cancers Article TSPAN1: A Novel Protein Involved in Head and Neck Squamous Cell Carcinoma Chemoresistance Yoelsis Garcia-Mayea 1,2 , Cristina Mir 1 , Laia Carballo 1 , Josep Castellvi 1 , Jordi Temprana-Salvador 1 , Juan Lorente 3 , Sergi Benavente 4 , Juana M. García-Pedrero 5,6 , Eva Allonca 5 , Juan P. Rodrigo 5,6 and Matilde E. LLeonart 1,7, * 1 Biomedical Research in Cancer Stem Cells, Vall d’Hebron Research Institute (VHIR), Autonomous University of Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; yoelsis.garcia@vhir.org (Y.G.-M.); cristina.mir@vhir.org (C.M.); laia.carballo@alumni.vhir.org (L.C.); joscastellvi@vhebron.net (J.C.); jtemprana@vhebron.net (J.T.-S.) 2 Genetic, Microbiology and Statistics Department, Faculty of Biology, University of Barcelona, Avenida Diagonal 643, 08014 Barcelona, Spain 3 Otorhinolaryngology Department, Hospital Vall d’Hebron (HUVH), Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; jlorente@vhebron.net 4 Radiotherapy Unit, Vall d’Hebron Research Institute (VHIR), Autonomous University of Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; sbenavente@vhebron.net 5 Department of Otolaryngology-Head and Neck Surgery, Central University Hospital of Asturias, University of Oviedo, ISPA, IUOPA, 33011 Oviedo, Spain; juanagp@ispasturias.es (J.M.G.-P.); ynkc1@hotmail.com (E.A.); jprodrigo@uniovi.es (J.P.R.) 6 Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Av. Roma SN, 33011 Oviedo, Spain 7 Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Vall d’Hebron Research Institute (VHIR), Passeig Vall d´Hebron 119–129, 08035 Barcelona, Spain * Correspondence: matilde.lleonart@vhir.org; Tel.: +34-934894169; Fax: +34-932746708  Received: 8 September 2020; Accepted: 30 October 2020; Published: 5 November 2020  Simple Summary: Therapy resistance in head and neck squamous cell carcinoma (HNSCC) patients is the main obstacle to achieve more effective treatments that improve survival and quality of life of these patients. Therefore, it is of vital importance to unravel the molecular and cellular mechanisms by which tumor cells acquire resistance to chemotherapy. We conducted a comparative proteomic study involving cisplatin-resistant cells and cancer stem cells with the aim of identifying proteins potentially implicated in the acquisition of cisplatin resistance. Through this study, we identified for the first time tetraspanin-1 (TSPAN1) as an important protein involved in the development, progression and chemoresistance of HNSCC tumors. Abstract: Sensitization of resistant cells and cancer stem cells (CSCs) represents a major challenge in cancer therapy. A proteomic study revealed tetraspanin-1 (TSPAN1) as a protein involved in acquisition of cisplatin (CDDP) resistance (Data are available via ProteomeXchange with identifier PXD020159). TSPAN1 was found to increase in CDDP-resistant cells, CSCs and biopsies from head and neck squamous cell carcinoma (HNSCC) patients. TSPAN1 depletion in parental and CDDP-resistant HNSCC cells reduced cell proliferation, induced apoptosis, decreased autophagy, sensitized to chemotherapeutic agents and inhibited several signaling cascades, with phospho-SRC inhibition being a major common target. Moreover, TSPAN1 depletion in vivo decreased the size and proliferation of parental and CDDP-resistant tumors and reduced metastatic spreading. Notably, CDDP-resistant tumors showed epithelial–mesenchymal transition (EMT) features that disappeared upon TSPAN1 inhibition, suggesting a link of TSPAN1 with EMT and metastasis. Immunohistochemical analysis of HNSCC specimens further revealed that TSPAN1 expression was correlated with phospho-SRC (pSRC), and inversely with E-cadherin, thus reinforcing TSPAN1 association with EMT. Overall, TSPAN1 emerges as a novel oncogenic protein and a promising target for HNSCC therapy. Cancers 2020, 12, 3269; doi:10.3390/cancers12113269 www.mdpi.com/journal/cancers Cancers 2020, 12, 3269 2 of 20 Keywords: cancer; HNSCC; resistance; cancer stem cells; apoptosis; autophagy 1. Introduction Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer type since approximately 60% of patients have locally advanced disease upon diagnosis and approximately half of affected individuals do not survive more than five years after diagnosis [1]. Unfortunately, chemotherapeutic choices for the treatment of primary HNSCC have not improved over the past 50 years. Current chemotherapeutic options for HNSCC patients comprise chemotherapy, based mainly on cisplatin (CDDP) and also 5-fluorouracil, taxanes or cetuximab in certain metastatic tumors [2,3]. Recently, immunotherapeutic agents (nivolumab and pembrolizumab) have also been approved for recurrent/metastasic HNSCC [4]. In general terms, the high aggressiveness of HNSCC has been closely associated with the large genetic complexity and intratumor heterogeneity as well as the presence of distinct cancer cell subpopulations within the tumors showing different cancer stem cell (CSC) properties and resistance levels. Resistance to stress, ultra-violet (UV) light, and chemotherapy or radiation treatment can be intrinsic or acquired. There is a general belief that resistance appears through natural selection of pre-existing mutant clones during stress. However, a novel type of resistance appears throughout the administration of chemotherapeutic agents, which seems to be related to treatment failure in cancer patients. Furthermore, the acquisition of resistance to one drug, usually favors the acquisition of resistance to other unrelated compounds, suggesting that the mechanisms of therapy resistance developed by cancer cells seem to follow a universal pattern against a wide variety of drugs [5,6]. CSCs are of particular relevance, emerging at the top level of the resistance scale since they are refractory to most, if not all, conventional chemotherapeutic treatments and radiotherapy. Accumulating studies show that various CSC markers and properties are crucial for tumor initiation, maintenance, expansion, and invasion, as well as mediators of treatment resistance, thereby allowing CSCs to survive in the most drastic conditions in vivo to further metastasize upon reaching an appropriate niche [7,8]. In particular, inhibition