic liposomes and EGFP/mCherry double positive population was sorted after 48 h into 96-well plates at one cell/well density. Obtained clones were PCR-screened for presence of SLC3A2 deletion and validated via sequencing of regions adjacent to targeted positions. Promising clones were further checked for absence of both Cas9 integration and off-target effects predicted using CCTop online tool http://crispr.cos.uni- heidelberg.de/ (3 off-targets with highest score were checked). siRNA-mediated
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