L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population

Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM−/CD133− cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

2 according to manufacturer's recommendation; Miltenyi Biotec, Bergisch Gladbach, Germany) were used as controls. All investigated cell lines were gated individually to exclude debris, followed by single cell gating to remove dead cells and doublets. Subsequently the cells were plated for clonogenic, spherogenic and 2D responsiveness assay (assays are described in details below).
Western blot: Whole cell lysates were obtained from sub-confluent cultures. Cells were lysed for Western blot analysis according to standard laboratory protocols. The protein concentration of cell lysates was determined by BCA Protein Assay Kit (Pierce, Perbio Science, Lausanne, Switzerland).
Equal amounts of protein (20 μg) were loaded and separated using SDS-PAGE, followed by blotting onto a polyvinylidene difluoride membrane (Amersham Biosciences, Otelfingen, Switzerland). The membrane was blocked with 5% (w/v) BSA (Sigma-Aldrich, Buchs, Switzerland) in TBST for 1 h and incubated with primary monoclonal antibodies L1CAM (0.5 µ g/µ l), E-cadherin (1:1000), vimentin Cell migration assay: Sub-confluent cells were counted and 8 x 10 4 to 1 x 10 5 cells were seeded with serum-free media in 12-well plates into the upper chamber of each insert. Incubation at 37°C for 24 h allowed cells to migrate to the chemo-attractant (growth medium containing 10% FCS). After incubation, medium in the interior part of the insert was removed and the insert was dipped in 0.2% crystal violet in 4% paraformaldehyde for 20 min. The insert was intensively washed and nonmigrated cell in the interior of the insert were removed using a cotton-tip swab. Five random areas of the inserts were photographed with an Olympus IX81 microscope and cell count was performed.
Single-guided RNA design and vector construction: Single guided RNAs (sgRNA) targeting exon 2 of L1CAM were designed using the web tool of the Zhang laboratory (http://crispr.mit.edu) [2].   Table   S1). The PCR was performed using Expand Tm High Fidelity PCR System (Roche Switzerland), 300 nM forward and reverse primer, 100 ng genomic DNA (gDNA), 10 mM dNTPs and nuclease-free water under following conditions: 94°C for 2 min followed by 10 cycles of 94°C for 15 sec, 58°C for 30 sec, 68°C for 3 min, and 20 cycles of 94°C for 15 sec, 58°C for 30 sec, 68°C for 1 min 30 sec finished with 1 cycle at 68°C for 7 min. Amplicons were visualized on 1% agarose gel and purified by Wizard SV gel and PCR Clean-Up System (Promega, Dübendorf, Switzerland) and sequenced using T7 primer (Supplementary Table S1) by Sanger DNA sequencing service from Microsynth AG (Switzerland). The desired L1CAM open reading frame was then introduced into pUltra (Addgene, #24129) via NheI and XbaI cloning procedure for further lentiviral transduction. All plasmids were partly sequenced using EGFP_F primer (Supplementary Table S1) by Sanger DNA sequencing from Microsynth AG (Switzerland).

SgRNA1 and sgRNA2 (Supplementary
Lentivirus production and transduction: HEK293T cells were cultured as described above. One day prior to transfection, 4 x 10 6 cells were seeded in a T75 cm 2 flask. Cells were transfected when they Immunohistochemistry: Biopsy samples of the tumors were immediately put in 10% neutral buffered formalin for 24 h. Formalin-fixed, paraffin-embedded (FFPE) tissue sections (2 μm thickness) were mounted on positively charged slides and dried overnight at 37°C. Drying was followed by the deparaffinisation of the slides with four xylene baths for 5 min each using the Tissue-Tek® Prisma® and Film® (Sysmex,Horgen, Switzerland). For rehydration, a degressive alcohol series using 100% ethanol, 95% ethanol, 70% ethanol, and distilled water was performed. Further Antibody specific protocols were used. For L1CAM immunohistochemical staining underwent an antigenretrieval pretreatment after rehydration by putting the slides into EDTA-buffer (basic buffer pH 9.0) and then into a pressure cooker for 20 min at 98°C, followed by rinsing with distilled water.    Clonogenic capacity (left graph), spherogenic capacity (middle graph) and radiation responsiveness (right graph) of IGROV1 wild type, ∆L1CAM and ∆L1CAM rescue cells. Each experiment has been performed three times in triplicates and data are expressed as means ± SD. One-way ANOVA; ***p < 0.001. (B) Representative pictures of 2D colonies and 3D spheres. (C) After seeding, for IGROV1 wild type, ∆L1CAM and ∆L1CAM rescue cells were treated with MTT for five consecutive days. Absorbance was measured at 540 nm. Error bars indicate SD. Two-way ANOVA; ***p < 0.001 (D) Statistical plots and representative photos and of migration assays for IGROV1 wild type, ∆L1CAM and ∆L1CAM rescue cells. Following 24 h incubation, cells that migrated through the insert were counted in five random fields. One-way ANOVA; * p < 0.03 and ***p < 0.001.          Color key [% of positive cells] 0-5% 5-25% 25-50% 50-75% >75%

Table S5
Human cancer cells derived from ovarian cancer patients' ascites and ovarian cancer cell lines show heterogeneous expression of different cell surface markers.
The expression of putative CSC markers was analyzed by flow cytometry. FIGO stage for each ovarian cancer patients' ascites is provided. The numbers indicate the percentage of antigen expressing-cells in the sample population  SD of three independent experiments. Due to the limited number of cells, the expression of markers in patient-derived samples was measured only once.