of key CSC markers such as Wnt, NANOG, CD44, CD133, CD55, ALDH1, OCT3/4, SOX2, KLF4 or NOTCH1 sensitizes cancer stem cells to chemotherapy [9,10]. Consistently, inhibition of CSC properties enhances the effects of chemotherapy [11]. Importantly, resistant phenotypes can be reverted to drug-sensitive phenotypes [12]. Consequently, in terms of applicability and clinical efficacy, cancer treatments should be capable of effectively targeting not only the bulk of cancer cells but also the subpopulations of drug-resistant cell clones, including CSCs. In metabolomic and proteomic studies, our group has recently demonstrated common regulatory pathways to distinguish resistant cancer cells (non-CSCs) and CSCs from parental cells using a breast cancer model [13]. Hence, it is of major relevance to focus on those proteins present in resistant cancer cells and CSCs in order to design more effective treatment strategies that might synergistically act with standard chemotherapy [14]. With the idea of unraveling crucial proteins involved in HNSCC chemoresistance, a proteomic study was performed using the following cell lines: CCL-138 (parental), CCL-138-R (resistant to CDDP) and CSCs (derived from parental CCL-138 cells). Based on this analysis, we identified tetraspanin-1 (TSPAN1) as one of the most significantly upregulated proteins in CCL-138-R cells and CSCs compared to parental CCL-138 cells. Tetraspanins are a family of cell surface receptors that comprise a total of 33 members involved in signal transduction [15]. The role of tetraspanins in cancer has emerged in the last decade, being involved in proliferation, metastasis and immune protection and, consequently, postulated as potential oncogenic proteins [16,17]. Recently, some tetraspanin members have also been linked to chemoresistance. For example, TSPAN9 has been found to suppress chemosensitivity to 5-fluorouracil [18]. Specifically, TSPAN1 has been proved to be important for the development of prostate, colon and gastric cancer [17,19]. For the first time, this study investigates the clinical and biological relevance of TSPAN1 in HNSCC, with a special Cancers 2020, 12, 3269 3 of 20 focus on laryngeal and pharyngeal tumors. For this purpose, the impact of TSPAN1 inhibition was functionally assessed both in vitro and in vivo using disease-relevant HNSCC models, and the clinical role of TSPAN1 was also further explored in a large series of HNSCC patient samples. 2. Materials and Methods 2.1. Cell Lines and Culture Conditions Fresh samples of normal and tumor tissue from patients surgically treated for squamous larynx carcinoma were obtained from the Otorhinolaryngology department of Vall d´Hebron University Hospital (HUVH) as described [20]. These patients had not previously undergone any radio- or chemotherapeutic treatment before surgical intervention. All of them signed the informed consent for the analysis and publication of the data derived from their samples. The present study was carried out following the instructions and requirements of the declaration of Helsinki along with the approval of the Ethical Committee of the HUVH (Ref: PR[AG]342/2016). Patient-derived cell lines were established in culture. The human pharyngeal cancer cell lines HTB-43 (FaDu) and CCL-138 (Detroit 562) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), while the laryngeal cancer cell line JHU029 Lucires-GFP (RRID: CVCL_5993) and the tongue cell line SSC-25 were kindly provided by Dr. S. Aznar-Benitah (Institute for Research in Biomedicine Barcelona, Spain). All of them were satisfactorily authenticated based on polymorphism of small repetitive tandem loci. The above-mentioned HNSCC cell lines and their respective Cancer Stem Cells (CSCs) subpopulations were cultured as previously described [20]. 2.2. Drug Assay To determine their respective IC50, JHU029, JHU029-R, HTB-43, HTB-43-R, CCL-138 and CCL-138-R, cell lines were seeded at 15 × 103 cells per well into 96-well plates, and the primary cell lines derived from patients at 10 × 103 cells per well. Twenty-four hours later, cells were exposed in triplicate to CDDP (range 0–150 µM) or dasatinib (Sigma-Aldrich, Merck, Madrid, Spain) (0–3 µM) for 48 h. Cellular viability was measured by MTS (AQueous MTS Reagent Powder, Promega Biotech Iberica, Madrid, Spain) based on the rate of metabolic activity of the previously treated cells. 2.3. Proteomic Study A comparative proteomic analysis of the following cell lines was performed: CCL-138, CCL-138-R and CCL-138 CSCs by using three biological replicates for each of them. The proteomic study has been carried out by the COS Centre (Centre for Omic Sciences, Tarragona, Spain). 2.4. Sample Preparation and LC-MS/MS Analyses The proteomic study was carried out by the technological infrastructures of the Center for Omic Sciences (COS, EURECAT, Tarragona, Spain). Cell lysis and protein solubilization was performed with modified Pierce RIPA Buffer (Thermo Scientific, Waltham, MA, USA). Thus, 200 µL of lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1% SDS and protease and phosphatases inhibitors) was added to cell pellets that were sonicated (Sonics & Materials, Vibra Cell, Illkirch, France) for three cycles of 10 s repeated six times with 15-s pause intervals. Afterwards, the suspension was centrifuged, the supernatants transferred to new tubes and protein concentration was determined by the Lowry method. Then, 65 µg of protein from each sample was reduced with 4 mM DTT for 25 min at 56 ◦ C and alkylated with 8 mM iodoacetamide for 30 min at 25 ◦ C in the dark and loaded in a polyacrylamide gel to remove detergents. Next, the gel slice containing unresolved proteins was cut into small pieces and digested overnight at 37 ◦ C with trypsin at an enzyme: protein ratio of 1:100. After digestion, a small aliquot of 2 µg was purified using C18 zip-tip (Merck Life Cancers 2020, 12, 3269 4 of 20 Science, Madrid, Spain) to check proper protein digestion by nanoLC-Orbitrap before following sample preparation steps. 2.5. Peptide 10-Plex TMT Labeling The remaining digested protein sample was desalted on C18 Sep-Pack column (Waters, Bedford, MA, USA) using 80% acetonitrile, 20% water with 0.1% formic acid for elution. The eluted peptides were dried in the Speed-Vac and labeled with TMT 10-plex labelling (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. To normalize all the samples, a pool was created by mixing an equally small aliquot of each sample and then, 40 µg of this pool and 40 µg of each of the nine individual samples were labeled. Next, a small aliquot of 5 µg from each labeled sample was mixed and purified using C18 zip-tip (Millipore, Burlington, MA, USA) and analyzed by nanoLC-Orbitrap to check the labeling reaction. 2.6. Peptide Fractionation The labeled peptides from each sample were mixed together and desalted on C18 Sep-Pack column (Waters, Bedford, MA, USA) using 80% acetonitrile, 20% water with 0.1% formic acid for elution. The TMT pooled sample was dried in the Speed-Vac and resuspended in rehydration buffer (5% Glycerol and 1% v/v IPG strip Buffer 3-10NL), and subsequently fractionated by isoelectrofocusing on an Off-Gel fractionator from Agilent Technologies through 12-well IPG strips (Nonlinear gradient from pH 3 to 10) according to the supplier’s protocol. Initially, 13-cm-long IPG strips were hydrated with 40 µL per well of the rehydration buffer. 200 µg of TMT pooled sample was loaded on the strip (150 µL of sample in each well). The samples were focused at 50 µA, with voltages between 500 and 4500 V for a total of 20 kVh. After separation, each one of the 12 fractions obtained was desalted on C18 Sep-Pack column (Waters, Bedford, MA, USA) using 80% acetonitrile, 20% water with 0.1% formic acid for elution. Eluted fractions were resuspended in 50 µL of 0.1% formic acid. 2.7. NanoLC-(Orbitrap) MS Analysis The 12 fractions obtained from Off-Gel fractionation method were separated on a trap nano-column (100 µm I.D.; 2 cm length; 5 µm particle diameter, Thermo Fisher Scientific, San José, CA, USA), and then separated onto a C-18 reversed phase (RP) nano-column (75 µm I.D.; 15 cm length; 3 µm particle diameter, Nikkyo Technos Co. LTD, Tokyo, Japan). The chromatographic separation was performed with a continuous acetonitrile gradient using Milli-Q water (0.1% FA) and ACN (0.1% FA) as mobile phase. A flow rate of 300 nL/min was used to elute peptides for real time ionization and peptide fragmentation on an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher). An enhanced FT-resolution spectrum (resolution = 30,000 FHMW) followed by two data dependent MS/MS scan events was performed. One consists of an HCD fragmentation (40% NCE) and FT-MS/MS acquisition (R = 15,000 FHMW) from most intense ten parent ions with a charge state rejection of 1 and dynamic exclusion of 0.5 min, which is used for peptide quantification. The other event consists of a CID fragmentation (35% NCE) and IT-MS/MS acquisition from the same most intense ten parent ions, which is used for peptide identification. The 12 raw data files obtained were analyzed by Multidimensional Protein Identification Technology (MudPIT) on Proteome Discoverer software v.1.4.0.288 (Thermo Fisher Scientific). For protein identification, all MS and MS/MS spectra were analyzed using Mascot search engine (version 2.5). Mascot was set up to search the SwissProt_2017_05.fasta database (554,515 entries), restricting for human taxonomy (20,202 sequences) and assuming trypsin digestion. Two missed cleavages were allowed and an error of 0.02 Da for FT-MS/MS fragment ion mass, 0.8 Da for IT-MS/MS fragment ion mass and 10.0 ppm for a FT-MS parent ion mass were allowed. TMT-10plex on lysine and N-termini were set as quantification modifications, oxidation of methionine and acetylation of N-termini were set as dynamic modifications, whereas carbamidomethylation of cysteine was set as static modifications. The false discovery rate (FDR) and protein probabilities were calculated by Percolator software. Cancers 2020, 12, 3269 5 of 20 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [21], dataset identifier PXD020159. 2.8. Protein Analysis Total protein extracts used for Western blot analysis were obtained from subconfluent cells and lysed with RIPA buffer (25 mM TrisCl, 150 mM NaCl, 1% Igepal, 1% sodium deoxycholate, 0.1% SDS, pH 7.5, plus 2 mM full cocktail of protease and phosphatases inhibitors, Thermo Scientific). The following primary antibodies were incubated overnight at 4 ◦ C: TSPAN1 (ab96070), ATG5 (ab109490) (Abcam, Cambridge, UK); LC3B (#3868), pScr (#6943), Src (#2109), pAKT (# 9271), AKT (#9272), PARP1 (#9542S), pERK1/2 (#9101L) (Cell Signaling Technology Europe Leiden, The Netherlands); SQSTM1 (p62) (SAB3500430), β-actin (A3854) (Sigma—Aldrich Química SL, Madrid, Spain); ERK1/2 (sc-514302), Vinculin (sc-73614) (Santa Cruz Biotechnology, Heidelberg, Germany). The membranes were revealed with Super Signal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Results were confirmed in at least three independent experiments. 2.9. Transfections Two hundred thousand HNSCC cells were transfected in reverse with 10 nM siRNA TSPAN1 (siTSPAN1) or 10 nM siRNA negative control (NC) (Integrated DNA Technologies) and 4 µL of Lipofectamine RNAimax (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) per well for 6-well plates in OPTIMEM serum-free medium (Gibco, Thermo Fisher, Waltham, MA, USA). Extracts were collected at 48–72 h after transfection. Furthermore, we used an additional siTSPAN1 (siTOOLs Biotech, Planegg, Germany) to demonstrate independently the biological effect of TSPAN1 inhibition. This siRNA complex pool (siPOOLs) consists of 30 optimally-designed siRNAs that has demonstrated to efficiently remove off-target effects and improve reliability of the results [22]. For the construction of the corresponding proliferation curves, six cell counts were performed every 1–3 days up to a maximum of 12 days. 2.10. Colony Formation Assay A total of 5 × 103 cells were seeded per well in triplicate in 6-well plates and the colony formation was allowed to grow for 10–14 days. The quantification of the colonies was performed by fixation with 4% paraformaldehyde, followed by staining with crystal violet (0.5% w/v in water) and quantified colorimetrically (once treated with 15% acetic acid), at 595 nm in an Epoch spectrophotometer (Biotek, Winooski, VT, USA). Results were confirmed in at least three independent experiments. 2.11. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from transfected cells at 72 h post-transfection using MirVana kit (Ambion, Austin, TX, USA). Subsequently, total RNA was treated with DNAse I using the DNA-free™ DNA Removal kit (Invitrogen, Thermo Fisher Scientific), according to the manufacturer’s instructions. The cDNA was obtained from PCR amplification of 500 ng of total RNA (RIN ratio ≥ 8) using the RevertAid H Minus First Strand cDNA Synthesis kit. The expression of TSPAN1 (Hs00371661_m1) was analyzed by qRT-PCR using the expression of IPO8 (Hs00183533_m1) as an endogenous gene and TaqMan™ Universal Master Mix II (Applied Biosystems, CA, USA; Thermo Fisher Scientific). Results were confirmed in at least three independent experiments (each sample run in triplicate). Apoptosis detection by flow cytometry and autophagy analysis by transmission electron microscopy (TEM), were respectively described elsewhere [20,23]. 2.12. Animal Models Five-week-old female mice from NMRI-FOXn1 nu/nu strain (Janvier Labs, Saint Berthevin Cedex, Le Genest-Saint-Isle, France) were used for in vivo studies. JHU029 and JHU029-R cell lines were Cancers 2020, 12, 3269 6 of 20 both labeled with luciferase and GFP as previously described [24], and transfected with siTSPAN1 vs. negative control (NC). Forty-eight hours post-transfection, 1 × 106 cells were injected in each flank of each animal, at a final volume of 200 µL of PBS and Matrigel (1:1, v:v) (Life Sciences, Corning, NY, USA). A total of 16 mice were included in the study, four mice for each of the following experimental groups: JHU029 NC, JHU029 siTSPAN1, JHU029-R NC, JHU029-R siTSPAN1. Tumor volume was measured every 2–4 days, for a total of 42 days, using an electronic calibrator. At the end point, the animals were analyzed by IVIS Spectrum (Perkin Elmer, Waltham, MA, USA). Mice were sacrificed by cervical dislocation. An autopsy was performed on all the animals to collect liver, spleen, kidneys, lungs, heart and bones, which were analyzed by IVIS Spectrum for ex vivo detection of micrometastases. 2.13. Image Platform Optical imaging studies were carried out by the Preclinical Imaging Platform technicians (Lab Animal Service, Campus Vall d’Hebron, Barcelona, Spain). For bioluminescence studies, animals were injected with an intraperitoneal injection of luciferin at a dose of 150 mg/kg body weight. Then, the animals were anesthetized prior to the scans with an isoflurane mixture (5% during induction, 2% in maintenance). Air flow was 0.8 L/min. After the image acquisitions, the animals were returned to their cages for recovery. All the procedures were performed following instructions from the institutional ethic committee (Ref. CEEA 42/15). Images were analyzed by the Preclinical Imaging Platform staff with Living Image® software, from Perkin Elmer. Study analysis consists in light radiance quantification. Signals from the light sources were isolated and characterized. The kinetic curve of bioluminescence chemical reaction was examined, searching for the highest signals in each light source. These signals are theoretically proportional to the number of cells involved in the bioluminescence reaction. Analysis units were photons (p)/second (s)/centimeter (cm2 ) /stereoradian (sr). These units are technically corrected and allow comparisons between different studies. 2.14. Patients Surgical tissue specimens, from 106 patients with laryngeal or pharyngeal squamous cell carcinoma who underwent surgical treatment at the Hospital Universitario Central de Asturias, were retrospectively collected in order to perform immunohistochemistry (IHC) studies. All experimental procedures were conducted in accordance to the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Hospital Universitario Central de Asturias (Ref. 141/19, project PI19/00560). Representative tissue sections were obtained from archival, paraffin-embedded blocks, and the histological diagnosis was confirmed by an experienced pathologist. Three morphologically representative areas were selected from each individual tumor block to construct five tissue microarray (TMA) blocks. In addition, each TMA also contained three cores of normal epithelium as an internal control. All patients had a single primary tumor, microscopically clear surgical margins and received no treatment prior to surgery. All patients were habitual tobacco smokers, 65 moderate (1–50 pack/year) and 41 heavy (>50 pack/year); 98 were habitual alcohol drinkers and 57 out of 106 patients (54%) received postoperative radiotherapy. The clinicopathologic features of HNSCC patients are summarized in Table S1. The stage of disease was determined after the surgical resection of the tumor according to the TNM system of the International Union against Cancer (7th Edition). The histological grade was determined according to the degree of differentiation of the tumor (Broders’ classification). 2.15. IHC The formalin-fixed, paraffin-embedded tissues were cut into 3-µm sections and dried on Flex IHC microscope slides (Dako, Glostrup, Denmark). The sections were deparaffinized with standard xylene and hydrated through graded alcohols into water. Antigen retrieval was performed using Envision Flex Target Retrieval solution (Dako, Glostrup, Denmark), high pH. Staining was done at room temperature on an automatic staining workstation (Dako Autostainer Plus), using the Dako Cancers 2020, 12, 3269 7 of 20 EnVision Flex + Visualization System (Dako Autostainer, Denmark) with the following antibodies: anti-TSPAN1 monoclonal antibody Clone HPA011909 (Sigma—Aldrich Química SL, Madrid, Spain) at 1:50 dilution, anti-E-Cadherin (BD Biosciences #610181, San Jose, CA, USA) at 1:4000 dilution and active SRC monoclonal antibody Clone 28 (Thermo Fisher Scientific #AHO0051) at 1:300 dilution. The SRC antibody, which detects the active protein, has been previously described [25]. Counterstaining with hematoxylin was the final step. Immunostaining was scored blinded to clinical data by two independent observers. TSPAN1 and E-cadherin were scored as follows: quantity scores from 0 to 3 were respectively assigned if 0%, 1% to 10%, 11% to 50%, and 51% to 100% of the tumor cells showed cytoplasmic staining, and the staining intensity was rated on a scale of 0 to 2 (0 = negative, 1 = weak, 2 = strong). The raw data were then converted to an Immunoreactive Score (IRS) by multiplying the quantity and staining intensity scores. For E-cadherin, an IRS score equal to or above the median (score 4) was considered high expression. TSPAN1 expression was dichotomized as negative (score 0) vs positive (scores 1 to 6) expression. In addition, nuclear expression of TSPAN1 was recorded as negative vs. positive. Active SRC staining showed a homogeneous distribution, and therefore a semiquantitative scoring system based on staining intensity was applied: low (0, 1+), moderate (2+), or high expression (3+). 2.16. Experimental Design and Statistical Rationale In order to discover new proteins involved in HNSCC chemoresistance, a comparative proteomic analysis was performed using CDDP-resistant cells (CCL-138-R) and CSCs in relation to control cells (CCL-138). For protein quantification, the ratios between each TMT-label against 126-TMT label were used. Protein quantification was normalized based on protein median and Log2 transformed for statistical analysis. Statistical analysis was performed by using Mass Profiler Professional software v. 14.5 from Agilent Technologies to find significant protein changes between the cell conditions assayed. Two different statistical analyses were performed to cover the different experimental conditions described. In first instance, One-way ANOVA and Post-Hoc analysis was applied to all cell groups. Next, an unpaired T-test for CCL-138 CSCs vs CCL-138 and CCL-138-R vs CCL-138 was applied to check differences between them. In both cases, a Benjamini-Hochberg p-value correction for multiple comparisons was applied to reduce false-positive findings. Only those proteins that were quantified in at least 2 out of 3 independent experiments were considered for statistical purposes. To determine the relevance of TSPAN1 expression in both HNSCC cell lines and patients including its possible relevance in the clinic, all the statistical analyses in patient samples and cell lines were performed using the SPSS 15.0 software package (SPSS Inc., Chicago, IL, USA). The χ2 test and Fisher’s exact test were used for comparison between categorical variables. For time-to-event analysis, Kaplan–Meier curves were plotted. Differences between survival times were analyzed by the log-rank method. Wilcoxon test was used as a non-parametric test for mean ranks comparisons. Unpaired Student’s t-test was performed to compare means between two groups. All tests were two-tailed. p values below 0.05 were considered statistically significant. 3. Results 3.1. TSPAN1 Protein Is Upregulated in CDDP-Resistant HNSCC Cells In order to identify proteins that might potentially have a relevant role in cancer chemoresistance, a proteomic analysis was performed to compare the proteins expressed in parental CCL-138 cells vs. CSCs and CCL-138-R cells (CDDP-resistant cells). A total of 1535 proteins were identified in these three cell groups (Table S2). One-way ANOVA showed that 476 proteins were differentially expressed among at least two of these groups (Table S3). A Post-Hoc analysis revealed which of these proteins were dysregulated in each group, by comparing CCL-138-R vs. CCL-138 (262 proteins), and CSC vs. CCL-138 (363 proteins) (Tables S4 and S5). Finally, 36 proteins were commonly and differentially expressed in CCL-138-R and CSCs compared to parental CCL-138 cells; 12 proteins were upregulated proteins), and CSC vs. CCL-138 (363 proteins) (Tables S4 and S5). Finally, 36 proteins were commonly and differentially expressed in CCL-138-R and CSCs compared to parental CCL-138 cells; 12 proteins were upregulated and 24 proteins downregulated (Figure 1A, Table S6). One of the most upregulated protein, a member of the tetraspanin family, named TSPAN1, was primarily selected for its potential clinical interest in various cancer models [26,27]. In order to explore the putative relevance of TSPAN1 Cancers 2020, 12,in3269 HNSCC, TSPAN1 levels were compared in the following HNSCC cell lines: CCL-138,8 of 20 JHU029, HTB-43 and SSC-25. The metastatic cell line CCL-138 derived from a pharyngeal cancer showed the highest TSPAN1 levels (Figure 1B, S8). In order to verify the proteomic results, TSPAN1 andexpression 24 proteins downregulated was (Figure blot analyzed by Western 1A, in Table the S6). One of the most aforementioned HNSCC upregulated protein, a that cell lines confirming member of the tetraspanin TSPAN1 family, named was upregulated TSPAN1, in CCL-138-R, was primarily JHU029-R selected and HTB-43-R cellsfor its potential compared clinical to their interest respective control cells in various cancer (Figure 1C, S8). models Moreover, [26,27]. TSPAN1 In order was upregulated to explore the putative in the second and relevance third generation of TSPAN1 in HNSCC, (G2 and TSPAN1 G3) of levels enriched were CSCs in compared inHTB-43 cells at protein the following HNSCC level and cell also CCL-138, lines: in G3 from JHU029, HTB-43 and CCL- and HTB-43 138 atThe SSC-25. mRNA level, as metastatic compared cell to parental line CCL-138 derivedcells (Figure from 1C,D). It should a pharyngeal cancerbe noted that showed HTB-43 TSPAN1 the highest cell line has the highest proliferative capacity in 3D conditions [28]. levels (Figure 1B, Figure S8). In order to verify the proteomic results, TSPAN1 expression was analyzed The functional relevance of TSPAN1 was assessed by siRNA depletion in HNSCC cells. by Western blot in the aforementioned HNSCC cell lines confirming that TSPAN1 was upregulated Transduced parental JHU029, HTB-43 and CCL-138 cells and their respective CDDP-resistant in CCL-138-R, JHU029-R and HTB-43-R cells compared to their respective control cells (Figure 1C, variants with siTSPAN1 robustly decreased TSPAN1 mRNA levels by ~90% in all cell lines (Figure Figure S8). Moreover, TSPAN1 was upregulated in the second and third generation (G2 and G3) of 1D). Cell proliferation decreased in TSPAN1-depleted cells (Figure 1E), and the most potent enriched CSCs reduction wasinobserved HTB-43incells at protein resistant leveland JHU029-R and also in G3 HTB-43-R cellsfrom HTB-43 compared and CCL-138 to parental at mRNA cells (Figures level, 1E as andcompared to parental S1). Consistently, cells colony (Figure capacity formation 1C,D). Itwasshould be noted that also significantly HTB-43 reduced cellTSPAN1 upon line has the highest proliferative capacity in 3D conditions [28]. depletion in CDDP-resistant and parental HNSCC cells (Figure 1F). Figure Figure 1. TSPAN1 1. TSPAN1 is isrevealed revealedas as aa protein protein involved involved ininheadheadand andnecknecksquamous squamous cell cell carcinoma carcinoma (HNSCC) chemoresistance and proliferation. (A) The proteomic study identified (HNSCC) chemoresistance and proliferation. (A) The proteomic study identified 36 proteins commonly 36 proteins commonlyinderegulated deregulated CSCs and in CSCs and CDDP-resistant CDDP-resistant cells compared cells compared with parentals with parentals (P) cells,(P) cells, TSPAN1 TSPAN1 was one of the most highly upregulated protein. (B) Western blot analysis of TSPAN1 expresion expresion was one of the most highly upregulated protein. (B) Western blot analysis of TSPAN1 in in four different four different HNSCC cells (CCL-138, JHU029, HTB-43 and SCC-25). (C) Western blot analysis HNSCC cells (CCL-138, JHU029, HTB-43 and SCC-25). (C) Western blot analysis confirmed proteomic confirmed proteomic data verifying increased TSPAN1 expression in CDDP-resistant cells; and CSC data verifying increased TSPAN1 expression in CDDP-resistant cells; and CSC G2 and G3 from the G2 and G3 from the HTB-43 cell line. (D) TSPAN1 mRNA levels in parental and CDDP-resistant HTB-43 cell line. (D) TSPAN1 mRNA levels in parental and CDDP-resistant HNSCC cells upon TSPAN1 HNSCC cells upon TSPAN1 depletion. Data were normalized to IPO8 (endogenous control), and depletion. Data were normalized to IPO8 (endogenous control), and relative to NC (negative control or relative to NC (negative control or scramble) cells assigned the value of 1. TSPAN1 mRNA level for scramble) CSC from cells assigned HTB-43 and the valuecell CCL-138 of 1. TSPAN1 lines was alsomRNA level assessed. for CSCtwo-tailed Unpaired from HTB-43 and t-test, Student’s CCL-138 *** cell lines was also assessed. Unpaired two-tailed Student’s t-test, *** (p < (p < 0.001). (E) Proliferation curves of cells transduced with siTSPAN1 vs. non-targeted siRNA control of 0.001). (E) Proliferation curves cells(NC) transduced are shown.with (F)siTSPAN1 vs. non-targeted Colony number quantificationsiRNA of HNSCCcontrol cell(NC) linesare shown. with transduced (F) Colony number siTSPAN1 quantification of HNSCC cell lines transduced with siTSPAN1 compared to the respective NC controls. Unpaired two-tailed Student’s t-test, * (p < 0.05), ** (p < 0.01), *** (p < 0.001). OD: Optical Density measured at 595 nm (Crystal violet). The functional relevance of TSPAN1 was assessed by siRNA depletion in HNSCC cells. Transduced parental JHU029, HTB-43 and CCL-138 cells and their respective CDDP-resistant variants with siTSPAN1 robustly decreased TSPAN1 mRNA levels by ~90% in all cell lines (Figure 1D). Cell proliferation decreased in TSPAN1-depleted cells (Figure 1E), and the most potent reduction was observed in resistant JHU029-R and HTB-43-R cells compared to parental cells (Figure 1E and Figure S1). Consistently, Cancers 2020, 12, x FOR PEER REVIEW 9 of 21 compared to the respective NC controls. Unpaired two-tailed Student’s t-test, * (p < 0.05), ** (p < 0.01), *** (p < 0.001). OD: Optical Density measured at 595 nm (Crystal violet). 3.2. TSPAN1 Cancers Inhibition 2020, 12, 3269 Induces Sensitivity of HNSCC Cells to Chemotherapeutic Agents 9 of 20 The effects of TSPAN1 depletion on drug response were analyzed in parental and resistant JHU029, HTB-43 capacity colony formation and CCL-138 cells.significantly was also These cell lines showed reduced levels upon of sensitization TSPAN1 depletiontoinCDDP that vary CDDP-resistant between and parental14–43% HNSCC IC50cells reductions (Figurein relation to the control cells. Sensitization to CDDP was observed 1F). by TSPAN1 depletion in all cell lines, with a higher effect in the respective resistant variants, and 3.2. TSPAN1cells HTB-43-R Inhibition Induces showing the Sensitivity of HNSCC Cells highest sensitization to Chemotherapeutic (Figure 2A,B). In order to Agents demonstrate that the phenotypic effects observed by TSPAN1 inhibition using the previous siRNA was not due to off- The effects of TSPAN1 depletion on drug response were analyzed in parental and resistant target effects, we tested an independent siRNA#2 against TSPAN1 (Integrated DNA Technologies, JHU029, HTB-43 and CCL-138 cells. These cell lines showed levels of sensitization to CDDP that Coralville, IA, USA). Moreover, a pool of 30 specific siRNAs against TSPAN1 (siPool) vary between 14–43% IC50 reductions in relation to the control cells. Sensitization to CDDP was (siTOOLsBiotech, Planegg, Germany) was also tested. TSPAN1 inhibition with these new siRNAs observed by TSPAN1 depletion in all cell lines, with a higher effect in the respective resistant variants, (siRNA#2 and siPool) decreased cell proliferation and sensitized HNSCC cells to the effects of CDDP, and HTB-43-R cells showing the highest sensitization (Figure 2A,B). In order to demonstrate that the corroborating our previous results (Figure S2) [28]. The role of TSPAN1 on CDDP response was also phenotypic effects observed by TSPAN1 inhibition using the previous siRNA was not due to off-target explored in biopsy-derived cell lines from laryngeal and pharyngeal cancer patients. We chose four effects, we tested biopsy-derived cell an independent lines with high siRNA#2 levelsagainst TSPAN1 of resistance to (Integrated CDDP (IC50DNA Technologies, values ranging 39–53 Coralville, μM). IA, USA). Moreover, a pool of 30 specific siRNAs against TSPAN1 (siPool) (siTOOLsBiotech, According to our previous report, IC50 values >15 μM were considered as highly resistant to CDDP Planegg, Germany) was biopsy-derived [20]. All four also tested. TSPAN1 inhibition cell lines showed with CDDP these new siRNAs sensitization (siRNA#2 upon TSPAN1 andinhibition, siPool) decreased with a cell proliferation and sensitized HNSCC cells to the effects of CDDP, corroborating range of 29–53% IC50 reductions in comparison with control cells (Figure 2 C,D). Therefore, our previous results TSPAN1 (Figure S2) sensitizes depletion [28]. The with role of TSPAN1 effect a stronger on CDDP response wascell in biopsy-derived alsolines explored than inin established biopsy-derived CDDP-cell lines from laryngeal and resistant HNSCC cell lines. pharyngeal cancer patients. We chose four biopsy-derived cell lines with high levels Next,of weresistance to CDDP investigated (IC50 values if the sensitizing ranging action 39–53 µM). of TSPAN1 According depletion couldtobeour previous extended report, to other IC50 values >15 therapeutic were as µM such drugs, considered dasatinib,asa highly resistant kinase dual SRC/ABL to CDDP [20]. All inhibitor four biopsy-derived actively tested in various cell lines clinical trials, including HNSCC [29,30] (www.clinicaltrials.gov). TSPAN1 inhibition sensitizedin showed CDDP sensitization upon TSPAN1 inhibition, with a range of 29–53% IC50 reductions comparison HNSCC cells with control cells to dasatinib (Figure (Figure S3). 2C,D). Hence,Therefore, the effect ofTSPAN1 TSPAN1depletion inhibitionsensitizes with a stronger on drug sensitization is effect in biopsy-derived not restricted to CDDP cell and,lines beyondthandasatinib, in established it could CDDP-resistant HNSCC also extend to other cell lines. chemotherapeutic drugs. Figure2.2.TSPAN1 Figure TSPAN1sensitizes sensitizes HNSCC HNSCC cells cells to to CDDP. CDDP. (A) (A) TSPAN1 TSPAN1 depletion depletionsensitizes sensitizesparental parentaland and resistantcells resistant cellsCCL-138, CCL-138,JHU029 JHU029and and HTB-43 HTB-43 to to CDDP. CDDP. AA representative representativeexperiment experimentisisshown shownfrom fromatat leastthree least three independent independent experiments. experiments. (B)(B) Table Table summarizes summarizes the IC50 the IC50 values values (µM)(μM) from from the shown the plots plots shown in panel A, the IC50 values are represented graphically as the percentage of sensitization in panel A, the IC50 values are represented graphically as the percentage of sensitization relative to relative control to control cells cellsa (assigned (assigned a value(C) value of 100%). of Sensitization 100%). (C) Sensitization of biopsy-derived of biopsy-derived HNSCC cell HNSCC cell lines from lines from four CDDP-resistant cancer patients: P33, P13 and P74 (laryngeal biopsies) four CDDP-resistant cancer patients: P33, P13 and P74 (laryngeal biopsies) and P23 (pharyngeal and P23 biopsy). A representative experiment is shown from at least three independent experiments. (D) Table summarizes the IC50 values (µM) from the plots shown in panel C, the IC50 values are represented graphically as the percentage of sensitization relative to control cells (assigned a value of 100%). Cancers 2020, 12, 3269 10 of 20 Next, we investigated if the sensitizing action of TSPAN1 depletion could be extended to other therapeutic drugs, such as dasatinib, a dual SRC/ABL kinase inhibitor actively tested in various clinical trials, including HNSCC [29,30] (www.clinicaltrials.gov). TSPAN1 inhibition sensitized HNSCC cells to dasatinib (Figure S3). Hence, the effect of TSPAN1 inhibition on drug sensitization is not restricted to CDDP and, beyond dasatinib, it could also extend to other chemotherapeutic drugs. 3.3. TSPAN1 Depletion Induces Apoptosis in HNSCC Cells TSPAN1 protein levels were effectively reduced by siTSPAN1 (Figure 3A, Figure 4A and Figure S8). Evidence of apoptosis induction upon TSPAN1 inhibition emerged from PARP cleavage analysis by Western blot (Figure 3A and Figure S8). The active form of PARP, cleaved PARP1 -indicative of apoptosis activation-, was found to increase in all TSPAN1-depleted cell lines. To corroborate these results, apoptosis was further evaluated by Annexin V through flow cytometry (FACS). Upon TSPAN1 inhibition, apoptosis increased in all cell lines except HTB-43-R, which was only observed in early apoptosis, (Figure 3B). In CCL-138 cells, the early and late apoptosis represent 1.4% and 4.4%, respectively (5.8% total), whereas these percentages slightly increased to 1.9% and 5.2% (7.1% total) in the corresponding TSPAN1-depleted cells. For CCL-138-R cells, early and late apoptosis represent 1.5% and 2.1%, respectively (3.6% total), while apoptosis further increased in TSPAN1-depleted cells to reach 2.2% and 4.1%, respectively (6.3% total). In parental JHU029 cells, early and late apoptosis were 7.7% and 15.4%, respectively (23.1% total apoptosis), robustly increasing in TSPAN1-depleted cells up to 3.5% and 53.9% (57.4% total). Similarly, early and late apoptosis in resistant JHU029-R cells represent 7.1% and 27.9%, respectively (35% total), and apoptosis was markedly induced in TSPAN1-depleted JHU029-R cells, reaching 7.4% and 42.2% (49.6% total). For parental HTB-43 cells, the early and late apoptosis represent 2.4% and 1.9% respectively (4.3% total), respectively increasing to 3.3% and 4.2% (7.5% total) by TSPAN1 depletion. For HTB-43-R cells, early and late apoptosis represent 4.0% and 6.1% respectively (10.1%) compared to 5.3% and 4.0% (9.3%) in TSPAN1-depleted cells (Figure 3B). These results show that TSPAN1 depletion induces apoptosis in HNSCC cells. 3.4. TSPAN1 Depletion Downregulates Several Signaling Cascades with SRC Kinase Signaling as a Central Node Based on our previous report linking HNSCC chemoresistance and autophagy [20], the possible relationship between TSPAN1 and autophagy was investigated. LC3 lipidated (LC3-II) (the most universal marker of autophagy activation) and p62 proteins decreased at protein level in cells depleted for TSPAN1 (ATG5 only decreased in parental CCL-138 cells) (Figure 4A and Figure S8). Autophagy inhibition was confirmed by TEM (Figure 4B,C). Therefore, this inhibitory effect on autophagy might be associated with the suppressive effect of TSPAN1 depletion on cell proliferation and apoptosis induction. Given the fact that TSPAN1 inhibition sensitized both parental and the CDDP-resistant derivatives of JHU029, HTB-43 and CCL-138 cells to dasatinib (Figure S3), we wonder whether phosphorylation of SRC kinase (p-SRC or active SRC), a direct target of dasatinib, could be a mediator of TSPAN1 function in our HNSCC models. In fact, p-SRC levels (an indicator of SRC activation) consistently decreased in all six cell lines tested: JHU029, JHU029-R, HTB-43, HTB-43-R, CCL-138, CCL-138-R upon TSPAN1 depletion (Figure 4A). Notably, p-SRC levels correlate well with TSPAN1 expression and, although expression is low in CSCs at G1, both proteins increase in CSCs at G3 (Figure S4A,B). Interestingly, dasatinib robustly inhibited SRC activity and its major target p-SRC in our HNSCC models; however, dasatinib did not alter the expression of various autophagy-related proteins or TSPAN1 (Figure S4B). These findings indicate that TSPAN1 inhibition efficiently targets SRC-dependent and independent signaling pathways, beyond merely mimicking the action of dasatinib as a SRC inhibitor. and 53.9% (57.4% total). Similarly, early and late apoptosis in resistant JHU029-R cells represent 7.1% and 27.9%, respectively (35% total), and apoptosis was markedly induced in TSPAN1-depleted JHU029-R cells, reaching 7.4% and 42.2% (49.6% total). For parental HTB-43 cells, the early and late apoptosis represent 2.4% and 1.9% respectively (4.3% total), respectively increasing to 3.3% and 4.2% (7.5% total) by TSPAN1 depletion. For HTB-43-R cells, early and late apoptosis represent 4.0% and Cancers 2020, 6.1% 12, 3269 respectively (10.1%) compared to 5.3% and 4.0% (9.3%) in TSPAN1-depleted cells (Figure 3B). 11 of 20 These results show that TSPAN1 depletion induces apoptosis in HNSCC cells. Cancers 2020, 12, x FOR PEER REVIEW 11 of 21 parental and their respective CDDP-resistant cells (-R) JHU029, HTB-43 and CCL-138, comparing NC vs. TSPAN1depleted cells. 3.4. TSPAN1 Depletion Downregulates Several Signaling Cascades with SRC Kinase Signaling as a Central Node Based on our previous report linking HNSCC chemoresistance and autophagy [20], the possible relationship between TSPAN1 and autophagy was investigated. LC3 lipidated (LC3-II) (the most universal marker of autophagy activation) and p62 proteins decreased at protein level in cells depleted for TSPAN1 (ATG5 only decreased in parental CCL-138 cells) (Figures 4A and S8). Autophagy inhibition was confirmed by TEM (Figure 4B,C). Therefore, this inhibitory effect on autophagy might be associated with the suppressive effect of TSPAN1 depletion on cell proliferation and apoptosis induction. Given the fact that TSPAN1 inhibition sensitized both parental and the CDDP-resistant derivatives of JHU029, HTB-43 and CCL-138 cells to dasatinib (Figure S3), we Figure wonder3. TSPAN1 Figure whether inhibition 3. TSPAN1 induces inhibition phosphorylation ofapoptosis induces apoptosis SRC kinasein HNSCC in(p-SRC HNSCC cells. cells. or (A)(A) active Western Western SRC), a blot blot analysis analysis direct target of TSPAN1 ofofTSPAN1 dasatinib, andcouldand PARP1bePARP1 cleavage acleavage mediator in in parental ofparental TSPAN1 and and CDDP-resistant CDDP-resistant function in our HNSCC HNSCC HNSCC models.cells Inupon cells upon fact,TSPAN1 TSPAN1 p-SRC depletion. levels In the In the depletion. (an indicator CCL-138-R of SRC CCL-138-R cell consistently activation) cell line, line, despite despite notnot observing decreased observing TSPAN1 inTSPAN1 inhibition all six cell atat lines tested: inhibition protein JHU029, protein level (but(but it was JHU029-R, level inhibited itHTB-43, at HTB- was inhibited at 43-R,the mRNA CCL-138, level), an CCL-138-R induction upon of PARP1 TSPAN1 was observed. depletion (FigureNote: the 4A). lower Notably, band the mRNA level), an induction of PARP1 was observed. Note: the lower band corresponds to cleaved corresponds p-SRC levels to cleaved correlate well withPARP1 PARP1 TSPAN1 (arrow). (arrow). Vinculinand, expression Vinculin was although was used used for protein for protein loadingisnormalization. expression loading low in CSCs (B) normalization. Apoptosis at (B) G1, analysis by both proteins Apoptosis FACS in increase analysis by FACS CSCs using at G3Annexin (FigureV expression kit. Representative S4A,B). Interestingly, scatterrobustly dasatinib plots of propidium inhibited iodide (y-axis) vs. SRC activity and Annexin its major using Annexin V expression kit. Representative scatter plots of propidium iodide (y-axis) vs. Annexin V-APC target p-SRC (x-axis) in ourto detect HNSCC early (right bottom models; however,quadrant) dasatiniband late did (right upper not alter thequadrant) apoptosis expression in of various V-APC (x-axis) to detect early (right bottom quadrant) and late (right upper quadrant) apoptosis in autophagy-related proteins or TSPAN1 (Figure S4B). These findings indicate that TSPAN1 inhibition parental andtargets efficiently their respective SRC-dependentCDDP-resistant cells (-R) and independent JHU029, signaling HTB-43 and pathways, CCL-138, beyond merelycomparing mimickingNC vs.the TSPAN1depleted cells. action of dasatinib as a SRC inhibitor. Figure Figure 4. Characterization 4. Characterization of of TSPAN1downstream TSPAN1 downstream effectors. (A) (A) effectors. Western blot analysis Western of proteins blot analysis of proteins modulated by TSPAN1 depletion in HNSCC cells, including various key proteins related to modulated by TSPAN1 depletion in HNSCC cells, including various key proteins related to autophagy and autophagy and SRC/AKT/ERK signaling pathways. (B) Representative electronic microscopy images SRC/AKT/ERK signaling pathways. (B) Representative electronic microscopy images in TSPAN1-depleted in TSPAN1-depleted cells vs. control cells (NC) in CCL-138 and CCL-138-R cells. TEM images cells vs.revealed control autophagosomes cells (NC) in CCL-138 and CCL-138-R cells. and autophagolysosomes, TEM images characteristic revealed features autophagosomes of autophagic cells. (C) and autophagolysosomes, Quantification ofcharacteristic features autophagic vesicles of each from autophagic cells. group. experimental (C) Quantification of autophagic Note that autophagy vesicles vesicles from each experimental decrease in CCL-138group. NoteTSPAN1 cells upon that autophagy depletion. vesicles Unpaireddecrease inStudent’s two-tailed CCL-138t-test, cells *** upon(p